Ab181286

Western blot was performed as described in our previous studies [2 (link)]. In brief, total protein was extracted using pre-cooled RIPA lysis buffer (Beyotime, Shanghai, China) containing PMSF and phosphatase inhibitor, and then total protein concentration was measured by a BCA protein quantification kit (Beyotime, Shanghai, China). After denaturation, equal amounts of total protein were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Subsequently, the PVDF membrane was sealed with 5% skim milk for 1 h, and then incubated with primary antibodies for overnight at 4 °C. After overnight incubation of the primary antibody, the PDVF membrane was incubated with the secondary antibody for 2 h at room temperature. Finally, immunoreactive protein bands were visualized and analyzed by the ECL kit (Pierce, Thermo Fisher Scientific, IL, USA) and ImageJ. Primary antibody information was as follows: cleaved caspase-3 (1:500, ab32042, Abcam), Bax (1:1000, ab182733, Abcam), Bcl-2 (1:500, ab182858, Abcam), VEGFA (1:1000, ab214424), collagen I (1:1000, ab260043, Abcam), collagen III (1:1000, ab7778, Abcam), MMP2 (1:500, ab181286, Abcam), MMP13 (1:1000, ab39012, Abcam), GAPDH (1:1000, ab8245, Abcam), and β-actin (1:2000, ab8227, Abcam).

Western blot was carried out as described previously [16 (link)]. The total proteins of AGS, MKN-45 and 293T cells were extracted by using RIPA lysis buffer (KeyGEN BioTECH, China). Afterwards, the total proteins were treated with 10% SDS/PAGE Resolving Gel Master Mix (P0670-250ml, Beyotime, China) for separation, followed by transfer onto polyvinylidene fluoride (PVDF) membranes (T2234, Thermo Fisher, USA). Then, 5% skim milk was adopted to block the membranes, which were then subjected to incubation with primary antibodies overnight at 4°C. After the washes, the membranes were incubated with secondary antibodies for 1 h at room temperature. The results were then visualized and recorded. β-Actin was used as an internal reference. Bio-repeats were implemented in triplicate. The primary antibodies used in this assay include Anti-β-actin, Anti-MMP2 (ab181286, Abcam, UK), Anti-MMP9 (ab228402, Abcam), Anti-USP10 (ab109219, Abcam) and Anti-RFC2 (ab174271, Abcam).

RIPA lysis buffer (Beyotime, Shanghai, China) was applied to extract protein and the protein concentration was estimated using bicinchoninic acid method. Protein extracts were separated by SDS-PAGE and then transferred to PVDF membranes (Beyotime, Shanghai, China). Membranes were incubated with primary antibodies against PTPRO (Abcam, ab231560, 1:1000), MMP2 (Abcam, ab181286, 1:1000), MMP9 (Abcam, ab228402, 1:1000), Ki67 (Abcam, ab16667, 1:1000), VEGF (Abcam, ab214424, 1:1000), ET-1 (Abcam, ab2786, 1:1000), sFIt-1 (Abcam, ab32152, 1:1000), ERp44 (Abcam, ab137611, 1:3000) and GAPDH (Abcam, ab181602, 1:10,000) overnight at 4°C. On the second day, membranes were exposed to a horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 h. Protein signals were developed with an enhanced chemiluminescence (ECL) detection kit (Beyotime, Shanghai, China). Protein expression was analyzed using Image J software with GAPDH as a loading control.

The extraction of total proteins was conducted using a RIPA lysis buffer (Beyotime, Shanghai, China), and a BCA kit (Beyotime, Shanghai, China) was employed to examine protein concentrations. Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). After blocking in 5% nonfat milk for 1 h at 37°C, primary antibodies against MMP2 (Abcam, ab181286, 1:1000), MMP9 (Abcam, ab76003, 1:5000), N-cadherin (Abcam, ab76011, 1:5000), β-cadherin (Abcam, ab32572, 1:5000), Vimentin (Abcam, ab92547, 1:5000), E-cadherin (Abcam, ab40772, 1:5000), TSTA3 (Abcam, ab155306, 1:3000) and GAPDH (Abcam, ab181602, 1:10,000) were applied to incubate the membranes at 4 °C overnight. The next day, the incubation of membranes with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, ab205718, 1:50,000) lasted 1.5 h. Protein bands were visualized using enhanced chemiluminescence reagents, and protein signals were analyzed with the application of Image J software.

Cells were lyzed with Radio-Immunoprecipitation Assay buffer (Aspen Biotechnology, China) to extract total proteins. The concentration of proteins was measured using Bicinchoninic acid Protein Assay kit (Thermo Fisher Scientific). Next, protein samples (30 μg) were loaded to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, USA). After being blocked in 5% skim milk for 1 h, the membranes were incubated with primary antibodies [LRP4, ab230188, Abcam, UK; proliferating cell nuclear antigen (PCNA), ab92552, Abcam; matrix metallopeptidase 2 (MMP-2), ab181286, Abcam; matrix metallopeptidase 9 (MMP-9), ab283575, Abcam; GAPDH, ab181602, Abcam] overnight at 4°C. After that, the membranes were incubated with horseradish peroxidase‐labeled goat anti‐rabbit secondary antibody (ab205718, Abcam) for 1 h at room temperature. Finally, the protein bands were visualized using an efficient chemiluminescenc (ECL) kit (Millipore), and the gray density of protein bands were evaluate using Image J software (National Institute Health, USA).

Protein was separated and resolved by sodium dodecyl sulfonate-polyacrylamide gel (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Beyotime). The membranes were blocked with 5% skim milk for 2 h. The primary antibodies as following: anti-matrix metalloprotein 9 (anti-MMP9, ab283575, 1:1000, Abcam, Cambridge, MA, USA), anti-MMP2 (ab181286, 1:1000, Abcam), anti-PGK1 (ab233135, 1:1000, Abcam), anti-β-actin (ab8227, 1:1000, Abcam). The PVDF membranes were incubated in the primary antibodies at 4℃ overnight and then incubated in the secondary antibody for 1 h. The protein bands were visualized by ECL reagent (Beyotime).