Phospho c jun ser63

Cells were harvested and homogenised in modified radioimmunoprecipitation assay buffer (modified RIPA) supplemented with 50 mM Tris‐HCl (pH 7.4), 1% NP‐40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), aprotinin (1 mg/ml), 1 mM phenylmethylsulphonyl fluoride and leupeptin (1 mg/ml). Primary antibodies against fibronectin (#15613‐1‐AP, ProteinTech), Collagen I (#14695‐1‐AP, ProteinTech), α‐SMA (GTX100904, GeneTex), phospho‐SAPK/JNK (Thr183/Tyr185) (#4668; Cell Signaling), SAPK/JNK (#9252; Cell Signaling), phospho‐NF‐κb p65 (Ser536) (#3033; Cell Signaling), NF‐κb p65 (GTX107678; GeneTex), phospho‐c‐Jun (Ser 63) (#2361, Cell Signaling), c‐Jun (#9165, Cell Signaling), phospho‐p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#4377; Cell Signaling), p44/42 MAPK (ERK1/2) (#9102; Cell Signaling), CD44 (ab157107; Abcam) and α‐tubulin (T6199, Sigma) were used for Western blotting. α‐tubulin was used as the loading control.

Recombinant human PDGF-BB was from R&D Systems (Minneapolis, MN). Antibodies to PDGFRα, PDGFRβ, phospho-PDGFRα/β Tyr849/Tyr857, c-Jun, phospho-c-Jun Ser63, c-Myc, EGR1, RUNX1, DDIT3, CYR61 and GDF15 were from Cell Signaling Technology (Danvers, MA); antibodies to Myb and NFAT5 were from Epitomics (Burlingame, CA); antibodies to SOX5 and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA); antibody to β-actin was from Sigma Aldrich (Sigma Chemical Company, St. Louis, MO); antibody to DIAPH3 was a generous gift from Henry Higgs, Dartmouth Medical School. The c-Myc TF ELISA kit was from Active Motif (Carlsbad, CA). SP600125 and 10048-F4 were from EMD Biosciences (Billerica, MA). iScript cDNA synthesis reagents were from BioRad Laboratories (Hercules, CA). Universal PCR master mix for qRT-PCR and gene-specific assays were from Applied Biosystems (now Life Technologies, Grand Island, NY). Primers for human transcripts were as follows: Hs00171022_m1 for CXCL12; Hs00998500_g1 for CYR61; Hs01107330_m1 for DIAPH3; Hs02758991_g1 for GAPDH; Hs00171132_m1 for GDF15; Hs01110250_m1 for HMOX-1; Hs00998018_m1 for PDGFRA; and Hs01019589_m1 for PDGFRB. Primers for mouse transcripts were Mm00487499_g1 for CYR61; Mm99999915_g1 for GAPDH; Mm00442228_m1 for GDF15; Mm00435546_m1 for Pdgfrb.

Cells were lysed in cell lysis buffer (#P0013, Beyotime Biotechnology, China). In total, 20 μg of protein samples was subjected to SDS-PAGE. Next, the proteins were transferred onto PVDF membranes (#ISEQ00010, Merck Millipore, Germany). Then, the membranes were blocked in 5% skim milk for 2 h and incubated with primary antibodies overnight at 4°C. The primary antibodies for GAPDH (1:1,000, # 5,174), Smad3 (1:1,000, # 9,523T), phospho-Smad3 (Ser423/425) (1:1,000, # 9,520), c-Jun (1:1,000, # 9,165), and phospho-c-Jun (Ser63) (1:1,000, # 2,361) were purchased from Cell Signaling Technology (Danvers, MA, United States). Then, the membranes were incubated for 2 h with secondary antibodies (1:2,000, # 7074, Cell Signaling Technology) at 25°C. After the membranes were washed in TBST, chemiluminescent signals were detected using the Bio-Rad Molecular Imager ChemiDoc^TM XRS + system (Bio-Rad, United States). GAPDH was used as the loading control.

The following antibodies were used: PR65 (sc-15355, Santa Cruz, CA, USA), PP2Ac (05-421, Millipore), FLAG M2 (F1804, Sigma Aldrich), HA (sc-7392, Santa Cruz), phospho-AKT (Thr308) (9275, Cell Signaling), phospho-AKT (Ser473) (9271, Cell Signaling), phospho-SRC (Tyr416) (6943, Cell Signaling), phospho-FAK (Tyr397) (3283, Cell Signaling), phospho-PP2Ac (Tyr307) (ab32104, Abcam), SRC (2108, Cell Signaling), p21 (2946, Cell Signaling), β-actin (sc-4778, Santa Cruz), phospho-SAPK/JNK (Thr183, Tyr185) (9251, Cell Signaling), phospho-c-Jun (Ser73) (9164, Cell Signaling), phospho-c-Jun (Ser63) (9261, Cell Signaling), and c-Jun (sc-1694, Santa Cruz).

Cells were treated with 5μg/ml anti-ErbB2 antibodies for 4h at 37°C. After washing, the cells were lysed in SDS lysis buffer and the cell lysates were subjected to SDS-PAGE and immunoblotted with antibodies against ErbB2, phospho-ErbB2-Tyr1221/1222, ErbB3, phospho-ErbB3-Tyr1289, AKT, phospho-AKT-Ser473, p44/42 MAPK, phospho-p44/42 MAPK-Thr202/Tyr204, SAPK/JNK, phospho-SAPK/JNK-Thr183/Tyr185, c-Jun, phospho-c-Jun-Ser63, PARP, Caspase-3, LC3A/B, Bak, Bax, Puma, Bid, Bim, Mcl-1, Bcl-xl, phospho-Bcl-2-Ser70, Bcl-2 (all from Cell Signaling Technology).