Anti pakt pe clone rea359

For cell culture, the following antibodies were used: anti-CD40 (rat IgG2a clone FGK45.5) from Miltenyi Biotec (Bergisch Gladbach, Germany) and anti-IgM F(ab´)₂ goat polyclonal antibody from Jackson ImmunoResearch (West Grove, PA, USA). For cell staining, the following antibodies were used: anti-CD19 FITC (clone 1D3/CD19), anti-GL-7 Pacific Blue (clone GL7), anti-Ki-67 Alexa 488 (clone 16A8), anti-pSTAT1 PE (clone A15158B), anti-pSTAT3 PE (clone 13A3-1), anti-pERK1/2 PE (clone 6B8B69), and anti-IL-6 PE (clone MP5-20F3) from BioLegend (San Diego, CA, USA), anti-CD19 PerCP-Cy5.5 (clone 1D3), and anti-CD21 APC (clone 7G6) from BD Biosciences (Mountain View, CA, USA), anti-CD138 PE (clone 30506), anti-CD23 PE-cyanine7 (clone B3B4) from Invitrogen (Carlsbad, CA, USA), anti-pSTAT5 PE (clone SRBCZX), anti-CD80 PE (clone 16-10A1), anti-CD86 PE (clone GL1), anti-BCL6 PE (clone BCL-DWN), and anti-IRF4 PE-Cy7 (clone 3E4) from eBioscience (San Diego, CA, USA), and anti-pAKT PE (clone REA359) from Miltenyi Biotec.

Differentiated B-GCs without PRL were allowed to settle for 8 h in basal medium. Subsequently, the cells were incubated with PRL for 30 minutes. The cells were fixed with 1x BD Phosflow Lyse/Fix Buffer 5x (BD Biosciences)/10 minutes, with BD Phosflow Perm Buffer III (BD Biosciences); the cells were permeabilized to determine intracellular STAT1 (anti-pSTAT1 PE, clone A15158B, BioLegend), STAT3 (anti-pSTAT3 PE, clone 13A3-1, BioLegend), and STAT5 (anti-pSTAT5 PE, clone SRBCZX, eBioscience) phosphorylation. To determine AKT (anti-pAKT PE, clone REA359, Miltenyi Biotec) and ERK1/2 (anti-pERK1/2 PE, clone 6B8B69, BioLegend) phosphorylation, cells were treated with IC Fixation Buffer (eBioscience) for 30 minutes at 4°C, washed with FACS buffer or Perm/Wash (BD Biosciences), and incubated with the antibodies for flow cytometric analysis for 30 minutes at 4°C. The samples were read using a MACSQuant Analyzer 10 flow cytometer and the data were analyzed with FlowJo 10 software.