P jak3

Cells were harvested after 5 days of incubation at 37 °C in humidified incubator and were lysed in ice-cold radioimmunoprecipitation (RIPA) lysis buffer (Catalog No. 89901, Thermo Scientific, Rockford, USA) containing protease and phosphatase inhibitor cocktails (Roche Diagnostic, Germany). Protein concentrations were determined by BCA protein assay Kit (Catalog No. 23225, Thermo Scientific, Rockford, USA) as described by manufacturer instructions. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (PVDF). After blocking, membranes were incubated with primary antibodies such as p-p38 (9211S, Cell Signaling), p38 (9212S, Cell Signaling), P-JNK (9251S, Cell Signaling), JNK (9252S, Cell Signaling), P-JAK1 (3331S, Cell Signaling), JAK1 (3332S, Cell Signaling), P-JAK3 (5031S, Cell Signaling), JAK3 (8863S, Cell Signaling) P-STAT6 (9361S, Cell Signaling), STAT6 (9362S, Cell Signaling), p-NF-κBp65 (SC-136548, Santa Cruz), NF-κBp65 (SC-109, Santa Cruz) and GAPDH (SC-32233, Santa Cruz) at 1:1000 dilutions for overnight at 4 °C. Membranes were then washed and incubated with horseradish-peroxidase conjugated secondary antibodies (1:5000 dilutions) for 2 h at room temperature. The blots were then detected with western blot detection kit (WesternBrightTMECL, USA).

Cells were lysed with RIPA buffer (50 mM Tris–HCl, pH 7.5, 1% Nonidet P-40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, l g/ml leupeptin, 1 mM sodium vanadate, and 150 mM NaCl). The cell lysate was resolved on a 8-12% SDS-PAGE gels and transferred to PVDF membrane (Millipore, Bedford, MA). The membrane was probed with primary antibodies specific to the following molecules: SOCS2, JAK2, p-JAK2, JAK3, p-JAK3, STAT3, p-STAT3, STAT5, p-STAT5 (Cell Signaling Technology); and β-actin (Santa Cruz). After incubation with peroxidase-conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch), the signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce).

CuE (C₃₂H₄₄O₈) was purchased from Baoji Herbest Bio-Tech Co., Ltd. (Baoji, Shanxi, China) and the purity was determined to be 99.07% based on HPLC. Fetal bovine serum (FBS) was from PAN-Biothech (Logan, UT, USA). Antibiotics and trypsin were from Macgene (Beijing, China). Matrigel was bought from BD Bioscience (Bedford, MA, USA). Antibodies against p-JNK, JNK, p-ERK, ERK, p-p38 MAPK, p38 MAPK, p-JAK3, JAK3, p-STAT3, STAT3, Cyclin A, Cyclin B1, CDK1 and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA). Rhodamine-conjugated phalloidin was obtained from Thermo Fisher Scientific (Waltham, MA, USA). De-ionized water was obtained from Milli-Q system (Millipore, MA, USA).

Antibodies to pSTAT3 (tyrosine 705), pJAK3, JAK3, and PTPN6 were purchased from Cell Signaling Technologies (Beverly, MA, USA) Antibody to β-Actin was purchased from Santa Cruz (Dallas, TX, USA). P-tyrosine (4G10) antibody was purchased from Millipore. The pharmacological inhibitors romidepsin and azacytidine were purchased from Sigma-Aldrich. JAK3 inhibitor WHIP-154 was purchased from Santa Cruz Biotechnology and JAK2 inhibitor ruxolitinib was procured from ChemieTek (Indianapolis, IN).

B cells were preactivated with anti-IgM, CD40L for 72 h, and then stimulated with TSLP or anti-TSLP antibody (proteintech) for 60 min. Total protein of B cells was extracted by Minute Total Protein Extraction Kit (Invent Biotechnologies, USA), and concentrations were determined by BCA Assay kit (Pierce Biotechnology, USA). Protease inhibitor cocktail (huaxingbio) and phosphatase inhibitor (Keygen Biotech) were added to the lysis buffer. Antibodies for immunoblotting: phosphorylation (p)-JAK1, JAK1, p-JAK2, JAK2, p-JAK3, JAK3, p-Stat1, Stat1, p-Stat3, Stat3, p-Stat5, Stat5, β-actin were all from Cell signaling Technology. Images were captured and analyzed on Chemiluminescent Imaging System. Total density of each protein band was determined and the ratio of target protein to β-actin density was calculated.

Wet-western blotting was performed^{61 (link)}. pJAK3, pJAK2, pJAK1, pSTAT5, pFOXO1, JAK1, JAK2, JAK3, STAT5, FOXO1, and GAPDH antibodies were obtained from Cell Signaling Technology. More information about the antibodies used in this study are provided in Supplementary Table 4. All originals uncropped gels are provided in Supplementary Fig. 15.

For PLA experiemtns^{25 (link),58 (link)}, the cytokine IL7 and the chemokine CXCL12 were labeled with PLA-PLUS and PLA-MINUS probes. For PLA experiments with JAK3 or pJAK3 the corresponding antibodies were used (Cell Signaling). The PLA probes were then subjected to ligation and polymerization reactions (Sigma-Aldrich). The cells were then examined for the frequency of signals per cell under the fluorescence microscope (Leica). Pictures were taken and quantified Image J and BlobFinder software.