Ab86607

Tissue protein of heart and spleen was lysed by the RIPA lysate (50:1) and tissue disrupter and quantified by the BCA method. The quantified homogenate was added to loading buffer, and the mixture was boiled and denatured at 99°C. Each lane was loaded with 10 ul of proteins. After electrophoresis at 100 V for 1–1.5 h, proteins were transferred to PVDF membranes at 300 mA for 1–1.5 h. Afterwards, the membrane blocked with 5% skim milk for 1–1.5 h at room temperature, incubated on a shaker, and washed with TBS-T. Western blot analysis was conducted using anti-AT1 (ab18801; Abcam, United States), anti-MCP-1 (ab25124, Abcam, United States), anti-CCR2 (PAI-27409), anti-TGF-β1 (3711s, Cell Signaling Technology, Germany), anti-Smad3 (ab28379, Abcam, United States), anti-MMP2 (ab86607, Abcam, United States), anti-Col III (ab7778, Abcam, United States), anti-VEGF (ab10972, Abcam, United States), anti-CD31 (ab24590, Abcam, United States) and anti-GAPDH (ab8245, Abcam, United States) at 4°C overnight. After incubation with the appropriate secondary antibodies at 37°C for 2 h at room temperature, the membrane treated with ECL for 1 min at room temperature. The final expression of each protein was normalized by GAPDH and grayscale analysis was performed using Image J software.

RIPA lysis buffer (Beyotime) and BCA protein assay kit (Beyotime) were utilized for total protein extraction and quantification, respectively. Then, equal amount of protein (20 μg/lane) was segregated by SDS-PAGE gel and then shifted into PVDF membrane (Solarbio). After blocked in 5% non-fat milk for 1 h, the membrane was probed with specific primary antibodies at 4 °C overnight, and subsequently probed with HRP-conjugated secondary antibody (1:20,000, ab205718 or ab205719, Abcam, Cambridge, MA, USA) for 2 h. The blots were exposed to enhanced BeyoECL Moon (Beyotime), and the bands density was assessed via Image J software (NIH, Bethesda, MD, USA). The primary antibodies obtained from Abcam included anti-B-cell lymphoma-2 antibody (Bcl-2; 1:1000, ab32124), anti-BCL2-associated X protein antibody (anti-Bax; 1:1000, ab32503), anti-matrix metalloproteinase 2 (MMP2) (1:1000, ab86607) antibody, anti-MMP9 (1:1000, ab137867) antibody, anti-PPM1A (1:500, ab14824) antibody, and β-actin antibody (1:5000; ab6276). Relative protein expression was normalized by internal reference β-actin.

Cells treated under specific conditions were lysed using a whole-cell lysis buffer (50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 5 mM EGTA, and a protease inhibitor cocktail). Equal amounts of protein extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes using standard techniques. Membranes were probed with specific antibodies against MMP2 (ab86607, Abcam, Cambridge, UK), MMP9 (sc-376861, Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin (sc-8426), vimentin (sc-6260), β-catenin (sc-7963), p85 (#sc-1637, Cell Signaling Technology, Danvers, MA, USA) Akt (#4691), p-Akt (#4060), and α-tubulin (sc-5286). The antibodies were diluted in TBS solution containing 2% skim milk and incubated overnight at 4 °C. Signals were detected using an Image Station 4000MM image analyzer (Kodak, Rochester, NY, USA).

DMEM (Gibco, Grand Island, NY, USA), antibiotic–antimycotic solution (Gibco, Grand Island, NY, USA), foetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), SYBR Green Master Mix (Takara Bio, Takara, Japan), PCR primers (Solarbio Life, Beijing, China), an RNase inhibitor (Sangon Biotech, Shanghai, China), Transwell chambers (Corning, Corning, NY, USA), Matrigel matrix (Corning, Corning, NY, USA), paclitaxel (Aladdin, Shanghai, China), and chromatography-grade acetonitrile (Merck, Darmstadt, Germany) were used. Antibodies specific for Bcl-xL (ab32370), cyclin D1 (ab134175), Jagged1 (ab109536), matrix metallopeptidase 2 (MMP-2) (ab86607), MMP-9 (ab137867), signal transducer and activator of transcription 3 (STAT3) (ab68153), phosphor-STAT3 (ab267373), survivin (ab76424), β-Actin (ab8226), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ab205719), and goat anti-rabbit IgG (ab205718) were purchased from Abcam (Cambridge, England). Antibodies specific for Src (#2109), phospho-Jak family (#32901), Jak1 (#3332), and IL-6 (#12153) were obtained from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were obtained from Shanghai Sinopharm (Shanghai, China), unless otherwise indicated.

Mouse monoclonal antibody against mouse β-actin (ab8277), mouse monoclonal antibody against mouse Mmp2 (ab86607), rabbit polyclonal antibody against mouse Mmp9 (ab38898), rabbit polyclonal antibody against mouse Acta2 (ab5694), rabbit monoclonal antibody against mouse Vim (ab92547), rabbit polyclonal antibody against mouse Col1a1 (ab34710), mouse monoclonal antibody against mouse Tgfb1 (ab27969), and rabbit polyclonal antibody against mouse Fn1 (ab2413) were purchased from Abcam (Cambridge, MA). Rabbit monoclonal antibody against mouse Reck (D8C7) was purchase from Cell Signaling Technology (Beverly, MA).