Anti phospho jak2 tyr1007 1008

The RPMI 1640 medium, fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were procured from Hyclone. Lipopolysaccharide (LPS, From E. coli, Cat no: L-2630) was procured from Sigma (MO, USA). Primers for real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and TRIzol (Cat no: 15596026) were purchased from Invitrogen. Revert Aid First-Strand cDNA Synthesis Kit (Cat no: k1621) and FastStart Universal SYBR Green Master (ROX) (Cat no: 4385610) were purchased from Roche. RIPA (Cat no: 9806S) and primary antibodies for Western blot analysis, including anti-IκB (Cat no: 76041S), anti-phospho-IκB (Ser32) (Cat no: 2859S), anti-NF-κB p65(Cat no: 8242S), anti- phospho-NF-κB p65(Ser536) (Cat no: 3033) anti-JAK2(Cat no: 3230S), anti-phospho-JAK2(Tyr1007/1008) (Cat no: 3771S), and anti-GAPDH (Cat no: 5174S), were purchased from Cell Signaling Technology. Secondary antibodies were obtained from Sigma. The Enhanced Chemiluminescence Solution Kit (Cat no: WBKLS0500) was obtained from Millipore (MA, USA). APC mouse anti-human CD16(Cat no: ab203883) and FITC mouse anti-human CD86(Cat no: ab213044) were purchased from Abcam (Cambs, UK). APC mouse anti-human CD40(Cat no: 555591) and FITC mouse anti-human CD23(Cat no: 561146) were purchased from BD Systems (BD Biosciences, USA).

Rabbit polyclonal anti-Ki67 (1:1000, 9027), anti-phospho-JAK2 (Tyr-1007/1008, 1:1000, 3776), anti-phospho-STAT3 (Tyr-705, 1:1000, 9145) were obtained from Cell Signaling Technology (MA, USA). Rabbit polyclonal anti-JAK2 (1:1000, A19629), and anti-STAT3 (1:1000, A11185) were purchased from Abclonal (Wuhan, China). Anti-NOX1 (1:1000, GTX103888) was collected from Genetex (CA, USA). Rabbit polyclonal anti-OPN (1:1000, ab214050) was obtained from Abcam (Cambridge, UK). The human recombinant OPN, FLLL32, NAC, and BHA were purchased from Sinobiological (Beijing, China), MCE (Shanghai, China), and Sigma-Aldrich (St Louis, USA), respectively.

Cells were lysed in NET buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA pH 8) and immunoblotted with the following antibodies: anti-STAT3 (#9139), anti-phospho-STAT3(Tyr705) (#9131), anti-phospho-STAT3(Ser727) (#9134), anti-IL1β (#12242), anti-phospho-JAK2(Tyr1007/1008) (#3771), anti-PAI-1 (D9C4) (#11907) from Cell Signaling; anti-NFκB p65 (sc-372), anti-USP18 (sc-98431) and anti-Actin (sc-1616) from Santa Cruz Biotechnology. Proteins of interest were detected with HRP-conjugated anti-mouse/rabbit/goat IgG antibodies from Santa Cruz Biotechnology and visualized with the Pierce ECL Western blotting substrate (ThermoScientific), according to the provided protocol. Autoradiography images were developed with a KODAK MIN-R processor. Full-length blots are included in Supplementary Figures 9/10.

Tris base, dithiothreitol, acrylamide, N,N’-methylenebisacrylamide, sodium dodecyl sulfate (SDS), glycine, ammonium persulfate, NP-40, TEMED, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-IKK2, anti-phospho-IKK2 (Ser180), anti-STAT3, anti-phospho-STAT3 (Tyr705), anti-JAK2, anti-phospho-JAK2 (Tyr1007/1008), anti-ubiquitin, anti-K63-linkage specific polyubiquitin, anti-lamin A/C, anti-actin, anti-p50, anti-IκBα and anti-phospho-IκBα (Ser32) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Tetraubiquitin, K48-linked and K63-linked polyubiquitin chains were ordered from Boston Biochem Inc. (Cambridge, MA, USA). The streptavidin-coupled Dynabeads were purchased from Invitrogen (Carlsbad, CA, USA). Anti-CD3 and anti-CD28 antibodies were purchased from BioLegend Inc. (San Diego, CA, USA). The recombinant TNF-α was ordered from Cell Guidance Systems (St. Louis, MO, USA).