Supersignal west femto agent

The total proteins from C2C12 cells were lysed in RIPA lysis buffer supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). The membranes were blocked with 5% BSA for 1.5 h at room temperature, and subsequently probed with primary antibodies overnight at 4 °C. The following dilutions were used for each antibody: myogenin (1:1000; Proteintech, Rosemont, IL, USA), MyHC1 (1:1000; Dshb, New Delhim, India), GAPDH (1:1000; Proteintech), PCNA (1:1000; Proteintech), cyclin E1 (1:1000; Proteintech), and Rpl7 (1:1000; Abcam, Cambridge, MA, USA). The membranes were then washed with PBS-Tween and incubated for 30 min with horseradish peroxidase-conjugated secondary antibodies (Proteintech). Protein bands were detected after treatment with SuperSignal West Femto agent (Thermo Scientific).

We asked if circLMO7 affect the translation of genes which were regulated with cell proliferation and differentiation. We collected cells from the different treatment groups, pelleted them by centrifugation (12 000 × g, 5 min) and lysed them in RIPA buffer (Solarbio, Beijing, China). We prepared total protein and determined protein concentrations using the Bradford method. Proteins were then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to nitrocellulose membranes and blocked with 5% skim milk powder solution for 1.5 h at room temperature. We incubated membranes overnight with the primary antibodies. We purchased anti-MyoD, anti-MyoG, anti-BCL-2, anti-BAX, anti-Caspase9, and anti-β-actin from Abcam (Cambridge, MA, USA). Afterwards, we washed the membranes with PBS-tween and incubated them for 1.5 h with horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, MA, USA). Protein bands were detected after treatment with SuperSignal West Femto agent of Thermo (Thermo Scientific, Karlsruhe, Germany).

The total proteins from C2C12 cells were lysed in RIPA lysis buffer supplemented with a protease inhibitor cocktail (4693124001; Roche, Mannheim, Germany). The membranes were blocked with 5% BSA for 1 h at room temperature and subsequently probed with primary antibodies overnight at 4 °C. the following dilutions were used for each antibody: METTL3, METTL14, WTAP, DNMT1, DNMT3A and DNMT3B (all 1:1000; Abcam, Cambridge, England). The membranes were then washed with PBS-Tween and incubated for 50 min with horseradish peroxidase-conjugated secondary antibodies (SA00001–1; Proteintech, Wuhan, China). Protein bands were detected after treatment with the SuperSignal west Femto agent (34094; Thermo Scientific, Waltham, USA).