Phospho stat2

Cells were seeded (2 × 10⁵) in a six well plate for 24 h and were treated with GE at 150 and 200 (µg/mL) for 24 h followed by washing with pre-cooled PBS. Ice cold RIPA buffer with protease/phosphatase was added for the cell lysis. The lysates were collected and centrifuged at 13,000× g for 30 min at 4 °C. The total protein concentration in cell lysates was measured by the Bradford method. The cell extracts were separated by SDS polyacrylamide gels (8–10%), and then, the protein was transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk, incubated with indicated primary and appropriate secondary antibodies (anti-rabbit IgG-HRP conjugates), and protein bands were detected using 1-Step Ultra TMB-Blotting Solution (Thermo Scientific). STAT1 (D1K9Y) Rabbit mAb, Phospho-STAT1 (Tyr701) (58D6) Rabbit mAb, STAT2 (D9J7L) Rabbit mAb, Phospho-STAT2 (Tyr690) (D3P2P) Rabbit mAb, JAK1 (6G4) Rabbit mAb, Phospho-JAK1(Tyr1034/1035) (D7N4Z) Rabbit mAb, TYK2 (D4I5T) Rabbit mAb, Phospho-TYK2 (Tyr1054/1055) (D7T8A) Rabbit mAb, β-Actin (13E5) Rabbit mAb and Anti-rabbit HRP-linked antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Densitometric analysis was performed by Image J software version 1.44.

The following antibodies were purchased from Cell Signaling (Danvers, MA, USA): JAK1 Antibody (#3332, 130kDa), Phospho-JAK2 (Tyr1007/1008) Antibody (#3771, 125kDa), β-Actin Antibody (#4967, 45kDa), Phospho-STAT1 (Tyr701) (58D6) Rabbit mAb (#9167, 84,91kDa), Phospho-STAT2 (Tyr690) Antibody (#4441, 113kDa), Phospho-STAT3 (Tyr705) Antibody (#9131, 79,86kDa), Phospho-STAT5 (Tyr694) Antibody (#9351, 90kDa), Phospho-STAT6 (Tyr641) Antibody (#9361, 110kDa), Ras Antibody (#3965, 21kDa), p44/42-MAPK (Erk1/2) Antibody (#9102, 42,44kDa), GSK-3β (27C10) Rabbit mAb (#9315, 46kDa), MMP-9 Antibody (#3852, 84,92kDa), Phospho-PKCα/β II (Thr638/641) Antibody (#9375, 80,82kDa); PKCα Antibody (#2056, 80kDa); Phospho-PKC (pan) (βII Ser660) Antibody (#9371, 78 a 85kDa); Phospho-PKD/PKCμ (Ser744/748) Antibody (#2054, 115kDa); PKD/PKCμ Antibody (#2052, 115kDa); PKCδ Antibody (#2058, 78kDa); Phospho-PKCδ/θ (Ser643/676) Antibody (#9376, 78kDa), PKCζ Antibody (#9372, 78kDa); PLCγ1 Antibody (#2822, 155kDa); anti-mouse, anti-rabbit and anti-goat IgGs antibodies. From Abcam (Cambridge, MA, USA): anti-PKC antibody (ab59363) was purchased. PepChip1-Kinomics^™ slides were obtained from Pepscan Presto BV (Lelystad, the Netherlands).
WP1066 (#573097; C₁₇H₁₄BrN₃O) and Tofacitinib (#PZ0017; C₁₆H₂₀N₆O · C₆H₈O₇) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell lysis, protein isolation and immunoblotting were performed as described previously [24 (link)]. Twenty micrograms of protein were resolved using 4–12% Bis-Tris polyacrylamide gels. Gels were transferred to PVDF membranes and subjected to blocking and incubation with primary and secondary antibodies. The primary antibodies used in this study included: phospho-STAT3 (Tyr705, Cell Signaling, Boston, MA and Abcam, Cambridge, MA), total STAT3 (Cell Signaling and Abcam), phospho-STAT1 (Cell Signaling), total STAT1 (Cell Signaling), phospho-STAT2 (Cell Signaling), phospho-STAT4 (Cell Signaling), phospho-STAT6 (Cell Signaling), and GAPDH (Cell Signaling). Protein bands were detected using Western Lightning ® Plus Enhanced Chemiluminescence substrate (Perkin Elmer, Waltham, MA) and HyBlot CL ® Autoradiography film (Denville Scientific, Holliston, MA). The cytokines used in this study included: interferon gamma (IFN-γ, Cell Signaling), interferon alpha (IFN-α, Cell Signaling), interleukin-4 (IL-4, Cell Signaling), interleukin-6 (IL-6, Cell Signaling), and oncostatin M (OSM, Cell Signaling).

Western blotting, cell counting kit 8 (CCK‐8), Transwell migration, and flow cytometry assays were performed as previously described (Patel et al., 2006). The primary antibodies used in this study were as follows: GAPDH (dilution 1:1000; Cell Signaling Technology, Inc.), XCR1 (dilution 1:600; Cell Signaling Technology, Inc.), JAK1 (dilution 1:600; cat. no. 2019; Cell Signaling Technology, Inc.), phospho‐JAK1 at Tyr1034/1035 (dilution 1:500; Cell Signaling Technology, Inc., JAK2 (dilution 1:1000; Cell Signaling Technology, Inc.), phospho‐JAK2 at Tyr1007 (dilution 1:1000; Cell Signaling Technology, Inc.), JAK3 (dilution 1:500; Cell Signaling Technology, Inc.), phospho‐JAK3 at Tyr980 (dilution 1:500; Cell Signaling Technology, Inc.), STAT 1–4 (dilution 1:500; dilution 1:500; Stat Antibody Sampler Kit, Cell Signaling Technology, Inc.), phospho‐STAT1 at Tyr701, phospho‐STAT2 at Tyr690, and phospho‐STAT4 at Tyr693 (dilution 1:500; Cell Signaling Technology, Inc.).

Human monocytes were lysed using RIPA buffer supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (Sigma-Aldrich). Lysates were separated by NuPAGE 4%–12% Bis-Tris Protein Gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes (Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 50% Odyssey Blocking buffer in PBS-T (0.1% Tween 20 in PBS) and incubated with appropriate antibodies overnight at 4°C. The following antibodies were used: STAT1, phospho-STAT1(Y701), STAT2, phospho-STAT2 (Y690), TBK1, phospho-TBK1 (Ser172), LAMP1 (D2D11) XP, SQSTM1/p62, phospho-S6 (Ser235/236), GAPDH, and β-actin (Cell Signaling Technology); IRF3 and phospho-IRF3 (S386) (Abcam); and LC3 and vinculin (Sigma-Aldrich). Antibody suppliers and catalog numbers are shown in Supplemental Table 10. Primary antibody incubations were followed by incubation with IRDye secondary antibodies for 1 hour at room temperature. Immunoblots were imaged using Odyssey CLx Imaging System (LI-COR Biosciences). Protein band intensity was quantified using ImageJ (NIH) and normalized to β-actin or vinculin. Detailed information about antibodies used in this study is summarized in Supplemental Table 10.