Ripa lysis and extraction buffer

Cellular protein in different groups was extracted with 1% PMSF a RIPA Lysis and Extraction Buffer (Beyotime, China). After the total protein was reacted with SDS-PAGE test buffer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to perform further examination. In this step, the proteins were transmembrane onto a polyvinylidene difluoride layer (Novus, USA). After being blocked for 1 h at room temperature, the layer was brooded with anti-Rabbit TRIM2 (1:10,000) (20356-1-AP, Proteintech, USA), Snail1 (1:1000) (#3879, CST, USA), Bmil (1:1000) (#2742, CST, USA), ALDH1 (1:1000) (#54135, CST, USA), CD133 (1:1000) (#64326, CST, USA), GAPDH (1:1000) (#2118, CST, USA), Cyclin D1 (1:1000) (#2978 CST, USA), PCNA (1:1000) (#13110 CST, USA), E-Cadherin (1:1000) (#31958, CST, USA), N-Cadherin (1:1000) (#13116, CST, USA), Vimentin (1:1000) (#5741, CST, USA), and MMP9 (1:1000) (#13667, CST, USA) overnight. Proteins were hatched with the corresponding secondary antibodies for 1 h at room temperature after treated with ECL Chemiluminescence Detection Kit (PromoCell, German). The bands were observed with Chemiluminescence Imaging (clinx Ltd., China).

Total proteins were prepared from fresh frozen tissue or cultured cells in radio immunoprecipitation assay (RIPA) lysis and extraction buffer (Beyotime Biotechnology, Shanghai, China) with protease and phosphatase inhibitors. Denatured proteins (20–50 mg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The following primary antibodies were used according to the manufacturer’s instructions: anti-DDIT4 (Dilution 1:500, Abcam, Cambridge, MA, USA) and anti-β-actin (Dilution 1:2000), anti-Ki67 (Dilution 1:1000), anti-p53 (Dilution 1:1000), anti-p-p53 (p-Ser6) (Dilution 1:1000), anti-p-p53 (p-Ser315) (Dilution 1:1000), anti-p21^Cip1 (Dilution 1:500), anti-p-p21^Cip1 (p-Thr145) (Dilution 1:500), anti-MEK1 (Dilution 1:1000), anti-p-MEK1 (p-Ser221) (Dilution 1:1000), anti-p42/44MAPK (Dilution 1:1000), and anti-p-p42/44MAPK (p-Thr202 and p-Tyr204) (Dilution 1:1000) (Cell Signaling Technology, Beverly, MA, USA). Densitometry of specific blotted bands was analyzed by ImageJ 1.48 software (Image-Processing and Analysis in Java; National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/), and the intensity values were normalized against the β-actin loading control.

Cellular protein in different groups was extracted with 1% PMSF a RIPA Lysis and Extraction Buffer (Beyotime, China). Sodium dodecy lsulfate–polyacrylamide gel electrophoresis was used to perform further examination. In this step, the proteins were transfered onto a polyvinylidene difluoride layer (Novus, USA). After blocking for 1 h at room temperature, the layer was brooded with anti-Rabbit USP18 (1:1000) (#4813, CST, USA), E-cadherin (1:1000) (# 3195S, CST, USA), Vimentin (1:1000) (# 5741S, CST, USA), N-cadherin (1:1000) (#13116S, CST, USA), CD133 (1:1000) (#64326, CST, USA), CD44(1:1000) (# 37259S, CST, USA), Snail1 (1:1000) (#3879, CST, USA), and GAPDH (1:1000) (#2118, CST, USA), overnight. Proteins were hatched with the corresponding secondary antibodies for 1 h at room temperature after being treated with ECL Chemiluminescence Detection Kit (PromoCell, German). The bands were observed with Chemiluminescence Imaging (Clinx Ltd., China).

Cellular protein in different groups was extracted with 1% PMSF a RIPA Lysis and Extraction Buffer (Beyotime, China). After the total protein was reacted with SDS-PAGE test buffer, sodium dodecy lsulfate-polyacrylamide gel electrophoresis was used to perform further examination. In this step, the proteins were transmembrane onto a polyvinylidene difluoride layer (Novus, USA). After being blocked for 1h at room temperature, the layer was brooded with anti-Rabbit USP43 (1:1000) (NBP2-88562, Novus Biologcials, USA), E-cadherin (1:1000) (#3195S, CST, USA), Vimentin (1:1000) (#5741S, CST, USA), N-cadherin (1:1000) (#13116S, CST, USA), CD133 (1:1000) (#64326, CST, USA), CD44 (1:1000) (#37259S, CST, USA), ZEB1 (1:1000) (#70512S, CST, USA), GAPDH (1:1000) (#2118, CST, USA), overnight. Proteins were hatched with the corresponding secondary antibodies for 1 h at room temperature after treated with ECL Chemiluminescence Detection Kit (PromoCell, German). The bands were observed with Chemiluminescence Imaging (clinx Ltd., China).

After being harvested, cells were lysed in radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Beyotime Biotechnology, China) at 4°C with protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology, China). Protein concentrations were detected using a bicinchoninic acid protein assay kit (Beyotime Biotechnology, China). Samples containing the same amount of protein were separated using 8%–10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and then electrotransferred onto polyvinylidene fluoride membranes (Bio-Rad Laboratories, USA), followed by blocking with the blocking buffer (Beyotime Biotechnology, China). These membranes were incubated at 4°C overnight with the primary antibodies (Abcam, USA) and then incubated with the secondary antibodies (Abcam, USA). Signals of the protein bands were detected using an enhanced chemiluminescence system (Millipore, Billerica, MA, USA). Target proteins were visualized with the FluorChem HD2 system (ProteinSimple, USA).