Table 1. List of proteins coimmunoprecipitated with L-PGDS.
| Accession number* | Protein name | Symbol | Peptide hit** |
|---|---|---|---|
| IPI00554929 |
Heat shock protein HSP 90-β |
Hsp90ab1 |
31 |
| IPI00110850 |
Actin, cytoplasmic 1 |
Actb |
25 |
| IPI00323357 |
Heat shock cognate 71 kDa protein |
Hspa8 |
12 |
| IPI00319992 |
78 kDa glucose-regulated protein |
Hspa5 |
7 |
| IPI00462072 |
α-enolase |
Eno1 |
7 |
| IPI00129526 |
Hsp90b1 endoplasmin |
Hsp90b1 |
5 |
| IPI00275539 |
Reticulon 4 |
Rtn4 |
4 |
| IPI00229534 |
Myristoylated alanine-rich C-kinase substrate |
Marcks |
4 |
| IPI00111560 |
Isoform 1 of protein SET |
Set |
3 |
| IPI00115679 |
Isoform 2 of neutral α-glucosidase AB |
Ganab |
3 |
| IPI00133903 |
Stress-70 protein, mitochondrial |
Hspa9 |
2 |
| IPI00604969 | Titin isoform N2-A | Ttn | 2 |
SWISS-PROT accession numbers are listed. **Number of peptide hit identified by LC-MS/MS analysis. In brief, NIH3T3 fibroblast cells were treated with the recombinant L-PGDS protein (1 μg/ml), and then crosslinked with 1% formaldehyde for 1 h and rinsed twice with PBS. Cells were lysed in triple-detergent lysis buffer (50 mM TRIS-HCl, pH 8.0; 150 mM NaCl; 0.02% sodium azide; 0.1% SDS; 1% Nonidet P-40; 0.5% sodium deoxycholate; and 1 mM phenylmethyl sulfonyl fluoride). The lysates were centrifuged for 20 min at 4°C, and the supernatants were collected. The protein concentration in the cell lysates was determined using the Quant-iT Protein Assay kit (Molecular Probes). To remove nonspecific binding proteins in the lysates, the samples were incubated in a ~30 μl packed volume of recombinant protein G-agarose (PGA) for 1 h at 4°C. After a brief centrifugation, supernatants were collected and then incubated with anti-L-PGDS antibody (1 μg/ml; Cayman Chemical) for 4 h at 4°C. PGA (30 μl) was then added and incubated for 4 h. Afterwards, L-PGDS-Ab-PGA complexes were washed three times with wash buffer (50 mM HEPES, 150 mM NaCl, 0.1% Triton X-100 and 10% glycerol). For the identification of coimmunoprecipitated proteins, the immunoprecipitation samples were separated by electrophoresis on a 10% polyacrylamide gel and visualized by silver staining. The protein band of interest was excised from the silver-stained gel for in-gel tryptic digestion. The excised gel slices were destained and shrunk by dehydration in acetonitrile and dried in a vacuum centrifuge. Proteins within the shrunken gel slices were then digested overnight with trypsin at a substrate/enzyme ratio of 10:1 (wt/wt) in 25 mM ammonium bicarbonate (pH 8.0). The enzyme reaction was terminated by the addition of 0.1% formic acid in water. Peptides from gel pieces were extracted by sonication for 10 min and supernatants containing the peptides were transferred to new tubes. Peptides were analyzed using a liquid chromatography (LC) and tandem mass spectrometry (MS/MS) system with reverse-phase LC, which consisted of a Surveyor MS pump (Thermo Electron), a Spark autosampler (Spark Holland), and a Finnigan LTQ linear ion-trap mass spectrometer (Thermo Electron) equipped with nanospray ionization sources. All MS/MS data were searched against the IPI mouse protein database (version 3.16) using the SEQUEST algorithm (Thermo Electron) incorporated into BioWorks software (version 3.2).