Antibody against β actin

Antibodies against PKG I, EGFR, p-EGFR (T693), ERK1/2, VASP, c-Raf, MEK1/2, p-VASP (Ser 239), p-c-Raf (Ser338), p-Erk1/2 (Thr202/Tyr204), MEK1/2 (Ser217/Ser221), and Grb2 were purchased from Affinity Biosciences (Affinity Biosciences, OH, USA). Antibodies against p-EGFR (Tyr1068) was purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Sos1 was purchased from Santa Cruz (Santa Cruz, CA, USA). Antibodies against p-Thr/ser was purchased from Abcam Biotechnology (Abcam, Cambridge, UK). Antibody against β-actin was purchased from Proteintech (Proteintech, Chicago, USA). EGF was purchased from PeproTech (Rocky Hill, NJ, USA), 8-Br-cGMP, and Rp-8-Br-cGMPS were purchased from Biolog Biotechnology (Bremen, Germany) (Table 1).

Fetal bovine serum (FBS) and RPMI 1640 medium were purchased from Clark (Richmond, NC, USA), Life Technologies (Grand Island, NY, USA); penicillin, streptomycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Sigma (St. Louis, MO, USA); the Annexin-V-Fluos Staining Kit was bought from Roche (Mannheim, Germany); the OxiSelect Comet Assay Kit was obtained from Cell Biolabs (San Diego, CA, USA); and the Pierce BCA Protein Assay Kit was acquired from Thermo Scientific (USA). The antibody against β-actin was obtained from Proteintech (Chicago, IL, USA). Caspase-8 and caspase-3 antibodies were received from Cell Signaling Technology (Boston, MA, USA). Active caspase-3 and caspase-9 antibodies were received from Abcam (Cambridge, UK). The horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit) was obtainedfrom Proteintech (Chicago, IL, USA). SA and SB were purchased from the Control of Pharmaceutical and Biological Products (Beijing, China). DCFH-DA Reactive Oxygen Species Assay Kit and RIPA lysis buffer were gained from Beyotime (Haimen, China).

Cell lysis, protein extraction, and Western blotting were performed as described [43 (link)]. The antibody against β-Actin was from Proteintech (Rosemont, IL, USA), and the antibody against VAMP3 was from Sangon. For reporter assays, 293T cells were seeded in a 24-well plate and transfected with 20 ng pCMV-luc-VAMP3, 5 ng pRL-CMV, and 400 ng of an miR-124 mimic or a control RNA, and luciferase activities were measured using the Dual-Luciferase Assay System (Promega) two days later.

Extracted the proteins from SW480 cells with the radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Merk Life Sciences, Darmstadt, Germany) supplementary with phosphatase inhibitor and protease inhibitor (Sigma-Aldrich, Shanghai, China). The concentration of the extracted proteins was assessed using a commercialized Bradford protein assay kit (BOSTER Biotechnology, Wuhan, China). Protein of the same quantity (50 μg) was electrophoresly separated on 12% SDS-PAGE gel, and the protein in gels was transferred to methanol-pretreated PVDF membranes (Millipore, Darmstadt, Germany). The membranes with separated proteins on them were then submerged with 5% skim milk solution at 25℃ for 90 min, and immersed in indicated diluted primary antibodies at 4 °C for at least 12 h, followed by incubating with indicated secondary antibodies at the dilution of 1:5000 at room temperature for 90 min. In the present study, the primary antibodies against PCNA, Ki67, MMP2, and MMP9 were offered by Abcam (Cambridge, UK). Antibody against β-actin (Proteintech, Wuhan, China) was the internal control. Diluted all primary antibodies at the ratio of 1:1000. The protein bands were observed by the chemiluminescence in the enhanced ECL immunoblotting system (Tanon, Shanghai, China) and analyzed by ImageJ.

Cell lysis, protein extraction, and Western blotting were performed as described [43 (link)]. The antibody against β-Actin was from Proteintech (Rosemont, IL, USA), and the antibody against VAMP3 was from Sangon. For reporter assays, 293T cells were seeded in a 24-well plate and transfected with 20 ng pCMV-luc-VAMP3, 5 ng pRL-CMV, and 400 ng of an miR-124 mimic or a control RNA, and luciferase activities were measured using the Dual-Luciferase Assay System (Promega) two days later.

Total protein was extracted using ice-cold RIPA buffer (Sigma-Aldrich) with protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentrations were determined by bicinchoninic acid Protein Assay Kit (Pierce, Rockford, IL, USA). Ten µg of proteins were separated by 10% SDS-PAGE and then electrotransferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% skim milk for 1 h at room temperature, and incubated with a human-specific anti-TrxR1 antibody (ProteinTech, Chicago, IL, USA) overnight at 4 °C. After washing, membranes were incubated with anti-rabbit antibody (Sigma-Aldrich) conjugated to horseradish peroxidase for 1 h at room temperature. Blots were visualized on X-ray film using a SuperSignal West Pico Chemiluminiscence Substrate (Pierce). For loading control, membranes were stripped and reused with an antibody against β-actin (ProteinTech). Densitometric analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) to calculate TrxR1/β-actin ratio.

Cells were collected, washed twice with phosphate-buffered saline, and incubated on ice for 20–30 min in radioimmunoprecipitation assay lysis buffer containing 1% benzene sulfonyl fluoride (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology), and proteins were separated by 6%–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology), transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, CA, USA), and incubated for 60 min in 5% non-fat milk followed by primary antibodies against E-cadherin (1:10,000; Abcam, Cambridge, UK) and vimentin (1:5000; Abcam). An antibody against β-actin (1:1000; Proteintech, Rosemont, IL, USA) was used as the loading control. The membrane was incubated for 2 h at room temperature with horseradish peroxidase-conjugated secondary antibodies and protein bands were detected by enhanced chemiluminescence. Relative density values were calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).