Antipain

Bestatin, Chymostatin, E-64, Leupeptin, Pepstatin, Phosphoramidon, Pefabloc SC, EDTA, Aprotinin, Antipain were purchased from Roche as part of a protease inhibitors set and used at the following concentrations: 74 μM Antipain; 130 μM Bestatin; 100 μM Chymostatin; 28 μM E-64; 1 μM Leupeptin; 1 μM Pepstatin; 600 nM Phosphoramidon; 4 mM Pefabloc SC; 1.3 mM EDTA; 0.3 mM Aprotinin. The additional compounds ATP (Boehringer), Bacitracin (Applichem), insulin from bovine pancreas (Sigma), PMSF (Sigma), 6bK [45 (link)] (Phoenix Pharmaceuticals and Tocris), TPEN (Calbiochem), and NEM (Roche) were purchased from companies indicated.

Cell were incubated for 10 minutes at 4°C in TNTE lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% [v/v] Triton-X-100) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 μg/ ml pepstatin A (Sigma), 100 μg/ml benzamidine chloride (Calbiochem), and 1 mg/ml trypsin inhibitor, 10 μg/ ml antipain, 50 μg/ml aprotinin and 10 μg/ml leupeptin (Roche Applied Biosciences)), and phosphatase inhibitors (10 mM sodium pyrophosphate and 25 mM sodium fluoride (EM Sciences), and 1 mM sodium orthovanadate (Alfa Aesar)). Lysates were centrifuged at 15,000X g for 10 minutes at 4°C, and small aliquots were subjected to protein concentration determination using Bradford-based protein assays (Bio-Rad Laboratories). Cell extracts were resolved by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and were transferred onto nitrocellulose membrane (Bio-Rad Laboratory). The blots were incubated with mouse anti-FLAG (Sigma), rabbit anti-PIAS1 (Epitomics) or rabbit anti-actin (Sigma), as the primary antibody and HRP-conjugated donkey anti-mouse or anti-rabbit IgG (Jackson Laboratories) as secondary antibodies, followed by ECL and signal detection using a VersaDoc 5000 Imager (Bio-Rad Laboratories). Densitometry was performed using Quantity One software (Bio-Rad Laboratories).

Cell lysates were obtained using standard techniques. Lysis buffer contained 25 mmol/L HEPES (pH 7.7), 400 mmol/L NaCl, 1.5 mmol/L MgCl₂, 2 mmol/L EDTA, 0.5% Triton X‐100, 0.1 mmol/L PMSF, 3 mmol/L DTT, phosphatase inhibitor cocktail (20 mmol/L β‐GP, 1 mmol/L Na₃VO₄; Roche), and protease inhibitor cocktail (10 µg/mL leupeptin, 2 µg/mL pepstatin, 50 µg/mL antipain, 1 × benzamidine, 2 µg/mL aprotinin, 20 µg/mL chymostatin; Roche). For immunoprecipitation, lysates were incubated with primary antibody overnight at 4°C. Agarose beads conjugated with A/G were then added and incubated for 2 hours at 4°C. The immunocomplexes were spun and washed three times with cold phosphate‐buffered saline (PBS) and once with lysis buffer. Immunocomplexes were then subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). For Western blotting, 50‐80 µg of total proteins were electrophoresed on 6%‐12% SDS‐PAGE. The proteins were transferred to nitrocellulose membranes, probed with specific primary antibodies, and then probed with the appropriate horseradish peroxidase‐conjugated secondary antibodies (GE Healthcare, Waukesha, WI) Proteins were detected using a chemiluminescence‐based kit (GE Healthcare).