Pplcγ2

Antibodies with the following specificities were purchased from BD Biosciences: pPLCγ2 (Alexa Fluor 488, pY759), pSYK (PE, pY348), pLCK/pLYN (Alexa Fluor 647, pY505). Phosphatase abundance was estimated by SHP-1 antibody (Santa Cruz Biotechnology). Detection of PBMC subsets was achieved with Pacific Blue-conjugated anti-CD3 (clone UCHT1) and PerCPCy5.5 conjugated anti-CD20 (clone H1), and CLL circulating tumor was confirmed for CD5 positivity with PE-Cy7-conjugated anti-CD5 (clone L17F12, all BD Biosciences). Intracellular reactive oxygen species was measured under resting and H₂O₂-stimulated conditions using 2′,7’-dichlorofluorescin diacetate (DCFDA) (Abcam). For assessment of intracellular species, cells were permeabilized following fixation with PFA with cold methanol at −20°C for 10 minutes. Intracellular staining was carried out at 4°C in PBS + 2% FBS. Cells were washed twice with PBS + 2% FBS and analyzed on an LSRII cytometer (BD Biosciences). Analysis was carried out using FlowJo software (TreeStar), Prism (Graphpad), and with custom tools written in R statistical computing language (R project) and MATLAB (Mathworks). Code is available upon request.

For phosphoflow experiments, 0.5 × 10⁶ cells were cultured in RPMI‐2% FCS at 37°C for 1 min for pPI3K p85 (Cell Signaling Technologies), 3 min for pSHP‐1 (Cell Signaling Technologies), 5 min for pSrc (Abcam), pLyn (Abwiz Bio, San Diego, CA, USA), pCD79a (Cell Signaling Technologies), pSyk, pSLP‐65, pPLCγ2, and pErk (all BD Biosciences) or for 3 h for pS6 and pAkt (both Cell Signaling Technologies) with 25 μg/mL anti‐IgM or antigen‐binding F(ab’)₂‐Igκ fragments (anti‐IgK; Jackson Immunoresearch). After fixation with the eBioscience FoxP3 staining kit Fix/Perm solution, cells were washed twice with the accompanying Perm/Wash solution (Invitrogen). Next, cells were incubated with varying combinations of monoclonal antibodies (Supporting information Table S1) and stained according to previously described procedures [31 (link)]. After staining for the phosphoproteins, samples were measured in MACS buffer on an LSR‐II (BD Biosciences) and analyzed using FlowJo v10 (BD Biosciences).