Figure 1.
Effect of Aβ and tau on HRD1 solubility in N2a cells.
A. ELISA of Aβ peptides, Aβ40 and Aβ42, in N2a cells stably expressing PS2 24 h after transfection with APP-FLAG or an empty vector (mock). Results are expressed as a ratio of Aβ peptide (pg/ml) to whole cell protein extract (mg). B. Total cell lysates of NP-40-soluble and -insoluble fractions analyzed by western blotting with the indicated antibodies. C. Statistical analysis of (B) NP-40 soluble HRD1. Data are normalized to β-actin levels, and results are expressed as the fold increase compared with protein expression levels in untransfected control (mean ± SEM; n = 3). Statistical analysis was performed with ANOVA, followed by Student’s t-test (mock vs. APP; *p<0.05). D-E. Western blotting of (D) NP-40-soluble and (E) NP-40-insoluble proteins in the total cell lysates of N2a cells 24 h after transfection with the four-repeat isoform of human tau (tau0N4R; 0N4R), mutant tau (tauP301L; P301L), or the empty vector (mock). Abbreviation: m, mock. A, APP. p.c., positive control, which was extracted by NP-40 from wild-type HRD1-expressing Flp-In-293 cell.
Figure 2.
Influence of Aβ or tau on HRD1 and SEL1L solubility in the cerebral cortex of Tg2576 or JNPL3 mice.
HRD1 and SEL1L levels in the cerebral cortex of Tg2576 (hemizygous human Swedish double-mutated APP transgenic; APPSWE) or JNPL3 (human mutated four-repeat tauP301L transgenic; TauP301L) mice. The cerebral cortex of each mouse at 16 to 20 months of age was examined. The total lysates of (A) NP-40-soluble and (B) -insoluble fractions were analyzed by western blotting with the indicated antibodies; statistical analysis of these results are expressed as a dot plot (• wt, n = 7; • APPSWE, n = 4; ○ TauP301L, n = 5). Data are normalized to the wt averages for each fraction. Statistical analysis was performed with ANOVA, followed by Bonferroni correction (Control vs. APPSWE and TauP301L; *p<0.05).
Figure 3.
Induction of HRD1 and SEL1L expression by ER stress-inducing agents.
A. Real-time PCR analysis of gene expression in N2a cells treated for 24 h with or without tunicamycin (Tm; an inhibitor of N-linked glycosylation) and thapsigargin (Tg; an inhibitor of Ca2+ ATPase). Data are normalized to the amount of β-actin; results are expressed as a fold increase compared with the non-treated control (mean ± SEM; n = 3). Statistical analysis was performed with ANOVA, followed by Bonferroni correction (Control vs. Tm and Tg; *p<0.05). B-C. Western blotting of (B) NP-40-soluble and (C) -insoluble fractions from N2a cells treated with or without Tm and Tg for 24 h using the indicated antibodies. The black arrow in soluble fraction indicates mature SEL1L, and the white arrow indicates immature SEL1L without glycosylation.
Figure 4.
Insolubilization of HRD1 and SEL1L proteins by oxidative stress-inducing agents.
A–C. SH-SY5Y cells were treated with or without H2O2 (A) and rotenone (B) for 24 h. The total cell lysates of NP-40-soluble (left panel) and -insoluble (right panel) fractions were analyzed by western blotting with the indicated antibodies. C. Decrease in soluble HRD1 due to oxidative stress under inhibition of de novo protein synthesis. SH-SY5Y normal cells were treated with 25 µg/ml CHX. At 5 min after treatment, the cells were additionally treated with or without 100 µM hydrogen peroxide (H2O2) for 6 h. The total cell lysates of NP-40 soluble fractions were analyzed by western blotting using the indicated antibodies. Data are normalized to the amount of β-actin; results are expressed as a fold increase compared with the non-treated control (mean ± SEM; n = 6). Statistical analysis was performed with ANOVA, followed by Student’s t-test (CHX vs. CHX+H2O2; *p<0.05). Abbreviation: NT, non-treated control.
Figure 5.
4-HNE modifications of HRD1 protein.
A–B. SH-SY5Y cells were treated with or without 4-HNE for 24 h. The total cell lysates of NP-40-soluble (A) and -insoluble (B) fractions were analyzed by western blotting with the indicated antibodies. C. Total cell lysates were immunoprecipitated (IP) with anti-HRD1/SYVN1 antibody. These IP samples were treated with or without 100 µM of 4-HNE at 37°C for 3 h and then analyzed by western blotting with the indicated antibodies. Black arrow, 4-HNE-modified HRD1; white arrow, unmodified HRD1 protein. Lane 1, total cell lysate; Lane 2, IP with anti-HRD1/SYVN1 antibody; Lane 3, IP with anti-HRD1/SYVN1 antibody treated with 4-HNE; Lane 4, IP with anti-mouse IgG.
Figure 6.
Induction of HRD1 aggresome formation by oxidative stress.
SH-SH5H cells treated with H2O2 (10 µM), rotenone (10 nM), and 4-HNE (15 µM) for 48 h and then subjected to immunofluorescence staining using HRD1 (red) and γ-tubulin (green) antibodies. Arrowheads, MTOC; arrows, HRD1 in aggresomes. Scale bars, 20 µm.