Anti bcl2 antibodies n 19 sc 492

Mock and p140 cells (1 × 10⁵) were seeded in 6-well plates. At 24, 48, 72 and 96 h, cells were trypsinized and quantified using a Burker chamber (5 squares for each sample were counted, giving the average of cell number). For anoikis assays, cells were detached and kept in suspension in serum-free media. For apoptosis detection, cells were stained with APC Annexin V probe (1:200) (Biolegend, San Diego, CA, USA) for 15 min at room temperature, followed by Propidium Iodide staining (P4864, Sigma-Aldrich Co, Italy). Flow cytometric analyses were carried out on a FACSCalibur using CellQuest Software (Becton Dickinson). WB for Bcl2 expression was performed with anti Bcl2 antibodies (N-19 sc-492, 1:1000) from Santa Cruz Biotechnologies, Palo Alto, CA, USA). At least three independent experiments, with three replicates for each condition, were performed.

Co-IP is an effective approach for quantifying protein–protein interactions in cells. Briefly, after incubation at room temperature overnight, 500 mg cellular protein were labeled with anti-Bcl-2 antibodies (N19; sc-492; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The protein-antibody complexes were then collected using protein A/G plus agarose beads (sc-2003; Santa Cruz Biotechnology). Following the final wash, the samples were boiled and centrifuged to pellet the agarose beads. The immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting using anti-Beclin-1 or -Bcl-2 antibodies. Western blotting was performed to analyze proteins in the supernatant, and antigens were visualized using a chemiluminescence detection kit (Amersham Corp., Arlington Heights, IL, USA). The data were analyzed using Odyssey 2.1 software (Odyssey; LI-COR).