Anti usp14 a300 920a

The immunoblotting was performed as previously described (29 (link)). To prepare the whole cell lysates, cells were collected and washed three times with ice-cold phosphate-buffered saline (PBS). Then, cells were lysed by adding RIPA lysis buffer (25 mM HEPES (pH 7.7), 135 mM NaCl, 3 mM EDTA, 1% Triton X-100, 25 mM β-glycerophosphate, 0.5 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol, 10 μg/ml leupeptin, 1 mM Benzamidine). After centrifuging the lysate at 13,000 g for 15 min at 4°C, protein concentration was measured by Bradford Assay. Cell lysates were resolved with SDS-PAGE and transferred to PVDF membranes. The membranes were probed with appropriate primary antibodies at 4°C overnight and the IgG horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. The proteins were detected using the ECL-Plus Western blotting detection system (Amersham Biosciences) and visualized by autography. Anti-USP14 (A300–920A) was purchased from Bethyl Laboratories. Anti-PARP (9532S), anti-Caspse3 (9662), anti-HSP70 (4872S), anti-HSP40 (4871S), anti-mouse (7076S) and anti-rabbit (7074S) were from Cell Signaling Technology. Anti-β-actin (A2228) was from Sigma-Aldrich. Anti-Ubiquitin (linkage-specific K48) was obtained from Abcam (ab140601).