Anti rabbit

Cells were lysed in Prusiner's buffer (Tris‐HCl 1 M; pH 7.5 containing NaCl (150 mM), EDTA (5 mM), Triton ×‐100 (0.5%), and deoxycholate and protease inhibitor cocktail). The homogenates obtained were then briefly sonicated. Aliquots of 10µg of total protein were then loaded onto 8%–16% SDS‐PAGE gels. After migration, proteins were wet‐transferred to PVDF membranes and immunoblotted using certain antibodies. Immunological complexes were detected with either anti‐rabbit or anti‐mouse IgG‐coupled peroxidase antibodies (ProteinTech Group, Inc.) by the electrochemiluminescence detection method (Roche Diagnostics S.A.S).

Total proteins from the animal heart apex or H9C2 cells were extracted using RIPA (Solarbio, Beijing, China), and protein concentration and western blotting were performed as previously described [40 (link)] The primary antibodies used in this study were anti-acetyllysine rabbit pAb (1 : 1000; Jingjie PTM BioLabs, Inc., China), anti-Atp5f1 monoclonal antibody (1 : 1000; Proteintech, Wuhan, China), anti-p21 monoclonal antibody (1 : 1000; Boster, Wuhan, China), HA-tagged monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), 6× His-tagged monoclonal antibody (1 : 10000; Proteintech, Wuhan, China), GAPDH monoclonal antibody (1 : 20000; Proteintech, Wuhan, China), and anti-α-tubulin monoclonal antibody (1 : 1000; Boster). Anti-rabbit (1 : 10000) and anti-mouse (1 : 10000) antibodies were purchased from Proteintech.

Whole cell lysates of lung cancer samples were prepared with a proteinase and phosphatase inhibitor cocktail (Roche, CA, USA). Protein concentrations of lysates were measured using the BCA method (Thermo Fisher Scientific, USA). 20 µg of extracted protein was loaded on to a 10% SDS-polyacrylamide gels and then transferred onto PVDF membranes (Millipore, USA). Non-specific binding was blocked on the PVDF membranes with PBS buffer containing 0.1% Tween-20, 2% BSA, and 5% nonfat dry milk. Blots were then incubated with anti-rabbit primary antibodies overnight at 4 °C. The next day, membranes were extensively washed, and then incubated with horseradish peroxidase-conjugated anti-goat (Proteintech) or anti-rabbit (Proteintech) IgG at room temperature for 1 h. Protein blots were probed with primary antibodies against CHD1L (Abcam#ab197019), p65 (Cell Signaling Technology #8242), p65-pSer536 (Cell Signaling Technology #3033), IκBα (Cell Signaling Technology#4814), and IκBα-pSer32 (Cell Signaling Technology #2859). Signals were visualized by chemiluminescence (Bio-rad, Hercules, California) and quantitated using a Quantity One system (Bio-Rad, Hercules, CA, USA).

Total RNA from muscle tissue was extracted as described previously. Total RNA samples were digested by RNase R (Geneseed, Guangzhou, China) and linear RNA were broken. The translation potential of circRNA was subsequently detected by rabbit reticulocyte lysate system (Thermo, USA). The manufacturer's instructions were followed. The translation products were subsequently analyzed by western blotting to determine whether the circNEB had translation products. Primary antibody used was rabbit antibody as described previously, and secondary antibody was anti‐Rabbit (Proteintech group, Wuhan, China).

Cell samples were lysed using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) supplemented with phenylmethylsulfonyl fluoride (PMSF) (Beyotime, Shanghai, China) and total protein was extracted. The total protein samples were separated by electrophoresis in SDS-polyacrylamide gels and then transferred to NC membranes (Pall Corporation, East Hills, NY, USA). After blocking the membrane in 5% skim milk for 2 h, the primary antibody was incubated overnight (4 °C) and the secondary antibody was incubated at room temperature for 1 h. Protein bands were exposed with chemiluminescent reagents (Thermo, Waltham, MA, USA). An anti-α-Tubulin antibody was used as the loading control. The protein expression was quantified using ImageJ (https://imagej.nih.gov/ij/, accessed on 18 June 2021). The following primary antibodies were used: CDC10 (1:1000, Abcam), CyclinD1 (1:500, Proteintech), CyclinB1 (1:500, Proteintech), α-Tubulin (1:1000, Proteintech), C/EBPα (1:500, Proteintech), and PPARγ (1:500, Proteintech). The secondary antibodies were anti-rabbit (1:10,000, Proteintech) and anti-mouse antibodies (1:10,000, Proteintech). The targeted proteins were detected using the Tanon-5200 (Tanon, Shanghai, China) instructions of the manufacturer.