Cd79a

ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), Akt (pan), phospho-Akt (S473), CD79b, CD79a, phospho-CD79a (Tyr182), SHP1, phospho-SHP1 (Tyr564), SHIP1, phospho-SHIP1 (Tyr1020), BTK, phospho-BTK(Tyr223), NF-kB p65, phospho–NF-kB p65 (ser536), IgM, Lyn, phospho-Lyn (Y507), LCK, CD86, and ZAP70 antibodies were from Cell Signaling Technology (Beverly, MA). Anti-SRC family (phospho-Y418)–phospho-Lyn (Y396), CD62L, and IgG antibodies were from Abcam (Cambridge, UK). Purified anti-human actin antibody was obtained from MP Biomedicals (Illkirch, France). Goat anti Rabbit IgG (H+L)–HRP conjugate and Goat anti Mouse IgG (H+L)–HRP conjugate, Goat F(ab′)2 anti-human IgM and Goat F(ab′)2 anti-human IgG were from Jackson Immunoresearch Laboratories (West Grove, PA). All antibodies utilized in the study were used in concentrations according to the manufacturer’s instructions. Ficoll-Paque PLUS from GE healthcare (Uppsala, Sweden), dimethyl sulfoxide (DMSO) from Merck (Darmstadt, Germany), RPMI, fetal calf serum (FCS), Dulbecco’s phosphate-buffered saline (PBS), L-glutamine, and penicillin-streptomycin from Biological Industries (Beit-Haemek, Israel) were utilized for cell cultures.

Western blotting was performed as described previously [47 (link)] using primary antibodies against the following proteins: p-tyrosine, p-Src family, p-Src, p-Lyn, Src, p-AKT (Ser473), and CD79A (Cell Signaling Technology); Lyn, Akt, mTOR, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA); PI3K p85 and p-mTOR (Ser2448) (Abcam).
Cell lysates containing 0.5 mg protein were incubated for 4 hr with 5 μg of anti-CD19 antibody followed by incubation with 50 μg of protein A-Sepharose beads (GE Healthcare, Pittsburgh, PA, USA) overnight at 4°C. Immunoprecipitates were collected and washed three times with PBST, resuspended in SDS sample buffer, and subjected to Western blot analysis.