Gapdh

Cells were lysed in RIPA cell lysis buffer (Beyotime, Shanghai, China) in the presence of protease inhibitor cocktail (Biotool, Huston, USA) and PMSF (Beyotime). Protein concentration was quantified by BCA protein assay kit (Beyotime). 50μg of the extracts were separated on 10% SDS-PAGE and transferred to PVDF membrane. Membrane was then blocked with 5% non-fat milk and incubated overnight with the following primary antibodies, respectively, which are Keratin10 (Abcam, Cambridge, USA), Involucrin (Proteintech, Wuhan, China), OCT4 (Proteintech), SOX2 (Proteintech), GSK-3β (Abcam), β-catenin (Proteintech), non-phospho β-catenin (Cell Signaling, Boston, USA) and LEF1 (Proteintech) followed by incubation with appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h. Hybridization signal was detected by enhanced chemi-luminescence (ECL) (ThermoFisher, MA,USA) according to the manufacturer's instructions. GAPDH (ZSGB-BIO, Guangdong, China) was used as reference protein and determined following the same procedure as above.

Proteins of the tumor and normal tissues near the tumor were extracted using standard protocols, and the contents were determined by the BCA protein assay kit (from BOSTER) and ELISA. The proteins were separated by 8% SDS-PAGE and transferred to PVDF membrane. The membranes were then incubated with the primary antibodies against the proteins, including APC (No.ab58, Abcam, Cambridge, USA), β-catenin (No.ab32572, Abcam, Cambridge, USA), TCF7L1 (No.ab133360, Abcam, Cambridge, USA), TCF7L2 (No.ab76151, Abcam, Cambridge, USA), LEF1 (No.ab137872, Abcam, Cambridge, USA), C-myc (No.sc40, Santa, California, USA), C-jun (No.ab32137, Abcam, cambridge, USA), CYCLIND1(No.ab134175, Abcam, cambridge, USA), MMP7 (No.ab205525, Abcam, cambridge, USA), GSK-3β (No.sc53931, Santa, California USA) and GAPDH (No.ta08, ZSGB-BIO, Beijing China) in 5% non-fat milk in TBST at room temperature for two hours. After washing for three times using TBST, the membranes were incubated with secondary antibodies (No.zdr5306 and 5307, ZSGB-BIO, Beijing China) at room temperature for two hours. Then the membranes were developed using the enhanced chemiluminescence plus reagent and imaged using the Bio-Rad gel imaging system [55 (link)]. Finally, the band values were read using the image J software.

Total protein from cells and gastric cancer tissues was extracted using radioimmunoprecipitation assay lysis buffer containing protease inhibitors. Protein samples (20 μg) were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (12% resolving gel) and then transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). Membranes were then blocked with 5% nonfat milk to block nonspecific binding for 3 h at room temperature and incubated with primary antibodies against claudin 1 (1 : 1000, Abcam, Cambridge, MA, USA), caspase-3 (1 : 1000, Abcam), Bcl-2 (1 : 1000, Proteintech, Rosemont, IL, USA), c-Myc (1 : 1000, Proteintech), cyclin D1 (1 : 1000, Beyotime, Shanghai, China), and GAPDH (1 : 1000, Goodhere, Hangzhou, China) at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2000, ZSGB-BIO, Beijing, China) at room temperature for 3 h. GAPDH was used as an internal control for the relative protein expression. Protein bands were quantified using ImageJ software (NIH).

Samples were trypsinized and collected in ice-cold PBS after 48 h of transfection, RIPA buffer was used to isolate the total protein from the EWS cells. Protein concentrations from whole cell lysates were quantified by BCA assay Kit (Beyotime, Jiangsu, China). The protein (20–30 µg) were separated by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and electro-transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Then membranes were blocked by 5% non-fat dry milk and incubated overnight at 4°C in the presence of VEGFA (Cell Signaling Technology, USA), and GAPDH (ZSGB-BIO, Beijing, China). Upon washed in Tris-buffered saline-Tween 20 (TBST), the membranes were incubated in the presence of respective secondary antibody (ZSGB-BIO, Beijing, China). Proteins were visualized by chemiluminescence (ECL) kit (Millipore, USA) as recommended by the manufacturer. GAPDH was used as control.

Cells and frozen tissues were treated with RIPA buffer (Sigma, USA). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gelelectro phoresis (SDS-PAGE) followed by transfer to PVDF membranes (Millipore, USA). The membranes were blocked with 5% fat-free milk (BD, USA) for 1.5 h at room temperature and incubated with primary antibodies at 4°C overnight. The membranes were then incubated with secondary antibodies for 1 h at room temperature. The following antibodies were used in this study: mouse anti-STIM1 mAb (Abcam, ab57834), rat anti-caspase12 (Millipore, MABC555), rabbit anti-caspase3 (CST, #9662), rabbit anti-cyclin D1 (CST, #2922), rabbit anti-P21 (CST, #2947), mouse anti-Bcl2 (ZSGB-BIO, ZS-7382), rabbit anti-BAX (CST, #5023). GAPDH and β-actin (ZSGB-BIO, China) were used as internal control.

Equal amount of protein was separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). Membranes were probed overnight at 4 °C with primary antibodies against human TIPE3 (1:300; BOSTER, Wuhan, China), p-AKT, p-ERK, AKT, ERK (1:1000, Cell Signaling Technology, Beverly, MA), and flag (1:10000, MBL, Nagano, Japan), GAPDH or β-actin (1:1000; ZSGB-Bio, Beijing, China), followed by secondary antibodies (1:2000; ZSGB-Bio) conjugated with peroxidase for 1 h at room temperature. Signals were detected by SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL).