Pstat6

After treatment cells were washed with phosphate-buffered saline (PBS) and lysed using 1% Triton-X lysis buffer containing 50 mM Tris, 100 mM NaCl, 1X Protease inhibitor cocktail EDTA free (Roche), 1X Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific), Zn²⁺ chelator ortho-phenanthroline (o-PA) (LifeSensors) and deubiquitylase inhibitor PR-619 (LifeSensors). Protein lysate were quantitated using Bradford reagent. For Cul5 neddylation experiments whole-cell lysates were prepared as described previously^{49 (link)}. Briefly, cells were resuspended in SDS sample buffer (62.5 mM Tris-HCl, 2% w/v SDS, 10% glycerol, 50 mM DTT, and 0.1% bromophenol blue), vortexed to reduce sample viscosity, denatured by boiling, and then cooled on ice. This is to prevent the de-neddylation during protein extractions. Samples were resolved on Novex Tris-Glycine Gels (Thermo Fisher Scientific) and then transferred onto PVDF membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was probed with the primary antibodies Cul5 (Bethyl Lab; 1:5000), pJak1 (CST; 1:1000), Jak1 (BD; 1:1000), pSTAT6 (CST; 1:1000), STAT6 (CST; 1:1000), IL4Rα (BD; 1:1000), pIL4Rα (BD; 1:1000), Actin (SCBT; 1:5000), Cul5 (Bethyl; 1:5000). The membrane was then incubated with an appropriate secondary antibody at a dilution of 1:5000 and the protein of interest was detected with Li-Cor odyssey imaging system.

The SCNP assay was performed as described previously [7 (link)]. Modulators and concentrations were as follows: 1000 IU/ml IFN-α (PBL); 50 ng/ml IL-4, 5 μg/ml anti-IgD (BD Biosciences); 50 ng/ml IL-2, 50 ng/ml IL-6, 50 ng/ml IL-27, 5 μg/ml R848 (Invivogen); 40 nM PMA (Sigma Aldrich), 3 μg/ml anti-CD3 (eBioscience), 10 μg/ml anti-mouse (Santa Cruz Biotechnology), 10 μg/ml anti-IgM (Southern Biotech). For TCR stimulation, cells were exposed to anti-CD3 for 12 min, with anti-mouse added for the last 2 min; the anti-IgM modulation time was 10 min; all other modulation times were 15 min. Staining was performed using Ab cocktails with each cocktail consisting of 5 Abs to detect phenotypic markers and 2 Abs to detect intracellular protein readouts. Abs used include anti-CD3, -CD4, -CD45RA, -CD20, -p-NF-κB, -c-poly(ADP-ribose) polymerase, -p-Stat1, -p-Stat3, -p-Stat5, -p-Stat6, -p-Erk, -p-ZAP70/Syk (BD Biosciences); -p-Akt, -p-S6 (CST); and -CD14 (Beckman Coulter). Flow cytometry data was acquired on FACS Canto II Flow Cytometers (BD Biosciences). All flow cytometry data were analyzed with WinList (Verity House Software). PBMC subpopulations were delineated according to the immunophenotypic gating scheme described previously [7 (link)].