Nonfat dry milk

Forty-eight hours after the transfection of MDA-MB-231 cells, cell lysates were collected using 100 μL of RIPA lysate (Applygen, Beijing, China) and 10 μL of PMSF per well of a six-well plate. The protein concentration of cell lysates was measured with a BCA protein assay kit (Applygen, Beijing, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to nitrocellulose (PVDF) (Millipore, Boston, MA, USA) membranes for Western blotting. The membrane was blocked with 5% nonfat dry milk (BD, Franklin, LA, USA) for 2 h, and then diluted with primary antibody caveolin-1 (Affinity, Shanghai, China, 1:1000). β-actin (ZSGB-Bio, Beijing, China, 1:1000) was used as an internal reference. After overnight incubation, the membranes were washed 3 times with TBST (supplemented with 0.1% Tween 20) and incubated with HPR-labeled goat anti-rabbit IgG (ZSGB-Bio, Beijing, China, 1:1000) dilution for 2 h at room temperature. After washing with TBST, it was reacted with a luminescent solution (TransGen Biotech, Beijing, China) and developed under a gel imaging system.

Cells were washed cold PBS and lysed on ice for 15 min with RIPA buffer (Vazyme) supplemented with protease inhibitor cocktail (Roche), after which the cell lysate was collected and centrifuged to eliminate the cell debris. The supernatant mixed with loading buffer was boiled at 100 °C for 5 min and subjected to SDS-PAGE separation. The protein was then transferred to PVDF membrane (Invitrogen) and blocked with 5% nonfat dry milk (BD Biosciences) for 1 h at room temperature. The blot membrane was incubated with primary antibody overnight at 4 °C, washed three times with TBST buffer and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Signals were detected using High-sig ECL Western Blotting Substrate (Tanon). The antibodies used for immunoblotting were anti-TET2 (1:1500, 21207-1-AP, Proteintech) and anti-GAPDH (1:5000, KM9002, SUNGENE BIOTECH).

Cells were harvested at the indicated times after infection, washed with PBS, and lysed with RIPA lysis buffer containing protease inhibitor cocktail (Beyotime Institute of Biotechnology, Beijing, China). Protein concentrations were determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein were separated on 12% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Beijing, China). The membranes were blocked using 5% non-fat dry milk (BD Biosciences) at room temperature for 2 h, washed, and probed using the specified antibodies. Caspase 3 (8G10) rabbit mAb, caspase 8 polyclonal antibody (mouse-specific), cleaved caspase-8 (Asp387) polyclonal antibody (mouse-specific), caspase 9 polyclonal antibody (mouse-specific), cleaved caspase-9 (Asp353) polyclonal antibody (mouse-specific), Apaf-1 (D5C3) rabbit mAb, Cyt C (136F3) rabbit mAb, PARP (46D11) rabbit mAb, Bcl-2 (50E3) rabbit mAb, Bcl-xL (54H6) rabbit mAb, Bim (C34C5) rabbit mAb, Puma polyclonal antibody (rodent-specific), α-Tubulin (11H10) rabbit mAb, COX IV (3E11) rabbit mAb, and corresponding horseradish-peroxide-conjugated secondary antibody were obtained from Cell Signaling Technologies (Danvers, MA, U.S.). Protein bands were visualized using Western Lightning Plus-ECL (Perkin Elmer, MA, U.S.). β-actin served as a loading control.

Forty-eight hours after the transfection of MDA-MB-231 cells, cell lysates were collected using 100 μL of RIPA lysate (Applygen, Beijing, China) and 10 μL of PMSF per well of a six-well plate. The protein concentration of cell lysates was measured with a BCA protein assay kit (Applygen, Beijing, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to polyvinylidene fluoride (Millipore, Boston, MA, USA) membranes for western blotting. The membrane was blocked with 5% nonfat dry milk (BD, Franklin, LA, USA) for 2 h, and then diluted with primary antibody YTHDF1 (Abcam, Cambridge, UK, 1:1000), E-cadherin (Affinity, Shanghai, China, 1:1000), N-cadherin(Affinity, Shanghai, China, 1:1000), Vimentin(Affinity, Shanghai, China, 1:1000). β-actin (ZSGB-Bio, Beijing, China, 1:1000) was used as the internal control. After overnight incubation, the membranes were washed three times with TBST (supplemented with 0.1% Tween 20) and incubated with HPR-labeled goat anti-rabbit IgG (ZSGB-Bio, Beijing, China, 1:1000) dilution for 2 h at room temperature. After washing with TBST, it was reacted with a luminescent solution (TransGen Biotech, Beijing, China) and developed under a gel imaging system.

Cells were washed with cold PBS and lysed in radioimmunoprecipitation lysis buffer (Thermo Fisher Scientific; catalog no.: 89901) containing a protease inhibitor cocktail (Bimake; catalog no.: B14001) and phosphatase inhibitor cocktail (Bimake; catalog no.: B15001). Lysates were denatured for 10 min in 5× SDS-PAGE sample loading buffer, separated by SDS-PAGE, and transferred to nitrocellulose Western blotting membranes (GE Healthcare; catalog no.: 10600001). Subsequently, the membrane was washed and incubated with PBS containing 0.2% Tween-20 (Sigma–Aldrich; catalog no.: P1379) and 5% nonfat dry milk (BD; catalog no.: 232100), incubated with the primary antibodies at room temperature, followed by horseradish peroxidase–conjugated secondary antibodies (Proteintech Group; catalog no.: SA00001-1), and detected with enhanced chemiluminescence (Share-bio; catalog no.: SB-WB012).