Phosphotyrosine py

The antibodies we used in this study include rabbit antibodies against mGluR1a (Millipore), Src with phosphorylated tyrosine 416 (pan-pY416, Cell Signaling Technology), Fyn (Cell Signaling Technology), Src (Cell Signaling Technology), phosphotyrosine (pY; Millipore), Fak (Cell Signaling Technology), paxillin (Cell Signaling Technology), FLAG (Cell Signaling Technology), or β-actin (Sigma-Aldrich), or mouse antibodies against mGluR1a (BD), Fyn (Santa Cruz Biotechnology), pY (PY20, BD), or transferrin receptors (TfRs; Thermo Fisher Scientific). The antibody against pY416 reacts with the following Src family members when autophosphorylated at a conserved activation residue: Y416 (chicken Src), Y419 (rat Src), and Y420 (rat Fyn). Pharmacological agents, including (S)-3,5-dihydroxyphenylglycine (DHPG), 3-methyl-aminothiophene dicarboxylic acid (3-MATIDA), 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2), 1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP3), and oxotremorine-M, were purchased from Sigma-Aldrich. All drugs were freshly prepared on the day of the experiments.

The surface distribution of the BCR and other molecules were analyzed using a TIRFm (TE2000U, Nikon, Melville, NY, USA). To image intracellular molecules, B cells were incubated with Alexa Fluor 546-conjugated monobiotinylated Fab’ fragment of anti-mouse IgG + M (AF546-mB-Fab’-anti-Ig) tethered lipid bilayers17 (link) at 37 °C for varying lengths of time. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.05% saponin, and stained for phosphotyrosine (pY) (Millipore, Billerica, MA, USA, Cat. No 05–321), phosphorylated Btk (pBtk, Y551; BD Biosciences, Cat. No 558034) and WASP (pWASP, S483/S484; Bethyl Laboratory, Montgomery, TX ,USA, Cat. No A300-205A), as well as for F-actin by AF488-phalloidin (Thermo Scientfic, Cat. No A12379). GFP/AF488, AF546, and interference refection images (IRM) were acquired sequentially at each time point. B-cell contact area was determined using TIRFm images and MATLAB software. Total (TFI) and mean fluorescence intensities (MFI) of AF546-mB-Fab’-anti-Ig in the contact zone were quantified using Andor iQ software (Andor Technology, Belfast, UK). Background fluorescence was subtracted. For each set of data with statistics, more than 60 individual cells from two or three independent experiments were analyzed.