Anti p38 mapk

Protein was extracted from the cells and quantified by a Total Protein Extraction Kit (Keygen BioTech, Nanjing, China). Western blotting was performed according to the manufacturer’s protocol. Anti-TPPP, anti-YY1 (#ab109228), anti-E-cadherin (#ab40772), anti-vimentin (#ab92547), anti-MMP3 (#ab52915), anti-MMP7 (#ab205525), anti-VEGF (#ab32152), anti-p38 (#ab170099), anti-MAPK (#ab205926), anti-p38 MAPK (phosphor, Thr180/Tyr182, #4511S), anti-PI3K (#4255S), anti-PI3K (phosphor, Ser249, #13857S), anti-AKT (#2920S), anti-AKT (phosphor, Thr308, #13038S), anti-β-actin (#3700S) and anti-YY1 (#ab12132) antibodies for ChIP were obtained from Abcam (Cambridge, MA) or Cell Signaling Technology (Danvers, MA, USA). β-Actin was used as an endogenous reference. Each blot was independently repeated three times.

Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Recombinant human PDGF-BB was purchased from ACROBiosystems, Inc. (Newark, DE, USA) and AS-IV was purchased from Tauto Biotech (Shanghai, China). Dimethyl sulfoxide (DMSO) and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-smoothelin, anti-α-smooth muscle actin (α-SMA), anti-desmin, anti-phospho-p38 mitogen-activated protein kinase (MAPK), anti-p38 MAPK, anti-matrix metalloproteinase (MMP)2, anti-MMP9 and anti-GAPDH antibodies, as well as a goat anti-mouse secondary antibody, were obtained from Abcam (Cambridge, UK).

The following antibodies were used for western blot (WB) and immunofluorescence (IF): anti-α-Tubulin (WB 1:10000, Abcam ref.: ab7291), anti-β-Actin (WB 1:20000, Sigma-Aldrich ref.: A5441), anti-Phospho p38MAPK (T180/Y182) (WB 1:1000, Cell Signaling ref.: #4511), anti-p38MAPK (WB 1:1000, Abcam ref.: ab170099), anti-Phospho VEGFR2 (Y1175) (WB 1:1000, Cell Signaling ref.: #2478), anti-VEGFR2 (WB 1:1000, Cell Signaling ref.: #3770), anti-Phospho c-Kit (Y719) (WB 1:1000, Cell Signaling ref.: #3391), anti-c-Kit (WB 1:1000, Cell Signaling ref.: #3074 and WB 1:1000, Santa Cruz ref.: sc-13508), anti-DUSP2 (WB 1:1000, IF 1:100, Sigma-Aldrich ref.: SAB4300841), PathScan® RTK Signaling Antibody Array Kit (Chemiluminescent Readout, Cell Signaling ref.: #7982).

Western blotting was performed on cytosolic cellular extracts. Cells were washed with cold phosphate-buffered saline and lysed for 15 min on ice in 0.5 ml of lysis buffer containing protease and phosphatase inhibitors. Cell lysates were clarified by centrifugation (4 °C, 15 min, 12,000 rpm), and protein was subjected to 10% sodium dodecyl sulfate-PAGE (SDS-PAGE) and transferred to a nitrocellulose membrane using a wet transfer system. Membranes were incubated with 5% skim milk dissolved in TBS plus 0.05% Tween 20 (TBST) for 1 h to block nonspecific protein-binding sites. Then, the membranes were incubated with anti-CCRL2 (Abcam, no. ab88632), anti-CX3CR1 (Abcam, no. ab8021), anti-p38 MAPK (Abcam, no. ab197348), anti-ERK (Abcam, no. 196883), anti-AKT (Abcam, no. ab8805), anti-phospho-ERK (Abcam, no. ab50011), anti-phospho-AKT (Abcam, no. ab81283), anti-phospho-p38-MAPK (Abcam, no. ab47363), and anti-GAPDH (Abcam, no. ab181602) antibodies at 4 °C overnight according to the manufacturer’s instructions. The membranes were then washed with TBST and incubated with a secondary anti-rabbit Ab conjugated to HRP (Cell Signaling Technology, no. 7074s) at room temperature. The signals were detected and analyzed by a chemiluminescence imaging system (ChemiScope5600, CLINX, Shanghai, China), and each experiment was performed in triplicate.