Figure 1.
Identification of a new putative human LAP1 isoform.
A-Transfection of SH-SY5Y cells with pSIREN-RetroQ vector coding for LAP1-specific shRNAs resulted in the knockdown of two LAP1 isoforms: LAP1B and putative LAP1C. Data are presented as mean ± SEM of at least three independent experiments. Statistically different from CMS transfected cells, *p<0.05, **p<0.01. C1, pSIREN-C1 (directed against exon 7/8 of LAP1), C2, pSIREN-C2 (directed against exon 10 of LAP1); CMS, pSIREN-CMS (control missense). B- Transfection of SH-SY5Y cells with Myc-LAP1B (0.5 or 1 µg). Ponceau S staining was used as loading control. NT, non-transfected; IB, immunoblotting.
Figure 2.
Gene structure and splice variants of human, rat and mouse TOR1AIP1.
A- Structure of the human, rat and mouse TOR1AIP1 gene (coding for LAP1). Non-coding sequences in exons are represented by open boxes and coding exons are represented by black filled boxes. Predicted exons 1b and 3b in human and rat genes, and exon 2b in human gene are represented by grey boxes and denoted by an asterisk. The in-frame ATG codons are indicated by arrows. B- Schematic representation of the alternative splicing patterns and resulting LAP1 transcripts variants (human, rat and mouse). The translation initiation codons (ATG) and the stop codons (TAA or TGA in human and mouse/rat sequences, respectively) are indicated in each transcript. Human LAP1 transcripts variants differ only in exon 3 (dark grey) by three nucleotides through an alternative 3′ splicing event.
Figure 3.
A- Localization of the primers used for RT-PCR on human TOR1AIP1 gene. The cDNA was synthesized from adult brain poly A+ RNA (Clontech) (B, C and D) or SH-SY5Y cell total RNA (E) using a reverse primer targeted to exon 10 (E10RV) or an oligo(dT)20 primer. Amplification of the cDNA was performed using specific primer pairs. B- cDNA amplification using primers E1FW (targeted to exon 1) and E10BRV (targeted to exon 10). C- cDNA amplification using primers E2FW (targeted to exon 1/2) and E10BRV (targeted to exon 10). D- cDNA amplification using primers E5FW (targeted to exon 5) and E10CRV (targeted to the middle of exon 10). E- cDNA amplification using primers E1FW (targeted to exon 1) and E10BRV (targeted to exon 10). 1kb, DNA size marker 1kb ladder (Invitrogen); 1, cDNA synthesized using E10RV primer; 2, cDNA synthetized using oligo(dT)20 primer.
Figure 4.
Northern blot analysis of LAP1 RNAs in SH-SY5Y cells.
A- Total RNA was isolated from SH-SY5Y cells and membranes hybridized with a biotinylated probe directed against exon 10 of LAP1. β-actin was probed as control. RNA size markers are depicted on the left. 1, RNA isolated from SH-SY5Y cells non-differentiated; 2, RNA isolated from SH-SY5Y cells differentiated with retinoic acid.
Table 1.
LAP1B and LAP1C peptides identified by liquid chromatography-mass spectrometry.
Table 2.
Human LAP1 transcripts and isoforms.
Figure 5.
Predicted promoter and alternative transcription initiation site of human LAP1C.
A- Localization of a predicted promoter in the TOR1AIP1 genomic sequence. The promoter region predicted using the NNPP program is underlined. The transcription initiation site predicted by the TSSG program is indicated by an arrow and the one predicted by the NNPP program is indicated by a double arrow. The putative TATA box predicted using the TSSG program is indicated by a box. B- Alignment of the predicted human promoter region with the homologous mouse and rat sequences.
Figure 6.
Expression and localization of HA-tagged LAP1C in human cells.
SH-SY5Y cells were transfected with HA-LAP1C (LAP1C). A- Immunoblotting analysis using a HA antibody, which detected the transfected HA-LAP1C, or with a LAP1 antibody that detected endogenous LAP1 isoforms and transfected HA-LAP1C. Ponceau S staining was used to check equal loading. B- Immunolocalization of HA-LAP1C. Specific primary antibody against HA tag was detected with Alexa Fluor 594-conjugated secondary antibody (red). DNA was stained with DAPI NT, non-transfected; IB, immunoblotting.
Figure 7.
Solubilization properties of human LAP1 isoforms.
Solubilization of LAP1 in Tris-HCl buffer or Tris-HCl buffer containing 1% triton X-100, 1% triton X-100 and 50 mM NaCl or 500 mM NaCl. Equal fractions of supernatant (SN) and pellet (P) were loaded. IB, immunoblotting.
Figure 8.
LAP1 expression in different cell lines and tissues.
A- Endogenous LAP1 isoforms were detected in HeLa, SKMEL-28 and SH-SY5Y human cells and in rat PC12 cell line and rat cortex lysates. B- Human tissue blot (Clontech) was immunoblotted with LAP1 antibody. 1, liver; 2, brain; 3, lung; 4, kidney; 5, spleen; 6, testis; 7, ovary; 8, heart, 9, pancreas. C- Endogenous expression of LAP1 isoforms was detected in primary cortical neurons for 14 DIV, using a LAP1 antibody. Synaptophysin and PP1γ were used as controls for expressing patterns. Ponceau S staining was used to check equal loading. D- Endogenous expression of LAP1 isoforms was detected in SH-SY5Y cells differentiated with retinoic acid for 10 DIV using a LAP1 antibody. β-tubulin and PP1γ were used as controls. Ponceau S staining was used to check equal loading.
Figure 9.
Human LAP1 post-translational modifications.
A- SH-SY5Y cells were incubated with 0, 0.25 or 500 nM okadaic acid (OA) for 3 hours. Lysates were incubated at 30°C for 1 hour with or without 100 ng of PP1γ1 protein. B-. LAP1 isoforms are phosphorylated on Ser 143, Ser 216, Thr 221, Ser 306 and Ser 310 residues. PP1 was found to be responsible for Ser 306 and Ser 310 residues dephosphorylation (indicated by arrows). Methionine residues 146, 302 and 553 residues were found to be oxidized. Post-translational modified residues and flanking regions were aligned against others species using ClustalW algorithm. The residues numeration is relative to the LAP1B sequence and the translation initiation site of LAP1C is indicated by a dashed line. IB, immunoblotting.