Animal experiments

All animal procedures were conducted in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan. Protocols were approved by the Animal Experimentation Committee of Kyoto University (MedKyo: 21504, 22093, 23078, 24111, 25125). Unless otherwise specified, all mice used in this study were of the C57BL/6J strain. Generation and genotyping of SKO and GlcAT-P gene knockout (PKO) mice have been described previously [2,5]. To collect tissues, euthanasia was performed either by cervical dislocation under deep isoflurane anesthesia or by CO₂ inhalation, in accordance with institutional guidelines. For perfusion fixation experiment, mice were anesthetized by intraperitoneal injection of a mixed anesthetic consisting of medetomidine hydrochloride (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg) to alleviate suffering.

Preparation of renal membrane fractions

Adult mouse kidneys were homogenized in five volumes of ice-cold homogenization buffer (0.25 M sucrose, 10 mM Tris-HCl [pH 7.4], 1 mM EDTA, and protease inhibitor cocktail; Cat#25955−11, Nacalai Tesque, Kyoto, Japan). Homogenates were centrifuged at 1,000 × g for 10 min to remove nuclei, and the supernatant was further centrifuged at 105,000 × g for 1 h. The resulting pellet was resuspended in 1% SDS and used as the renal membrane fraction.

Western and lectin blotting

Membrane fractions were separated by SDS-PAGE (7% or 10%) and transferred onto nitrocellulose membranes. Precision Plus Protein™ All Blue Prestained Standards (Cat#1610373, Bio-Rad, Hercules, CA, USA) were used as molecular weight markers. Membranes were blocked with 5% skim milk in phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST), incubated with primary antibodies (1 µg/mL), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (1 µg/mL). For lectin blotting, membranes were blocked with Blocking One (Cat#03953–95, Nacalai Tesque) and incubated with HRP- or biotin-conjugated lectins diluted in 5% Blocking One in PBST. When biotin-conjugated lectins were used, HRP labeling was carried out according to the manufacturer’s instructions provided with VECSTAIN ABC Kit (Cat#PK-4000, Vector Laboratories, Newark, CA, USA). Signals were developed using SuperSignal™ West Pico Chemiluminescent Substrate (Cat#34580, Thermo Fisher Scientific, Waltham, MA, USA) and visualized with a LuminoGraph II system (ATTO, Tokyo, Japan). Band intensities were quantified using ImageJ software. Primary antibodies: M6749 monoclonal antibody (mAb; kindly provided by Dr. H. Tanaka, Kumamoto University [21]), anti-β-actin mAb (Cat#M177-3, MBL, Tokyo, Japan), anti-APN mAb (Cat#ab108310, Abcam, Cambridge, UK), and anti-MEP1A polyclonal antibody (pAb; Cat#PA5–81444, Thermo Fisher Scientific). Secondary antibodies: HRP-conjugated anti-mouse IgM pAb (Cat#62–6820), HRP-conjugated anti-mouse IgG pAb (Cat#62–6520), and HRP-conjugated anti-rabbit IgG pAb (Cat#65–6120), purchased from Thermo Fisher Scientific, respectively. Lectins: HRP-conjugated RCA120 lectin (from Lectin Set-HRP 1; Cat#J4S1, J-OIL MILLS, Tokyo, Japan), Biotin-conjugated MAM lectin (specific for α2–3-linked sialic acids; Cat#J210, J-OIL MILLS), and SiaFind α2–6 Specific Reagent Biotinylated Kit (specific for α2–6-linked sialic acids; Cat# SK2601B, Lectenz Bio, Athens, GA, USA). A polyclonal antibody against GlcAT-S (anti-GlcAT-S pAb) was generated using the same method previously employed for GlcAT-P [22], by immunizing a rabbit with the recombinant catalytic region of human GlcAT-S expressed and purified from Escherichia coli.

Real-time quantitative PCR

Total RNA from kidneys was extracted using TRIzol (Cat#15596018, Thermo Fisher Scientific), and cDNA synthesized from 2 µg RNA using oligo(dT) primer (Cat#3805, Takara Bio, Shiga, Japan) and SuperScript™ III Reverse Transcriptase (Cat#18080093, Thermo Fisher Scientific). qPCR was performed using StepOnePlus system (Thermo Fisher Scientific) and TB Green Premix Ex Taq II (Cat#RR820A, Takara Bio) according to the manufacture procedure. Primer pairs were used as follows: B4galt1, 5’-agcaactcgactatggcatc-3’, 5’-tccatcggaatgaggtccac-3’; Mgat4a, 5’-acaaaccggtcaacgtggag-3’, 5’-ccctctgcaacaccatactc-3’; Mgat4b, 5’-aagcctcaagacgtaccagc-3’, 5’-gcagcacctccacagaagtg-3’; Mgat5, 5’-cccttgtgtatggcaaagtg-3’, 5’-gtcacgtccactgagaatgc-3’; B3gat2 (mouse GlcAT-S), 5’-gacgatgacaacacgtacag-3’, 5’-taccaacggacgttcatagc-3’; B3gat1 (mouse GlcAT-P), 5’-gctcatcttgcagcgaagtc-3’, 5’-ccctcattgaccagcactgg-3’; Ugdh, 5’-agtgagctacagaccattgg-3’, 5’-aacttcggaatctcaccagg-3’; Uxs1, 5’-ccactcacagtctatggctc-3’, 5’-cacttccactaccaacaagg-3’; Gapdh, 5’-acttcaacagcaactcccac-3’, 5’-atgtaggccatgaggtccac-3’. Each primer was used at 0.4 µM. Reactions were run in duplicate for three biological replicates. Relative expression was calculated by the ΔΔCt method, using Gapdh as an internal control.

Immunofluorescence staining

Mice were deeply anesthetized and perfused transcardially with PBS, followed by 4% paraformaldehyde in PBS. Then, kidneys were immersed in 30% sucrose, sectioned at 40 µm using a microtome, and blocked with 3% bovine serum albumin (BSA) in PBS (BSA/PBS). Sections were incubated with M6749 mAb, anti-megalin pAb (Cat#ab76969, Abcam), and anti-MEP1A pAb (2.5 µg/mL in 3% BSA/PBS, respectively) for 2 h, followed by FITC-conjugated anti-mouse IgM pAb (1:400 in 3% BSA/PBS; Cat#31992, Thermo Fisher Scientific) or Alexa Fluor™ 488-conjugated anti-rabbit IgG pAb (1:400 in 3% BSA/PBS; Cat#A21206, Thermo Fisher Scientific) for 1 h. All steps were conducted at room temperature. Images were acquired using a FLUOVIEW FV1000 imaging system (Olympus, Tokyo, Japan).

UDP-GlcA extraction

UDP-GlcA was extracted from mouse kidney tissues or cultured cells based on a protocol from a previous report [23]. Briefly, ~ 25 mg of tissues or cells were homogenized in 500 µL of 60% methanol. After additional sonication, they were mixed with 225 µL chloroform. After centrifugation at 18,000 × g for 3 min at 4 °C, the upper aqueous layer was collected, dried in a vacuum evaporator, and reconstituted in ultrapure water.

ELISA for UDP-GlcA quantification

UDP-GlcA levels were quantified using a modified ELISA method based on a previous report with slight modification, originally developed for the quantification of UDP-N-acetylglucosamine [23]. Briefly, microplates were coated with 0.01% poly- L-lysine (Cat#P2636, Sigma-Aldrich, St. Louis, MO, USA) for 4–6 h at room temperature, followed by 0.5 µg asialorosomucoid (ASOR) overnight at 4 °C. ASOR is a glycoprotein in which terminal sialic acids on N-glycans have been removed, thereby exposing LacNAc structures that serve as acceptor substrates for GlcA transfer. In this study, ASOR was prepared according to a previously reported method [24]. A reaction mixture containing 0.1 M MES (pH 6.5), 0.2% Nonidet P-40, 10 mM MnCl₂, 2.5 mM adenosine triphosphate (ATP), and 10 µg/mL recombinant GlcAT-P (prepared according to a previous procedure [25]), along with samples and/or UDP-GlcA standards (Cat#217−00953, FUJIFILM Wako, Osaka, Japan), was added and incubated at 37 °C for 1 h. Note that recombinant GlcAT-P was used in this assay, as the binding to ASOR was not clearly observed with GlcAT-S and had been reliably analyzed only with GlcAT-P in the previous surface plasmon resonance experiments [26]. Plates were blocked with 1% BSA in Tris-buffered saline (TBS) and sequentially incubated with M6749 mAb (1 µg/mL in 1% BSA/TBS containing 0.1% Tween-20) and HRP-conjugated anti-mouse IgM pAb (1:5000, in 1% BSA/TBS containing 0.1% Tween-20). The reaction was developed using SureBlue™ TMB 1-Component Microwell Peroxidase Substrate (Cat#5120−0081, LGC Clinical Diagnostics, Milford, MA, USA), terminated with 1 M HCl, and absorbance at 450 nm was measured using an Infinite M200 plate reader (Tecan, Zürich, Switzerland). UDP-GlcA concentrations were determined based on absorbance values calibrated against a standard curve, and normalized to the wet weight of tissue or cells used in each assay, yielding values expressed in pmol/mg.

Expression plasmids and cell culture

The rat GlcAT-S expression plasmid, pEF-BOS/GlcAT-S, was constructed as previously described [27]. HK-2 and HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (Cat#08456−65, Nacalai Tesque) supplemented with 10% fetal bovine serum (Cat#172012, Sigma-Aldrich) at 37 °C in 5% CO₂. Cells at ~80% confluence in 6-cm dishes were transfected with plasmids using Lipofectamine 3000 (Cat#L3000008, Thermo Fisher Scientific), following the manufacturer’s protocol, and cultured for 48 h. For glucose treatment, D-glucose was added at final concentrations of 5 mM or 25 mM at the time of transfection. For UDP-GlcA depletion, 4-methylumbelliferone (4-MU; Cat#M1381, Sigma-Aldrich) was added to a final concentration of 1 mM at 24 h post-transfection, and cells were incubated for an additional 24 h. Dimethyl sulfoxide (DMSO) alone was used as a vehicle control.

Statistical analysis

Student’s t-test was used for comparisons between two groups. For comparisons among four groups, one-way ANOVA followed by Holm-Sidak post hoc test was applied by using OriginPro (OriginLab, Northampton, MA, USA). All source data for the graphs are included in S1 File.

nsHNK-1 glycan expression is more abundant in female kidneys

Sex differences in renal expression of nsHNK-1 glycans were investigated by western blotting analysis of renal membrane fractions from male and female mice. nsHNK-1 glycan expression was approximately three-fold higher in female kidneys than in males (Fig 1A and 1B), revealing a significant sex difference in renal nsHNK-1 glycan expression. Since a similar trend was observed in another mouse strain (S1A Fig), the higher renal expression of nsHNK-1 glycans in female mice appears to be a conserved mechanism in mammals, at least in mice. The spatial distribution of nsHNK-1 glycan expression in the kidney was further characterized by performing immunofluorescence staining on renal sections from male and female mice (S1B Fig). Focusing on the cortical region, nsHNK-1 glycan was predominantly localized to proximal tubules in both sexes. However, in female kidneys, the expression extended more prominently into the outer layer of the cortical region. This distribution pattern closely resembled that of megalin, a well-established marker for proximal tubules that is expressed throughout the S1, S2, and S3 segments [28,29]. In contrast, MEP1A, another proximal tubule marker primarily localized to the S3 segment corresponding to proximal straight tubules [28,30], exhibited an expression pattern that overlapped with the male distribution of nsHNK-1 glycan but not with the expanded outer localization observed in females. These findings suggest that nsHNK-1 glycan expression may be relatively abundant in the S1 and S2 segments of the proximal tubules in the female mice.

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Fig 1. Nonsulfated HNK-1 glycan is highly expressed in female mouse kidney.

(A) Renal membrane fractions from wild-type mouse kidneys were immunoblotted with the M6749 monoclonal antibody and anti-β-actin monoclonal antibody. β-actin was used as a loading control. All other blots in this figure were performed using the same loading amounts as in (A). (B) Relative expression levels of nonsulfated HNK-1 glycans (M6749) were quantified based on the band intensities in (A), normalized to β-actin. (C, E) Renal membrane fractions were blotted with RCA120 (C) and MAM (E) lectins. (D, F) Band signals in (C) and (E) were quantified relative to β-actin. (G) Immunoblots of renal membrane fractions using antibodies against APN and MEP1A. Bands marked by closed and open triangles correspond to the bands shown in (A). (H, I) Expression levels of nonsulfated HNK-1 glycans associated with APN (H) and MEP1A (I) were quantified from the band intensities indicated by closed and open triangles. All graphs represent mean ± SEM (male, n = 3; female, n = 3). Statistical analysis was performed using Student’s t-test. *: p < 0.05, ***: p < 0.001, n.s.: not significant. “M” and “F” in figures indicate Male and Female, respectively.

https://doi.org/10.1371/journal.pone.0335730.g001

To explore the possibility that increased expression levels of LacNAc structure may cause high expression of nsHNK-1 glycan in females, lectin blotting was conducted using RCA120, which binds terminal LacNAc structures (Fig 1C and 1D). Given that terminal sialic acid also modifies LacNAc and can potentially compete with GlcAT-S-mediated glucuronylation, blotting using sialic acid-specific lectins was additionally performed to assess sialylation levels (Fig 1E and 1F). A comparison between the sexes revealed that sialic acid levels were unaltered in both α2−3 and α2−6 sialylation (Fig 1E and 1F, S2C and S2D Fig). Likewise, LacNAc levels showed no significant sex difference (Fig 1C and D). Consistent with this finding, the mRNA level of B4galt1, which is involved in galactosylation of nonreducing terminus of N-glycan [31], did not show significant differences between sexes (S1E Fig). Furthermore, the mRNA levels of Mgat4a, Mgat4b, and Mgat5, which contribute to N-glycan branching [32], were also comparable between sexes, suggesting that the extent of glycan branching is unlikely to account for the high nsHNK-1 glycan expression in females. These findings suggest that glycan expression in female kidneys is not broadly increased compared with males. Rather, nsHNK-1 biosynthesis is selectively enhanced in female kidneys.

To determine whether high nsHNK-1 glycan expression in female kidneys is caused by increased levels of its known carrier proteins, western blotting was performed for APN and MEP1A, both previously identified as major carriers of nsHNK-1 glycans [4,9]. APN expression showed no significant sex difference, whereas MEP1A levels were slightly lower in females (Fig 1G). When normalized to the expression levels of their respective carrier proteins, nsHNK-1 glycan expression levels were approximately four-fold higher on APN and two-fold higher on MEP1A in female kidneys compared with male kidneys (Fig 1H and 1I). These results indicate that the high nsHNK-1 glycan expression in female kidneys is not attributable to increased expression of carrier proteins but reflects enhanced biosynthesis of nsHNK-1 glycan.

Sex difference in renal nsHNK-1 expression is independent of GlcAT-S and GlcAT-P levels

mRNA and protein levels of GlcAT-S (B3gat2) were examined to assess whether the abundant expression of nsHNK-1 glycans in female kidneys is caused by high expression of GlcAT-S. Quantitative PCR using cDNA from male and female mice kidneys exhibited no significant difference in B3gat2 transcript levels between sexes (Fig 2A). Rather, a slight decrease was observed in females. Similarly, GlcAT-S protein levels showed no significant sex difference (Fig 2B and 2C), and the signal was completely absent in kidneys from SKO mice, confirming the specificity of the antibody (Fig 2B). These results suggest that the higher expression of nsHNK-1 glycans in female kidneys is not explained by GlcAT-S expression.

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Fig 2. No sex difference in the expression of GlcAT-S and GlcAT-P.

(A) B3gat2 mRNA expression levels relative to Gapdh were determined by qPCR (male, n = 3; female, n = 3). (B) Renal membrane fractions prepared from wild-type (WT) and GlcAT-S gene knockout (SKO) mouse kidneys were immunoblotted with an anti-GlcAT-S polyclonal antibody. (C) GlcAT-S protein expression levels relative to β-actin were quantified based on the immunoblot in (B). Band intensities of β-actin were identical to those in Fig 1A (male, n = 3; female, n = 3). (D) B3gat1 mRNA expression levels relative to Gapdh were determined by qPCR (male, n = 3; female, n = 3). (E) Renal membrane fractions from WT, SKO, and GlcAT-P gene knockout (PKO) mouse kidneys were immunoblotted with the M6749 monoclonal antibody and anti-β-actin monoclonal antibody. All graphs show mean ± SEM. Statistical analysis was performed using Student’s t-test. n.s.: not significant. “M” and “F” in figures indicate Male and Female, respectively.

https://doi.org/10.1371/journal.pone.0335730.g002

Although GlcAT-P is considered absent in the kidney, a previous proteomics study detected GlcAT-P peptides in rat collecting ducts [33]. Therefore, to address its potential involvement in the sex difference of nsHNK-1 glycan expression, the expression of B3gat1 was assessed. No significant difference in B3gat1 mRNA levels between sexes was observed (Fig 2D). Renal membrane fractions from both sexes of WT, SKO, and PKO mice were then compared to further evaluate the involvement of GlcAT-P in nsHNK-1 glycan biosynthesis (Fig 2E). In SKO mice, nsHNK-1 glycan expression was undetectable in both sexes, indicating that GlcAT-P is not sufficient to support nsHNK-1 glycan synthesis. Moreover, in PKO mice, nsHNK-1 glycan expression remained markedly higher in female kidneys compared with male kidneys, with an expression pattern resembling that observed for WT mice. These results collectively indicate that GlcAT-P does not contribute to the sex difference in renal nsHNK-1 glycan expression.

Sex difference in renal UDP-GlcA levels implies higher utilization in female kidneys

GlcAT-S uses UDP-GlcA as the donor substrate to add glucuronic acid to the nonreducing terminus of LacNAc. However, UDP-GlcA is also used by UGTs, a family of enzymes that catalyze glucuronidation reactions [11]. Notably, previous RNA sequencing data from isolated mouse proximal tubules showed that Ugt1a2, a member of the UGT family, is expressed at significantly higher levels in females compared with males [19,20]. Additionally, urinary and blood levels of UDP-GlcA have been reported to differ between males and females [34]. These findings suggest that sex differences in UDP-GlcA availability contribute to the high expression of nsHNK-1 glycans in female kidneys.

Quantifying UDP-GlcA levels was achieved by developing a simple and reproducible ELISA-based assay based on a previous report with slight modifications (Fig 3A) [23]. In this method, ASOR was immobilized on a microplate as the substrate for GlcA modification. UDP-GlcA was then added with purified recombinant GlcAT-P, which was expressed in Escherichia coli. The reaction was performed at 37 °C, allowing GlcAT-P to transfer glucuronic acid from UDP-GlcA to LacNAc, thereby generating nsHNK-1 structures in vitro. The resulting nsHNK-1 glycans were detected with the M6749 mAb, and UDP-GlcA levels were estimated based on the HRP-dependent signal intensity. The feasibility of this assay was validated by generating a standard curve using serial dilutions of the UDP-GlcA trisodium salt. A strong linear correlation was observed between the UDP-GlcA concentration and absorbance, confirming that the assay reliably quantifies UDP-GlcA levels (Fig 3B, S2A Fig). Note that the absorbance remained unchanged upon addition of kidney extracts to the assay, indicating that interfering factors derived from kidney tissue are negligible in this measurement system (S2B Fig).

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Fig 3. Low UDP-GlcA levels in female mouse kidneys.

(A) Schematic overview of the ELISA-based assay developed to quantify UDP-GlcA levels. This method utilizes immobilized asialorosomucoid (ASOR) as a GlcA-accepting substrate and purified recombinant GlcAT-P to enzymatically transfer GlcA from UDP-GlcA to N-acetyllactosamine structures in vitro. The resulting nsHNK-1 glycans are detected using the M6749 monoclonal antibody, and UDP-GlcA levels are estimated based on HRP-derived signal intensity. (B) Validation of the method described in (A). A standard curve was generated using serial dilutions of UDP-GlcA trisodium salt. (C) UDP-GlcA levels in kidneys from wild-type (WT) and GlcAT-S gene knockout (SKO) mice were measured using the ELISA-based assay described in (A) (WT male and SKO male, n = 6; WT female and SKO female, n = 5). Statistical analysis was performed using one-way ANOVA followed by Holm-Sidak post hoc test. *: p < 0.05, ***: p < 0.001. n.s.: not significant. The statistical test results shown in the graph are as follows: WT male vs. WT female, ***; WT male vs. SKO male, n.s.; WT male vs. SKO female, n.s.; WT female vs. SKO female, *. (D) Schematic of the UDP-GlcA biosynthetic pathway. Abbreviations: UDP-Glc, UDP-glucose; UDP-Xyl, UDP-xylose; UGDH, UDP-glucose dehydrogenase; UXS1, UDP-glucuronate decarboxylase 1. (E) Ugdh and Uxs1 mRNA expression levels relative to Gapdh in WT mouse kidneys were determined by qPCR (male, n = 3; female, n = 3). Statistical analysis was performed using Student’s t-test. n.s.: not significant. All graphs show mean ± SEM. “M” and “F” in figures indicate Male and Female, respectively.

https://doi.org/10.1371/journal.pone.0335730.g003

Using this assay, renal UDP-GlcA levels were measured (Fig 3C). In WT mice, UDP-GlcA levels were significantly lower in females than in males, suggesting greater use of this compound in female kidneys. This sex difference was abolished in SKO mice, indicating that GlcAT-S-mediated consumption contributes to the low UDP-GlcA levels in females. Notably, UDP-GlcA levels were significantly high in SKO females compared with WT females, and although these levels in SKO females tended to be slightly lower than those in WT males, the difference was not significant. Additionally, to explore whether sex differences in UDP-GlcA levels arise from the biosynthetic pathway of UDP-GlcA, the expression of UDP-glucose dehydrogenase and UDP-glucuronate decarboxylase 1, which are key enzymes involved in UDP-GlcA metabolism (Fig 3D) [10,35], was examined. No significant differences in their mRNA expression were observed between males and females (Fig 3E). These findings suggest that the biosynthetic level of UDP-GlcA does not differ substantially between sexes, supporting the notion that UDP-GlcA is actively consumed in a GlcAT-S-dependent manner in female kidneys.

UDP-GlcA availability mediates nsHNK-1 glycan expression

The influence of intracellular UDP-GlcA levels on nsHNK-1 glycan expression was investigated using an in vitro approach. Increasing the glucose concentration in culture media increases UDP-GlcA levels through enhanced flux in the nucleotide sugar biosynthetic pathway [36]. Consistently, culturing HK-2 cells under high-glucose (HG, 25 mM) conditions caused a significant increase in intracellular UDP-GlcA compared with normal glucose (NG, 5 mM) conditions (Fig 4A). HK-2 cells essentially lack endogenous GlcAT-S. Thus, GlcAT-S was transfected into the cells to allow nsHNK-1 glycan synthesis. Under these conditions, a significant increase in nsHNK-1 glycan expression was observed in HG-cultured cells relative to NG (Fig 4B). This glucose-dependent upregulation of nsHNK-1 glycans was also reproduced in HEK293 cells (S3 Fig), suggesting that this regulatory mechanism is preserved across distinct kidney-derived cell types. 4-MU was added to the culture media to assess whether this effect was dependent on UDP-GlcA availability. 4-MU acts as a glucuronidation substrate, reducing intracellular UDP-GlcA levels through its consumption by UGT enzymes [37]. In the presence of 4-MU, the difference in nsHNK-1 glycan expression between NG and HG conditions was no longer detectable (Fig 4C). Collectively, these results demonstrate that glucose-induced increases in UDP-GlcA levels are sufficient to drive nsHNK-1 glycan expression, underscoring intracellular UDP-GlcA availability as a key metabolic determinant of nsHNK-1 glycan biosynthesis in the kidney.

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Fig 4. High UDP-GlcA levels increase nsHNK-1 glycan expression.

(A) HK-2 cells were cultured for 48 hours in medium containing either 5 mM glucose (normal glucose, NG) or 25 mM glucose (high glucose, HG). UDP-GlcA levels were then quantified using the ELISA-based assay described in Fig 3A. Graphs represent mean ± SEM (NG, n = 3; HG, n = 3). Statistical analysis was performed using Student’s t-test. *: p < 0.05. (B) Cells were transfected with or without GlcAT-S and subsequently cultured under NG or HG conditions for 48 h. Cell lysates were immunoblotted with the M6749 monoclonal antibody, anti-β-actin monoclonal antibody, and anti-GlcAT-S polyclonal antibody. (C) GlcAT-S-expressing cells were incubated under NG and HG conditions. After 24 h of incubation, 4-methylumbelliferone (4-MU) was added to the culture medium at a final concentration of 1 mM. Cell lysates were then immunoblotted with the M6749 monoclonal antibody, anti-β-actin monoclonal antibody, and anti-GlcAT-S polyclonal antibody.

https://doi.org/10.1371/journal.pone.0335730.g004