Anti mmp 3

The total protein was extracted using RIPA reagent (Beyotime), and the protein concentration was detected using BCA detection kit (Beyotime). 40 μg protein sample/lane was subjected to 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were sealed with 5% skimmed milk at room temperature and then incubated at 4℃ overnight with the corresponding primary antibody. Following that the membranes were incubated for 2 h at room temperature with the corresponding secondary antibody. Protein bands were developed using ECL (Millipore), and images were taken and analyzed using Bio-Rad ChemiDoc XRS + (Bio-Rad, CA, USA). Primary antibodies (Anti-GJA1 (1:1000), anti-Collagen II (1:1000), anti-MMP-3 (1:2000), anti-MMP13 (1:2000), anti-ADAMTS4 (1:1000), anti-ADAMTS5 (1:1000), anti-GAPDH (1:5000)) and HRP labeled secondary antibodies (1:5000) were obtained from Abcam (Cambridge, USA). GAPDH was the normalization.

For preparation of protein extracts, 12 pairs of cancer and adjacent normal tissues were crushed with a mortar under ice cold conditions and lysed with RIPA lysis buffer together with protease inhibitors. Cells were collected and lysed with RIPA lysis buffer together with protease inhibitors. After centrifugation at 12,000 rpm at 4°C for 20 min, supernatants were collected and protein concentration was determined using the Pierce™ BCA protein assay (Thermo, Waltham, MA, USA). Proteins were separated by electrophoresis on a 10% SDS-polyacrylamide gel, electroblotted onto a PVDF membrane, and blocked by 5% nonfat dry milk for 1 h. Membranes were then washed in TBST three times for 5 min and then incubated with anti-MMP1 (Abcam, Cambridge, MA, USA), anti-MMP2 (Abcam), anti-MMP3 (Abcam, USA), anti-MMP7 (Abcam, USA), anti-MMP8 (Abcam, USA), anti-MMP9 (Invitrogen, Waltham, MA, USA), anti-MMP11 (Bioss, Beijing, China), anti-MMP12 (Abcam, USA), anti-MMP14 (Abcam, USA), anti-MMP17 (Abcam, USA), anti-MMP19 (Bioss, China), anti-MMP28 (Abcam, USA), anti-Collagen (Abcam), anti-TIMP2 (Bioss, China), or anti-GAPDH (Abcam). Subsequently, the membranes were washed with PBST and incubated for 1 h with goat anti-rabbit IgG (Abcam). Finally, membranes were washed three times and immunoreactivity was determined by using a Chemi DOC™ XRS+ system (BioRad Laboratories, Hercules, CA, USA).

Proteins were extracted using RIPA buffer (Sigma) and protease inhibitor cocktail (Sigma), boiled in sample buffer (BioRad), and separated by SDS-PAGE using 6 % stacking gel and 12 % separating gel (cell sample: 50 μg/lane; medium sample: 20 μl/lane). PVDF (0.45 μm; Millipore, Billerica, MA, USA) blots were prepared and incubated at 4 °C overnight with the primary antibodies (1:1000; anti-MMP3, Abcam; anti-TIMP1, R&D; anti-GAPDH, Abcam), followed by enzyme-conjugated secondary antibodies (GE, Marlborough, MA, USA), and detection was carried out with the ECL Kit (Pierce, Thermo Fisher, Grand Island, NY, USA) and visualized using a FOTO/Analyst1 Fx CCD imaging system (Fotodyne, Hartland, WI, USA). Images were analyzed by NIH Image J 1.45s. Each blot was repeated at least in duplicate, and representative scans are presented.

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.

Total protein extracts were generated using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with PhosSTOP and Complete Phosphatase/Protease Inhibitor Cocktails (Roche Diagnostics GmbH, Mannheim, Germany). Protein extracts (20–25 μg per sample) were loaded onto SDS-PAGE gels and transferred electrophoretically to PVDF membranes and immunodetection of proteins was performed using ECL™ Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK). The following primary antibodies were used: anti-Myc, anti-HIF1a, anti-4EBP1, anti-eIF4E, anti-YBX1, anti-Snail, anti-CA9, anti-Smad2/3, anti-β-catenin (Cell Signaling), anti-phospho Smad2 (Millipore), anti-β3 integrin, anti-MMP3, anti-N-cadherin (Abcam), anti-CycD1, anti-vimentin (Santa Cruz Biotechnology), anti-BMP2, anti-CCDC103, anti-TTC30B, anti-EIF3G, anti-RPL11 (CusaBio), anti-Cx31 (Alpha Diagnostics), anti-eIF4E2 (GeneTex), anti-αv integrin and anti-β-actin (1:500; Calbiochem, Darmstadt, Germany). Anti-mouse and anti-rabbit HRP secondary antibodies were from Pierce. Bound antibodies were visualized with an enhanced chemiluminescence detection kit (Amersham Pharma-Biotech).

Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin solution, and fetal bovine serum (FBS) were purchased from Gibco (Life technologies Korea, Seoul, Korea). 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoli-um bromide (MTT) and 2′,7′-di-chlorodihydrofluoresceindiacetate (H2DCF-DA) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Anti-Nrf2, Anti-LaminB1, Anti-Procollagen1, Anti-MMP1, Anti-MMP3 and Anti-β actin antibody, Alexa Flour 488 goat anti-rabbit IgG and fluoroshield mounting medium with DAPI were purchased from Abcam (Cambridge, MA, USA). RNeasy Mini Kit was purchased from QIAGEN (Hilden, Germany). iScript™ Reverse Transcription Supermix and SsoFast™ EvaGreen^® Supermix were purchased from Bio-Rad (Hercules, CA, USA).