P jak

The STAT3 inhibitor SH-4-54 was obtained from Selleck Company. Antibodies for vimentin, Snail, E-cadherin, p-JAK, JAK, p-STAT3, STAT3, and β-actin were all obtained from Cell Signaling Technology (Louis Park, MN, USA). The IL-17 antibody was from Novartis Cosentyx. Recombinant human IL-17 was from Sigma-Aldrich.

OSU-A9 was provided by one of the co-authors, Dr. J.-R. Weng [9 (link)]. Identity and purity (≥99%) was verified by proton nuclear magnetic resonance, high-resolution mass spectrometry and elemental analysis. Primary antibodies for Akt, p-Akt (Ser473), ERK, p-ERK (Thr202/Tyr204), PARP, JNK, p-JNK (Thr183/Tyr185), p38 MAPK, p-p38 MAPK (Thr180/Tyr182), Caspase-3, MAPKAPK-2, p-MAPKAPK-2 (Thr334), JAK, p-JAK (Tyr1007/Tyr1008), STAT3, p-STAT3 (Ser727) were from Cell Signaling Technologies (Beverly, MA) whereas β-actin was from Sigma-Aldrich (St. Louis, MO). The p38 mitogen-activated protein kinase inhibitor SB203580 was purchased from Sigma-Aldrich. pCMV-Flag and STAT3-CA-Flag were purchased from Addgene (Cambridge, MA). The JAK2^V617F-MSCV-IRES-GFP vector was kindly provided by Dr. Yen, Jeffrey J.Y. (Academia Sinica, Taiwan).

The fresh ventricular tissue was homogenized by a rotor-stator homogenizer in ice-cold lysis buffer (Beyotime, Shanghai, China). After boiling with loading buffer (Beyotime), denatured proteins were separated in SDS-PAGE gel, and transferred onto PVDF membrane. The membrane was blocked with nonfat milk, followed by incubation with primary antibody of p-JAK, JAK, p-STAT3, STAT3, CD31, GAPDH (Cell Signaling, Danvers, MA, USA) and VEGF (Santa Cruz) at 4 °C overnight. HRP-conjugated secondary antibody (Cell Signaling) was used to incubate the membrane for another 1 h the next day. Immobilon solution (Millipore, Billerica, MA, USA) was poured on the membrane to develop the band captured by FluorChem Image System (Alpha Innotech, Santa Clara, CA, USA).

IMDM, penicillin, and streptomycin were purchased from Hyclone (Logan, UT, USA). Bovine serum albumin, PMA, U73122, PD98059, PF-02341066, and A23187 were purchased from Sigma (St. Louis, MO, USA). Anti-mouse IL-6 antibody and biotinylated anti-mouse IL-6 antibody were purchased from BD Biosciences (BD Pharmingen, San Diego, CA, USA). p-STAT3, STAT3, p-JAK, JAK, p-PLCγ1, PLCγ1, p-AKT, AKT, p-ALK, ALK, β-actin, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 antibodies were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). TNF-α, CSF2, IL-6, IL-6R, IL-8, and GAPDH oligonucleotide primers were purchased from Bioneer Corp. (Daejeon, Korea).