Anti p tyr

Cells were lysed using cell extraction buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and protease inhibitor cocktail from Roche). Soluble proteins were subjected to immunoprecipitation with anti-γ-tubulin (Santa Cruz), anti-Flag (Sigma-Aldrich), anti-p-Tyr (Merck), anti-GCP5 (Santa Cruz) antibodies, or anti-mouse IgG (Sigma-Aldrich). An aliquot of the total lysate (5%, v/v) was included as the input. Protein samples were separated by SDS-PAGE and transferred onto PVDF membranes. anti-γ-tubulin (Sigma-Aldrich), anti-c-Abl (Santa Cruz), anti-Arg (Santa Cruz), anti-GCP5 (Santa Cruz), anti-GCP2 (Novus), and anti-GCP3 (Proteintech) antibodies were used as primary antibodies to detect the corresponding proteins. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma-Aldrich) or anti-rabbit IgG (Sigma-Aldrich) was used as the secondary antibody. HRP-conjugated anti-p-Tyr (Merck), anti-Flag (Sigma-Aldrich), anti-Myc (Sigma-Aldrich), anti-GST (Santa Cruz), and anti-β-actin (Sigma-Aldrich) antibodies were used directly to detect the corresponding proteins. The protein bands were visualized using an ECL detection system (Millipore) at the final step with a chemical imaging analyzer (GE Healthcare).

For flow cytometry, anti-B220-PE/Cy7 (RA3-6B2), anti-CD19-BV510 (GD5), anti-IgD-PE (11-26c.2a), anti-CD23-APC/Cy7 (B3B4), anti-IgM-APC (RMM-1), anti-CD21-PerCP/Cy5.5 (7E9), and anti-CD24-PE (30-F1) were purchased from Biolegend. For magnetic cell separation, biotinylated anti-IgM (RMM-1), anti-B220 (RA3-6B2), anti-Gr1 (RB6-8C5), anti-CD23 (B3B4), and anti-CD3e antibody (145-2C11) were purchased from BD Pharmingen. For microscopy, anti-α-smooth muscle actin (ab5694, Abcam), anti-IgM-Cy3 (EMD Millipore), anti-IgD-FITC (11-26c, Invitrogen), anti-MARCO-FITC (Bio-rad), and anti-B220 (RA3-6B2, eBioscience) were used. For western blot, anti-NFkB2, anti-p-p38 (T180/Y182), anti-pAkt (S473) from Cell Signaling Technology, anti-β-actin (Invitrogen), anti-BAFFR and anti-ST6Gal-1 (R&D Biosystems), and anti-pTyr (EMD Millipore) were used.

The following antibodies were used: anti-Src (05–184; Merck, Darmstadt, Germany), anti-Yes (610375; BD Biosciences, San Jose, CA, USA), anti-Lyn (sc-7274; Santa Cruz Biotechnology, Dallas, TX, USA), anti-Csk (610080; BD Biosciences), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2275-PC-100; R&D Systems, Minneapolis, MN, USA), anti-p-Tyr (05–1050; Merck), anti-Src family phospho-Y418 (p-SFKs A-loop; ab40660; Abcam, Cambridge, UK), anti-p-Src Y530 (sc-166860; Santa Cruz Biotechnology), anti-p-Lyn Y508 (CSB-PA000691; Cusabio, Wuhan, China)), anti-Fyn (sc-434; Santa Cruz Biotechnology), anti-E-cadherin (#3195; Cell Signaling Technology, Danvers, MA, USA), anti-vimentin (V6630; Sigma-Aldrich), anti-Cbl-b (sc-8006; Santa Cruz Biotechnology) and anti-c-Cbl (sc-1651; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-mouse IgG (#7076; Cell Signaling Technology) and anti-rabbit IgG (711-035-152; Jackson Immunoresearch, West Grove, PA, USA) antibodies were used for Western blotting.