Anti mmp9 antibody

The indicated cells were harvested and lysed using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA). The concentration of protein was determined by the bicinchoninic acid assay (BCA; Thermo Fisher Scientific). Equal amounts of proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to the PVDF membranes (Roche, Indianapolis, IN). The membranes were blocked with 5% nonfat milk and then incubated with the primary antibodies: anti-INAVA antibody and anti-FGF1 antibody (ABCAm), anti-MMP9 antibody (Cell Signaling Technology), and anti-α-tubulin antibody (Sigma-Aldrich). The bands were detected using enhanced chemiluminescence (ECL) detection kit (Pierce, Rockford, IL) according to the manufacturer’s instructions.

Western blot assays were performed as we previously described [21 (link)]. The membranes were incubated with anti-BCL-2 antibody (#2876), anti-Bax antibody (#2772), anti-MMP-9 antibody (#3852), and anti-GAPDH antibody (#2118), all from CST (Cell Signaling Technology, Danvers, MA, USA), and subsequently, with the appropriate horseradish peroxidase-conjugated secondary antibody (Beyotime, Zhejiang, China).

RPMI 1640, Lympholyte^®-H, and pre-stained protein SHARPMASS VI MW marker were purchased from Euroclone SpA (Milan, Italy). Anti-MMP9 antibody, anti-P-ERK 1/2 antibody, anti-ERK1/2 antibody, horseradish peroxidase (HRP)-linked anti-rabbit and anti-mouse secondary antibodies, protease inhibitor cocktail (100×), and phosphatase inhibitor cocktail (100X) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-IkBα and anti-β-actin antibodies were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Dibutyryl-cAMP, the potentiator ivacaftor (VX770), gelatin, Triton X-100, and BRIJ^®35 Detergent Calbiochem^® were purchased from Sigma-Aldrich (Milan, Italy). ECL Select™ Western Blotting Detection Reagent, ECL Western Blotting Detection Reagent, and Amersham™ Protran^® Premium 0.45-µm nitrocellulose were obtained from GE Healthcare (Chicago, IL, USA). Brillant Blue R-250 and Acrylamide/Bis Solution were obtained from Bio-Rad Laboratories Srl (Segrate, MI, Italy).

Protein expression of N-cadherin, vimentin, Fibronectin E-cadherin and MMP-9 was performed with immunoblot analysis in cell lysates. After total protein quantification by BCA kit, an equal quantity 30–40 μg protein of all samples was separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and then electrophoretic transferred onto PVDF membrane. Membranes were experienced blockage with 5 % skim milk in TBS buffer, incubation with primary antibodies for target gene, and subsequent incubation with horseradish peroxidase-conjugated secondary antibody. Between each step, membranes was washed with TBS suppled with 20 % Tween20. In the present experiment, anti-E-cadherin antibody, anti-N-cadherin antibody, anti-Vimentin antibody, anti-Fibronectin antibody and anti-MMP9 antibody were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-GAPDH antibody and anti-AFAP1 antibody were purchased from Proteintech (Wuhan, China). The signal was finally visualized using an ECL detection reagent on Bio-Rad ChemiDoc MP. Visualization of GAPDH expression was used as a loading control.

Proteins (30 μg) from each sample were subjected to SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) (10% acrylamide, w/v) and transferred to PVDF (polyvinylidene fluoride) membranes (Amersham Biosciences, Uppsala, Sweden). Nonspecific binding sites were blocked with 5% (v/v) nonfat milk in 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 0.1% (w/v) Triton X-100 at 20°C at room temperature for 1 h. Immunological detection was performed using the anti-MMP9 antibody at 1:1000 dilution (Cell Signaling Technology, Inc, Danvers, MA, USA), anti-S100A9 at 1:250 dilution (HPA004193, Sigma-Aldrich), and anti-clusterin at 1:100 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing three times, blots were further incubated with a horseradish peroxidase-conjugated secondary antibody (1: 5000 dilution) at room temperature for 1 h. Protein intensities were determined using densitometry (LabWorks, UVP, Inc., Upland, CA, USA). Differences with p values<0.05 were considered to be statistically significant.

Total protein was extracted from tumor samples or cells. The protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking via 5% skimmed milk, membranes were incubated (overnight, 4°C) with specific primary antibodies as follows: anti-HOXB7 antibody, anti-NANOG antibody, anti-EPCAM antibody, anti-ERK antibody, anti-phosphorylated ERK antibody, anti-c-Myc antibody, anti-E-cadherin antibody, anti-N-cadherin antibody, anti-Slug antibody, anti-Vimentin antibody, anti-α-SMA antibody (Abcam); anti-MMP2 antibody, anti-MMP9 antibody, anti-GAPDH antibody, anti-SMAD3 antibody, anti-phosphorylated SMAD3, anti-p38 antibody, anti-phosphorylated p38, anti-AKT antibody, anti-phosphorylated AKT antibody (Cell Signaling Technology, Danvers, MA, USA). After washing, membranes were incubated 2 h with secondary antibodies (Sigma-Aldrich). The protein intensity was determined and measured by Image Lab software (5.2.1 Version, Bio-Rad Laboratories Co. Ltd, CA, USA). After normalization to GAPDH protein units for each sample, the semi-quantitate results for either tumor or adjacent samples were obtained as a ratio.

After transfection, cells were harvested and lysed in NP-40 lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; and 1% NP-40) supplemented with protease inhibitors (Roche). The clarified cell lysate (usually 50 μg protein) was applied onto 10% SDS-PAGE gels and then transferred to PVDF membranes. The membranes were probed with the primary antibodies followed by incubation with HRP-conjugated secondary antibodies. The proteins were visualized by ECL reagents (GE Healthcare). Monoclonal antibodies used in this study were anti-AGR2 antibody (1:1,000; abcam), anti-α-DG antibody (1:1,000; Cell Signaling), anti-β-DG antibody (1:1,000; Santa Cruz), MANDAG for β-DG (1:200; DSHB, University of Iowa), anti-MMP2 antibody (1:1,000; abcam), anti-MMP9 antibody (1:1,000; Cell Signaling), and anti-β-actin antibody (1:20,000; abcam).