Anti rtn3 sc 374599

For Western blot analysis of human proteins, 10 μg (fractions) or 50 μg (cell lysates) protein was loaded onto 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immobillion-P transfer membrane (Millipore, Darmstadt, Germany) for immunoblotting. Primary antibodies used were anti-TOM20 (sc-11415; Santa Cruz Biotechnology), anti-RTN3 (sc-374599; Santa Cruz Biotechnology), anti-GM130 (PA1-077; ThermoFisher Scientific), anti-Bcl2 (Ab131448; Abcam), anti-Mcl-1 (sc-819; Santa Cruz Biotechnology), anti-Bcl-xl (sc-56021; Santa Cruz Biotechnology), anti-Bax (4F11; Enzo), anti-Bak (556396; Pharmingen), anti-Bid (AF860; RnD Systems).

Cells were seeded in 24-well plates onto glass sterile coverslips and allowed to attach overnight. The indicated treatment was applied, then cells were fixed by 4% paraformaldehyde. The labeling with antibodies was realized according to the manufacturer’s instructions (Duolink, Sigma-Aldrich). Primary antibodies used were anti-TOM20 (612278; Pharmingen), anti-Bcl2 (ab131448; Abcam), and anti-RTN3 (sc-374599; Santa Cruz Biotechnology). Image acquisition was performed with a Zeiss Apotome microscope (Zeiss Axiovert 200-M inverted microscope and AxioVision 4.6 program, Carl Zeiss Gbmh). Data were analyzed by the ImageJ 1.46r software and spots were quantified by the Object 3D Counter plugin. The images shown are representative of at least three independent experiments with at least two technical replicates by experiment. Quantification was made from at least 100 cells by conditions.