Ab280375

Protein samples were prepared with radio-immunoprecipitation assay (RIPA) buffer (Beyotime). The concentrations of protein samples were analyzed using the BCA method. Protein samples (35 μg/lane) were loaded onto 10% separating gel and blotted onto a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk for 1 h at room temperature, the membrane was incubated with the primary antibodies overnight at 4 °C, including anti-c-myc (ab32072, Abcam, Cambridge, MA, USA), anti-N-cadherin (ab280375, Abcam), anti-vimentin (ab92547, Abcam), anti-METTL3 (ab195352, Abcam), anti-SOX2 (ab92494, Abcam), anti-TSG101 (ab125011, Abcam), anti-CD63 (ab134045, Abcam), anti-CD81 (ab59477, Abcam), and anti-β-actin (ab8226, Abcam). Afterwards, the membrane was incubated with the secondary antibody (Abcam) for 1 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Pierce, Waltham, MA, USA), and the intensities of protein bands were analyzed using the Image Lab analysis software (National Institutes of Health, Bethesda, MD, USA). Western blot assay was conducted three times.

Ice-cold RIPA buffer (Cell Signaling, USA) was used to lyse the cells. Total protein lysates (50 μg) were separated by denaturing 10% SDS-PAGE and electrotransfered onto a nitrocellulose membrane (Millipore, USA) for subsequent blotting with primary antibodies. The primary antibodies were: IMP4 (1 : 1000, ab181046; Abcam), Bcl-2 (1 : 1000, #15071; CST), Bax (1 : 1000, ab32503; Abcam), PARP (1 : 1000, ab191217; Abcam), E-cadherin (1 : 1000, 20874-1-AP; Proteintech, Wuhan), N-cadherin (1 : 1000, ab280375; Abcam), Vimentin (1 : 1000, ab20346; Abcam), glucose transporter 1 (GLUT1; 1 : 1000, ab115730; Abcam), hexokinase II (HK2; 1 : 1000, ab209847; Abcam), cleaved fructose phosphate kinase P (PFKP; 1 : 1000, ab32561, Abcam), pyruvate kinase M2 (PKM2; 1 : 1000, ab85555; Abcam), lactate dehydrogenase A (LDHA; 1 : 1000, #3582; Cell Signaling, USA), MEK1 (1 : 1000, ab32091; Abcam), p-MEK1 (1 : 1000, ab96379; Abcam), ERK (1 : 1000, ab184699; Abcam), p-ERK (1 : 1000, ab201015; Abcam), p21 (1 : 1000, ab109520; Abcam), p53 (1 : 1000, ab75754; Abcam), cyclin D1 (1 : 1000, ab16663; Abcam), and GAPDH (1 : 1000, ab181603; Abcam), according to the manufacturers' protocols. After incubation with secondary antibodies for 60 min, protein bands were visualised using ECL kit (Beyotime).

The total proteins were extracted by RIPA lysis buffer (Beyotime, Shanghai, China), with the protein concentration determined by a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). The equal amount of proteins (30 μg/lane) were separated by SDS-PAGE and then transferred to PVDF membrane (Millipore, Billerica, MA, USA). After being blocked with 5% skimmed milk at ambient temperature for 1 h, the membrane was incubated overnight at 4°C with primary antibodies including anti-ALX4 (SC-33643, 1: 1000, Santa Cruz Biotechnology, Shanghai, China), anti-E-cadherin (ab76055, 1:1000, Abcam, Shanghai, China), anti-N-cadherin (ab280375, 1:1000, Abcam, Shanghai, China), anti-Vimentin (ab16700, 1:1000, Abcam, Shanghai, China), and anti-GAPDH (ab9484, 1: 1000, Abcam, Shanghai, China). After the membranes were washed by TBST, the membranes were incubated with horseradish peroxidase (HRP)-coupled secondary antibody (ab205719, 1: 2000, Abcam, Shanghai, China) for 0.5 h at room temperature. Ultimately, Clarity Max™Western ECL substrate (Bio-Rad, Hercules, CA, USA) was adopted to develop the protein bands.