Rabbit anti mouse ig hrp

Cells were lysed in lysis buffer (1% Triton X100, 50 mM Tris pH 7.5, 300 mM NaCl, 1 mM EGTA, 1 mM DTT, and 1 mM NaVO₄) with protease inhibitors for 30, cleared by centrifugation, and quantified using BCA assay (Biovision). For detection of LC3, cells were washed with PBS and lysed in Phosphosafe (Novagen) for 5 min, then scraped, and cleared by centrifugation. Proteins were separated on SDS-PAGE and blotted onto PVDF membrane (Immobilon), blocked with blocking solution (LI-COR), and incubated with primary and secondary antibodies. Signals were detected on an Odyssey scanner (LiCor) or ChemiDoc (BioRad) or iBright FL-1000 (Thermo Fisher). The following antibodies were used: anti-SQLE (Proteintech, 12544-1-AP, Dilution 1:1000), anti-ZEB1 (Novus Biologicals, NBP-05987, Dilution 1:2000), anti-LC3 A/B (CST, #8457, Dilution 1:2000), anti-PARP1 (Abcam, ab32064, Dilution 1:1000), anti-b-actin (CST, #8457, Dilution 1:1000), and anti-vinculin (Sigma, V9131, Dilution 1:15.000). Secondary antibodies were from Dako: Polyclonal Swine Anti-Rabbit ig-HRP (Ref P0399) and Polyconal Rabbit Anti-Mouse ig-HRP (Ref P0260) both used in a dilution of 1:1000.

Non-stimulated and stimulated B cells were harvested as described in the figure legends. Twenty micrograms of total cell extracts were separated by SDS-PAGE and analyzed by western blot, using polyclonal rabbit anti-human Dock10 (Abcam) antibodies and polyclonal rabbit anti-α-tubulin or monoclonal mouse anti-α-tubulin (both from Abcam) antibodies, with HRP-conjugated swine anti-rabbit Ig (Dako) or rabbit anti-mouse Ig-HRP (Dako). The intensities of the bands are specified relative to that of α-tubulin.

Lysis of the cell pellets was performed in 50 mM tris (pH 8.0), 150 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride (PMSF), one tablet protease inhibitors (EDTA free, Roche) per 10 ml, and deoxyribonuclease (DNase; 30 μg/ml) (Sigma-Aldrich). After Western blotting and wet transfer in ethanol-based transfer buffer onto nitrocellulose membranes (Cytiva), proteins were detected by chemoluminescence (Advansta, K-12049-D50) using rat anti–caspase-2 (11B4, Enzo Life Science), rabbit anti-p19ARF (sc-32748, Santa Cruz Biotechnology), mouse anti-p53 (1C12, Cell Signaling), rabbit anti-p21 (ab7960), mouse anti-HSP90 (sc-13119, Santa Cruz Biotechnology), rabbit anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (14C10, Cell Signaling), and mouse anti-hBCL2 (clone S100). Goat anti-rabbit Ig/horseradish peroxidase (HRP) (Dako, P0448) or rabbit anti-mouse Ig/HRP (Dako, P0161) was used as secondary reagent, respectively.