In-gel digestion of protein samples. The gel pieces were washed with ddH 2 O and destained in 30 mM potassium ferricyanide (Sigma-Aldrich) and 100 mM sodium thiosulfate (Sigma-Aldrich) for 20 min until they became colorless. Disulfide bonds were reduced by treatment with 10 mM dithiothreitol (DTT, Amersham Biosciences) in 100 mM ammonium bicarbonate (JT Baker) for 1 h at 56°C. Iodoacetamide (Sigma-Aldrich) was then used to alkylate the cysteine residues. The samples were diluted with 50 mM ammonium bicarbonate, dehydrated with acetonitrile (ACN, Echo Chemical), and subjected to 0.001% trypsin (Biological Industries) digestion overnight at 37°C. The next day, the peptide samples were acidified with 5% formic acid (Acros Organics), and analyzed by LC/MS/MS using a Thermo Scientific LTQ XL mass spectrometer (Thermo Scientific).
Co-immunoprecipitation. HONE-1 cell lysates (1500 µg) were incubated with 4 µg of anti-caprin-1 antibody (ProteinTech Group) or GAR (Perkin-Elmer) in PBS containing 10 µM biotinylated tylophorine at 4°C for 4 h with constant agitation. The lysates were then incubated with washed protein G agarose (Millipore) at 4°C for 1.5 h. The beads were washed 5 times with PBS. Caprin-1-bound proteins were then eluted with Laemmli buffer, analyzed by 10% SDS-PAGE, and subjected to western immunoblot analysis. 