Gapdh rabbit monoclonal antibody

Cells were lysed with RIPA Lysis buffer by incubation on ice for 10 min. After centrifugation at 12,000 g for 10 min, the supernatants were collected and the concentrations of proteins were quantified by the BCA protein assay kit (Promega, USA). The protein samples were denatured by boiling for 5 min, loaded onto 10% SDS-PAGE gel for electrophoresis (at 120 V for 2 h), and then transferred (at 300 mA for 1 h) to a nitrocellulose membrane. After blocking with 5% non-fat dried milk at room temperature for 1 h, the membrane was incubated overnight at 4 °C with Bcl-2 or the internal control GAPDH rabbit monoclonal antibody (Cell Signaling Technology, USA), then washed and incubated with the secondary anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, USA) for 1 h. Finally, membranes were again washed and visualized using Pierce^™ ECL Western Blotting Substrate (Thermo Scientific, USA). Quantification of Western blot bands was performed using ImageJ software (National Institutes of Health), and the data were expressed as relative Bcl-2 levels vs. PBS-treated cells.

HEK293T cells were pelleted, washed with PBS, lysed on ice with lysis buffer (20 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 10% glycerol, 1 mM TCEP, phosphatase inhibitor cocktails (Sigma P5726 and Calbiochem 524624), and protease inhibitors (pepstatin, leupeptin, PMSF, and aprotinin), and clarified by centrifugation. Protein content was quantified by BCA and diluted to 2 mg/mL in lysis buffer. 40 μL samples of lysate were incubated with compounds or DMSO at desired concentrations and incubated at room temperature for 1 hour. Samples were then supplemented with 1:1 Ub-Pa/Ub-VME probe at a final concentration in the sample of 2 μM and incubated at room temperature with shaking for 30 minutes. Reactions were quenched with 4x LDS sample buffer (Thermo Fisher B0007) supplemented with 10% BME, vortexed vigorously, and heated to 95°C for 5 minutes. Samples were resolved by SDS-PAGE and analyzed by Western blot with the indicated antibodies (Primary antibodies used: USP28 rabbit monoclonal antibody from Abcam, catalog number: ab126604. GAPDH rabbit monoclonal antibody from Cell Signaling, catalog number: 2118S. Secondary antibody used: IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody from LI-COR, catalog number: 926–32211)