Pierce ecl substrate western blot detection system

Whole cell extracts were extracted using RIPA and protein concentrations were determined by BCA assay. Equal aliquots of 50 μg total proteins were separated by SDS‐PAGE. Then proteins were transferred to a PVDF membrane. Membranes were blocked with 5% bovine serum albumin in TBST and then incubated in primary antibodies overnight at 4°C followed by secondary antibodies for 1 h at 37°C. Rabbit polyclonal primary antibody against CXCR4 (sc‐9046), mouse monoclonal antibodies against RhoA (sc‐418), RhoGEF (sc‐166301), ROCK (sc‐17794), PI3K (sc‐166365), PAK (sc‐166174), PKN (sc‐136037), GAPDH (sc‐166545), and the horseradish peroxidase‐conjugated secondary antibodies against rabbit‐IgG (sc‐2004) and mouse‐IgG (sc‐2005) were purchased from Santa Cruz Biotechnology. The primary rabbit polyclonal antibody against ARHGAP5 was purchased from Sigma‐Aldrich. Finally, signal was visualized through a chemiluminescent detection system (Pierce ECL Substrate Western blot detection system, Thermo, Rockford, IL, USA).

Proteins were extracted from cultured cells, quantitated using a protein assay (bicinchoninic acid [BCA] method; Beyotime, Shanghai, China). Proteins were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membrane, blocked in 4 % dry milk at room temperature for 1 hour, and immunostained with primary antibodies at 4 °C over-night using anti-MTA1 (1:2000, Abcam, Cambridge, MA), anti-E-cadherin (1:1000; Abcam, Cambridge, MA), and anti-GAPDH (1:1000, Kangchen,China). The results were visualized via a chemiluminescent detection system (Pierce ECL Substrate Western blot detection system; Thermo, Rockford, IL) and exposed in Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA).

The proteins extracted from GC cell lines were quantified using a protein assay (Bio-Rad Laboratories, CA, USA). The protein samples (30–50 μg) were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane. β-actin was used to ensure equal loading. The results were visualized using a chemiluminescent detection system (Pierce ECL Substrate Western Blot Detection System, Thermo Scientific, IL, USA) and exposure to autoradiography film (Kodak XAR film).

Proteins were extracted from mouse tissues and cells using RIPA buffer containing fresh protease and phosphatase inhibitors, and quantified using a protein assay (Bio-Rad Laboratories, Hercules, CA). Protein samples (30 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was conducted using antibodies against PTPRO (Proteintech group, #12161), LC3 (Cell signaling technology, #12741), p62 (Cell signaling technology, #5114), PI3K (Cell signaling technology, #4249), p-PI3K(Y458/Y199) (Bioworld, BS4605), p-AKT(S473) (Cell signaling technology, #9271), p-AKT(T308) (Cell signaling technology, #9275), AKT (Cell signaling technology, #9272), p-s6k (Cell signaling technology, #9205), s6k (Cell signaling technology, #9202), p-MDM4(S367) (Abcam, ab122926), MDM4 (Abcam, ab76362), MDM2 (Abcam, ab3110), p53 (Cell signaling technology, #2527), Cyclin D1 (Santa Cruz, sc-20044), Bcl-2 (Santa Cruz, sc-130307). The results were visualized using a chemiluminescent detection system (Pierce ECL substrate western blot detection system, Thermo Scientific, 32106) and exposure to autoradiography film (Kodak XAR film).

Total proteins were prepared and quantified using a protein assay (BCA method, Thermo, USA). Proteins were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) transferred to polyvinylidene fluoride (PVDF) membrane, blocked in 5% dry milk at room temperature for 1 hour and immunostained with antibodies at 4°C overnight using anti-SOX4 (1∶1000, CST, USA), anti-N-cadherin (1∶1000, CST, USA) and anti-E-cadherin (1∶1000, CST, USA). Anti-GAPDH (1∶5000, sigma,USA) was used as a loading control. Results were visualized through a chemiluminescent detection system (Pierce ECL Substrate Western blot detection system, Thermo, Pittsburgh, PA) and then exposed in Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA).

To get protein, tissue samples and cultured cells were dissolved by RIPA regent plus phenylmethanesulfonylfluoride (Beyotime, Nantong, China). Consistently, 30 mg of the protein was loaded each lane, fractionated by SDS PAGE, transferred onto a PVDF membrane. And then the membrane was incubated at 4 °C overnight with human-specific antibody of Myd88 (Abcam, London, UK), p-NF-κB and NF-κB (CST, Boston, MA, USA), p-AKT/AKT (Abcam, London, UK), GAPDH (CST). The results were visualized by a chemiluminescent detection system (Pierce ECL substrate western blot detection system; Thermo Scientific, Waltham, MA, USA) and exposure to autoradiography film.