Rabbit anti-mouse IgG F(ab’)2 (Zymed Labortories) and goat anti-mouse IgM F(ab’)2 (Southern Biotechnology Associates) were used to signal via the BCR on A20 B lymphoma cells and primary B cells, respectively. Antibodies used for western blot analyses were anti-β-actin (AC-40; Sigma); anti-Syk, anti-Btk, anti-PLCγ2, anti-phospho-Syk (mouse Tyr519/520 or Tyr323), anti-phospho-Btk (Ser180; 3D3), and anti-phospho-PLCγ2 (Tyr759) from Cell Signaling Technology; rabbit anti-Gα12 (S-20), anti-Gα13 (A-20), anti-Gαq (E-17), HRP-labeled goat anti-mouse IgG and donkey anti-rabbit IgG from Santa Cruz Biotechnology.
The following fluorescently-labeled monoclonal antibodies were used for flow cytometric analysis: B220 (RA3-6B2; BD Pharmingen and eBioscience), CD69 (H1.2F3; eBioscience), CD86 (GL1; eBioscience), Igλ (JC5-1; Southern Biotech), CD23 (B3B4; BD Pharmingen) and CD21 (7G6; BD Pharmingen). NP-specific B cells were gated on 7AAD-negative viable cells and identified by flow cytometry after staining with Alexa647-coupled NP-CGG (Biosearch Technologies). Flow cytometric analyses were performed on either a FACSCalibur (BD Bioscience) or a BD LSRII (BD Bioscience) and analyzed with FlowJo v8.8.6 software (Tree Star, Inc.).
The following fluorescently-labeled monoclonal antibodies were used for flow cytometric analysis: B220 (RA3-6B2; BD Pharmingen and eBioscience), CD69 (H1.2F3; eBioscience), CD86 (GL1; eBioscience), Igλ (JC5-1; Southern Biotech), CD23 (B3B4; BD Pharmingen) and CD21 (7G6; BD Pharmingen). NP-specific B cells were gated on 7AAD-negative viable cells and identified by flow cytometry after staining with Alexa647-coupled NP-CGG (Biosearch Technologies). Flow cytometric analyses were performed on either a FACSCalibur (BD Bioscience) or a BD LSRII (BD Bioscience) and analyzed with FlowJo v8.8.6 software (Tree Star, Inc.).