α btk

Mouse α-HS1, mouse α-LYN and mouse α-SYK antibodies were purchased from BD Biosciences; mouse α-phosphoERK (Y204), rabbit α-ERK2 from Santa Cruz Biotechnology; mAb rabbit α-phosphoHS1 (Y397)-clone D12C1, rabbit α-phosphoLYN (Y507), rabbit α-VAV1, rabbit α-phosphoVAV (Y174); mAb rabbit α-phosphoLYN (Y396)-clone EP503Y from Epitomics. α-pPLC-γ -Y1271 and α-pPLC-γ, x-phosphoBTK (Y223) and α-BTK from Cell signaling. Primary antibodies were used at 1:1000 dilution. Anti-mouse IgG HRP-linked and anti-rabbit IgG HRP-linked (1:5000 dilution) were purchased from GE Healthcare.

Protein lysates were prepared from diseased splenic and lymph node tissues of treated and untreated Eμ-TCL1 and Eμ-TCL1;p53^R172H/+ mice in NP-40 lysis buffer supplemented with protease and phosphatase inhibitors as previously done.^{16 (link)} Fifty micrograms of soluble protein was separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% milk in phosphate-buffered saline plus 0.1% Tween 20 (PBST) for 1 h at room temperature, membranes were incubated with either α-BTK (Cell Signaling Technology, Davers, MA, USA; 1:1000 dilution), α-ERK (Cell Signaling Technology, 1:1000 dilution), α-pBTK-(Tyr223) (Cell Signaling Technology, 1:1000 dilution), or α-pERK(Thr202/Tyr204) (Cell Signaling Technology, 1:1000 dilution) antibodies at 4 °C overnight. Membranes were then washed with PBST and incubated with α-rabbit horseradish peroxidase-conjugated secondary antibodies and visualized with ECL plus (GE Bioscience, Pittsburgh, PA, USA).