Anti jak2 d2e12

Immunoblotting was performed as previously described (20 (link)). For examination of the protein involved in pro-survival signaling pathway, cell lysis from empty-, JAK2-, or JAK2^V617F-expressing macrophages were extracted and subpackaged equally for three groups (each group included empty, JAK2 and JAK2^V617F lysates), followed by separation on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. After semi-dry transfer, the membranes were sequentially probed with the indicated antibodies. Anti-p-JAK2 (T1007/1008) (#3776), anti-JAK2 (D2E12) (#3230), anti-p-STAT1 (T701) (#7649), anti-STAT1 (D1K9Y) (#14994), anti-p-STAT3 (Tyr705, D3A7) (#9145), anti-STAT3 (D3Z2G) (#12640), anti-p-STAT5 (Tyr694, C11C5) (#9359), anti-STAT5 (D206Y) -(#94205), anti-p-p38 (Thr180/Tyr182, D3F9) (#4511), anti-p38 (D13E1) (#8690), anti-p-AKT (Ser473, D9E) (#10019), anti-AKT (#9272), anti-p-JNK (Thr183/Tyr185, 81E11) (#4668), and anti-JNK (#9252) antibodies were purchased from Cell Signaling Technology. Anti-HK1 (19662-1-AP), anti-HK2 (22029-1-AP), anti-PKM1 (15821-1-AP), anti-flag (Sigma), and anti-β-actin (66009-1-Ig) antibodies were purchased from ProteinTech.

Sub-confluent cells were lysed in Laemmli buffer (LB) and 45 µg of total proteins were subjected to SDS-PAGE and western blotting following standard methods. Protein detection was performed by using the following primary antibodies, diluted as indicated: anti-human MET (3D4, 1:3000, Invitrogen Corp., Camarillo, CA), anti-pMET Tyr^1234/1235 (D26, 1:1000), anti-JAK1 (6G4, 1:1000), anti-pJAK1 Tyr^1022/1023 (1:1000), anti-JAK2 (D2E12, 1:1000), anti-pJAK2 Tyr^1007/1008 (1:1000) anti-STAT1 (1:1000), anti-pSTAT1 Tyr⁷⁰¹ (58D6, 1:1000), anti-PD-L1 (E1L3N, 1:1000), anti-GAPDH (D4C6R, 1:1000) (all from Cell Signaling Technology, Beverly, MA), anti-IFNGR1 (EPR7866, 1:1000, Abcam, Cambridge, UK) and anti-Vinculin (hVIN-1, 1:1000, Sigma Life Sciences). Secondary HRP-conjugated goat anti-mouse IgG (1:20,000) or anti-rabbit IgG (1:20,000) (from Jackson ImmunoResearch, Cambridge, UK) and ECL System (Promega, Madison, WI) were used for protein detection.

Human E-selectin/Fc, human ICAM-1/Fc, human VCAM-1/Fc and human CXCL12 were from R&D Systems (R&D Systems, Minneapolis, MN, USA); goat anti-human IgM was from SouthernBiotech (SouthernBiotech, Birmingham, AL, USA); fluorescein isothiocyanate (FITC) goat secondary antibody to mouse was from Sigma (Sigma-Aldrich, St. Louis, MO, USA); KIM127 mouse monoclonal antibody was from American Type Culture Collection (ATCC, Rockville, MD, USA); 327A mouse monoclonal antibody was kindly provided by Dr. Kristine Kikly (Eli Lilly and Co., Indianapolis, IN, USA); goat F(ab’)₂ anti-human IgM was from Southern Biotech (Southern Biotech, Birmingham, AL, USA); Tyrphostin and rabbit polyclonal anti-actin antibody were from Sigma; Ibrutinib was from Selleck Chemicals (Selleck Chemicals, Houston, TX, USA); PE mouse anti-BTK (pY223) antibody was from BD Biosciences (BD Biosciences, San Jose, CA, USA); rabbit monoclonal anti-JAK2 (D2E12) was from Cell Signaling Technology (Danvers, MA, USA); siRNAs (ON-TARGETplus SMARTpool) were from Thermo Fisher Scientific (Fremont, CA, USA).