Ab15148

The protein concentration of samples was detected by the BCA kit (Auragene, Changsha, P.R. China). Proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Invitrogen). PVDF membranes were blocked with 5% dry milk-TBST for 30 min at 37°C. The blots were then incubated with an antibody [anti-Btbd7 antibody (sc-241937; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-cadherin antibody (ab15148; Abcam, Cambridge, MA, USA), anti-N-cadherin antibody (ab12221; Abcam), anti-vimentin antibody (ab8978; Abcam), anti-CD45 antibody (ab10558; Abcam), anti-CD44 antibody (ab157107; Abcam), CD133 polyclonal antibody (18470-1-AP; Proteintech, Chicago, IL, USA), OCT4 polyclonal antibody (11263-1-AP; Proteintech), SOX2 polyclonal antibody (11064-1-AP; Proteintech), anti-ALDH1A1 antibody (ab52492; Abcam), and anti-Nanog antibody (ab109250, Abcam)] overnight at 4°C. Following three washes, the membranes were then incubated with a secondary antibody for 40 min at 37°C in TBST. Signals were visualized by ECL chemiluminescence detection kit (Auragene).

Protein samples were prepared in RIPA lysis buffer (Abcam, ab156034 with protease inhibitor cocktail). The protein concentration of the samples was tested with a BCA protein assay kit (Abcam, ab102536). Aliquots of 20 μg protein was fractionated with 10% SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked in 5% skimmed milk, dissolved in PBST (0.5% Tween-20) buffer for 2 h at room temperature, and incubated with the following primary antibodies: rabbit anti-TSG101 (Abcam, ab125011, 1/1000), mouse anti-HSP70 (Abcam, ab181606, 1/1000), rabbit anti-E-cadherin(ab15148) (1/500), or rabbit anti-Vimentin (ab92547) (1/1000) at 4°C overnight. Secondary antibodies (1/1000) were subsequently incubated with the membranes at room temperature for 1 h. Positive staining was visualized on X-ray films with Pierce enhanced chemiluminescent visualization reagents.

Transfected SK-BR-3 and BT-549 cells were lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). Proteins (50 µg per lane) were separated via SDS-PAGE on a 10% gel then transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Inc.). The membranes were blocked with 5% dry milk in Tris-buffered saline (TBS) with 0.2% of Tween-20 at 4°C overnight. The membranes were subsequently incubated with primary antibodies against E-cadherin (1:500; ab15148; Abcam), N-cadherin (1:500; ab18203; Abcam), Vimentin (1:500; ab8978; Abcam), and GAPDH (1:250; ab9485; Abcam) at room temperature for 3 h. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; ab6721; Abcam) at room temperature for 1 h. The protein signals were detected using an Enhanced Chemiluminescence Western Blotting Substrate kit (Thermo Fisher Scientific, Inc.) and quantified using ImageJ software v1.46 (National Institutes of Health). GAPDH was used as an internal control.

Proteins were extracted using RNA immunoprecipitation (RIP) assay lysis buffer (Beyotime, Beijing, China) and quantified by bicinchoninic acid (BAC) method, then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and shifted onto a polyvinylidene fluoride (PVDF) membrane. Immunoblotting used antibodies against CD9 (1: 5000, ab68418, Abcam, Cambridge, MA, USA), CD63 (1: 2000, ab68418, Abcam), E-cadherin (E-cad; 1: 1000, ab15148, Abcam), vimentin (1: 5000, ab92547, Abcam), ERα (1: 3000, ab13504, Abcam), GAPDH (1: 10 000, ab181602, Abcam) and the secondary horseradish peroxidase (HRP)-conjugated antibody (1: 1000, ab9482, Abcam). The protein bands were visualized using electrochemiluminescence (ECL).

At 48 h post-transfection, OS tissues and cells were collected and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing phenylmethanesulfonyl fluoride (PMSF; Beyotime) to obtain total protein. The protein concentrations of the total cellular lysates were quantified using a bicinchoninic acid (BCA) protein assay kit (Beyotime). An equal amount (40 µg) of protein was resolved by 810% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After that, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (0.2 μm, Beyotime). Next, all membranes were blocked using the 5% non-fat milk and then probed with the following primary antibodies: antibodies against hexokinase II (HK2) (1:5000, ab227198, 102 kDa), E-cadherin (1:500, ab15148, Abcam), Vimentin (1:2500, ab92547, Abcam), β-actin (1:2500, ab8227, Abcam), MAPK7 (1:200, ab92547, Abcam) and GAPDH (1:2000, ab37168, Abcam). Subsequently, all membranes were maintained in HRP-conjugated anti-rabbit/mouse IgG (Sangon Biotech). Finally, all protein bands were observed using the enhanced chemiluminescence (ECL) system (GEHeathcare, Waukesha, WI, USA). The protein abundances were normalized with GAPDH and β-actin, and ImageJ software was applied to evaluate the density.

Proteins were collected from cultured cells in Radio Immuno Precipitation Assay (RIPA) lysis buffer (Beyotime, Shanghai, China). The concentrations of proteins were detected via BCA protein kit (Beyotime). Aliquots of protein were separated by 12% SDS-PAGE and resolved proteins were transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% milk in PBS with 0.1% Triton X-100 and incubated with different primary antibodies: anti-RACK1 antibody (ab62735, 1:1,000, 30 kDa, Abcam, Cambridge, MA, USA), anti-E-cadherin antibody (ab15148, 1:500, 135 kDa, Abcam, USA), anti-N-cadherin antibody (ab18203, 1:1,000, 125 kDa, Abcam), anti-Snail antibody (ab82846, 1:500, 68 kDa, Abcam), anti-phosphorylation (p)-β-catenin antibody (ab27798, 1:500, 86 kDa, Abcam), anti-β-catenin antibody (ab32572, 1:5,000, 92 kDa, Abcam), anti-c-Jun antibody (ab32137, 1:1,000, 43 kDa, Abcam) and anti-GAPDH (ab181602, 1:10,000, 36 kDa, Abcam, USA) overnight at 4°C. The membranes were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Proteintech, Rosemont, IL, USA). Protein bands were detected with enhanced chemiluminescence (ECL, Thermo Fisher Scientific) and visualized by Quantity one (Bio-Rad Laboratories Inc., Hercules, CA, USA).