Mouse anti ubc9

For immunoblot analysis, HeLa cells or subcellular fractions were mixed with Laemmli buffer (125 mM Tris–HCl [pH 6.8], 4% SDS, 20% glycerol, 100 mM dithiothreitol [DTT], 0.02% bromophenol blue), sonicated, boiled for 5 min, and protein content was resolved by SDS polyacrylamide gel electrophoresis. Proteins were then transferred on PVDF membranes (GE Healthcare) and detected after incubation with specific antibodies using Pierce ECL 2 Western Blotting Substrate (Fisher Scientific). The following primary antibodies were used for immunoblot analysis: mouse anti‐actin (A5441; Sigma‐Aldrich; 1:10,000 dilution), mouse anti‐LAMP1 (555798 ; BD Pharmingen; 1:500 dilution), mouse anti‐Ubc9 (610748 ; BD Transduction Lab; 1:1,000 dilution), and goat anti‐cathepsin D (sc‐6486 ; Santa‐Cruz; 1:500 dilution). Anti‐mouse and anti‐goat HRP‐conjugated antibodies were used as secondary antibodies (Abliance; 1:8,000 and 1:4,000 dilution, respectively). All displayed immunoblots are representative of at least three independent experiments.