P jak2 tyr1008

The mesangial cells from different treatments were fixed and permeabilized to incubate with antibodies including phosphorylated p38 (p‐p38) MAPK (T180/Y182), p‐ERK1/2 (p‐ERK) (T202/Y204) (BD Biosciences), p‐JAK2 (Tyr1008) (Cell Signaling Technology, Danvers, MA, USA), p‐STAT1 (Ser727), p‐STAT3 (Tyr705) (BioLegend), and p‐STAT4 (Tyr693) (Thermo Fisher Scientific) at 4°C for 15 minutes. After washing, the mean fluorescence intensity (MFI) was measured by flow cytometry. The expression of IL‐12Rβ2 (BD Pharmingen) and gp130 (R&D Systems, Minneapolis, MN, USA) on mesangial cells were also determined by flow cytometry assay.³ While, the MFI of p‐STAT1 (Ser727), p‐STAT3 (Tyr705), and p‐STAT4 (Tyr693) in mesangial cells from PBS, IL‐35p, and IL‐35n treatments could determine the activity of IL‐35‐regulated singling pathway. The percentage and absolute number of CD4⁺CD25⁺FoxP3⁺ Tregs from lupus mice with different treatments were exanimated by flow cytometry.², ³

Chemical reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise specified. RPMI1640, fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Trypsin/EDTA was purchased from GE Healthcare Life Sciences (Logan, UT, USA). E-Cadherin rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AMPK-α rabbit polyclonal antibody, rabbit monoclonal antibodies specific to p-AMPK-α (Thr172), JAK2, p-JAK2 (Tyr1008), and occludin, and mouse monoclonal antibodies detecting STAT3, p-STAT3 (Tyr705), cleaved caspase 1, and IL-1β were purchased from Cell Signaling Technology Inc. (Boston, MA, USA). NOX2 and ICAM-1 rabbit monoclonal antibodies, and TGF-β, TNF-α, NOX1, p-p47phox, pro-caspase-1, and claudin 2 rabbit polyclonal antibodies were purchased from Abcam (Cambridge, MA, USA). Rabbit polyclonal antibodies specific for IL-10 or IL-6 were from Abbiotec (San Diego, CA, USA), and NLRP3 rabbit polyclonal antibody was purchased from Novus Biologicals (Centennial, CO, USA). Tofacitinib citrate salt was obtained from L.C Laboratories (Woburn, MA, USA). VAS2870 was purchased from Sigma-Aldrich (St. Louis, MO, USA). BJ-3105 was synthesized by Byeong-Seon Jeong as reported [47 ].