Anti flag l5

Samples in SDS loading dye were reduced by heating at 95°C with 2-mercaptoethanol. Samples were loaded on a Bolt 4–12% Bis-Tris Plus gel (Invitrogen), electrophoresed in MOPS running buffer, transferred to a PVDF membrane, and immunoblotted. Immunoblots were probed with 0.1 μg/mL mouse anti-phosphotyrosine PY20 (Southern Biotech), 0.5 μg/mL anti-SHIP1 (Invitrogen), 0.5 μg/mL anti-GRB2 (Cell Signaling Technologies),1 μg/mL anti-SOS1 (Cell Signaling Technologies), 0.5 μg/mL anti-FLAG L5 (BioLegend), or 0.5 μg/mL anti- FCRL1 350G10 which was generated in our lab. Secondary antibodies including 0.5 μg/mL anti-rat IgG (Cell Signaling Technologies, Danvers, MA, USA), 0.5 μg/mL anti-rabbit IgG (Cell Signaling Technologies), or 0.1 μg/mL anti-mouse IgG2b (Southern Biotech) were used according to the isotype and host species of primary antibody. Blocking and diluted antibody solutions consisted of 1% BSA in HEPES-buffered saline with 1 mM EDTA and 0.1% TWEEN-20. Blots were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and imaged using a ChemiDoc gel imager (Bio-Rad).