Bs 1630r

Total protein was extracted from transfected cells using a lysis buffer containing 1% phenylmethyl sulfonyl fluoride, and the concentration of total protein was determined using a protein concentration assay Kit (Pierce, Thermo Fisher Scientific Inc.). Protein samples were separated using 10% SDS–PAGE gel at a rate of 20 µg per well and then transferred to polyvinylidene difluoride membranes. The membranes were washed with 1 × TBST and incubated with the following primary antibodies: anti–DGAT1 (ab100982; Abcam, Cambridge, UK), anti–DGAT2 (ab237613; Abcam), anti–peroxisome proliferator-activated receptor γ (PPARγ) (bs-4509R; Bioss, Beijing, China), anti–CCAAT/enhancer-binding protein α (C/EBPα) (bs-1630R; Bioss), anti–C/EBPβ (bs-1396R; Bioss), and anti-lipin 1(LPIN1) (bs-7533R; Bioss) overnight at 4 °C under constant shaking. This was followed by incubation with the secondary antibody: horseradish peroxidase-conjugated affinipure goat anti-mouse IgG (Jackson, Anhui, China) for 2 h at room temperature (approximately 25 °C) after washing with PBS. The endogenous control was β-Actin (AB8226; Abcam). Protein bands were photographed using a multifunctional imaging system (Azure 600; Azure Biosystems, Dublin, CA, USA).

Total proteins were extracted using radioimmunoprecipitation assay buffer (RIPA) with 1% PMSF (Solarbio, Beijing, China) after transfection for 48 h or induction for six days after transfection. The extracted protein was mixed with protein loading buffer (denaturation) at a ratio of 4:1 and denatured at 100 °C for 10 min. Then, protein was subjected to 10% polyacrylamide gel electrophoresis at 80 V for 30 min and 120 V for 90 min and transferred to a 150 V ice bath for 40 min. The skim milk powder was blocked for 1 h, and the primary antibody was incubated overnight. The antibodies were then rinsed with PBS-Tween three times for 5 min each. The fluorescent secondary antibody was incubated in the dark for 2 h and washed with PBST three times for 5 min each. The primary antibodies used were against cyclin D1 (1:1000, bs-20596R, Bioss), proliferating cell nuclear antigen (PCNA; 1:1000, bs-0754R), peroxisome proliferator-activated receptor γ (PPARγ; 1:1000, bs-4590R), CCAAT/enhancer-binding protein alpha (C/EBPα; 1:1000, bs-1630R), phospho-beta-arrestin (ARRB1; 1:1000, bs-3048R), and GAPDH (1:5000, bs-2188R). The secondary antibody used was goat anti-rabbit IgG H&L/AP (1:5000, bs-0295G-AP). Finally, we present the results of Western blotting using Image Studio Lite ver. 5.2 (LI-COR Inc., Lincoln, NE, USA) and quantified with the ImageJ program (Bio-Rad, Hercules, CA, USA).

Transfected cells were lysed in RIPA buffer containing 1% phenylmethylsulfonyl fluoride (PMSF). A BCA protein kit (Pierce, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to determine the total protein concentration. Protein samples were separated using 10% SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4 °C with the following antibodies: anti-DGAT1 (ab189994, Abcam, Cambridge, UK), anti-DGAT2 (ab59493, Abcam, Cambridge, UK), anti-PPARγ (bs-4509R, Bioss, Beijing, China), anti-C/EBPα (bs-1630R, Bioss, Beijing, China), anti-SREBF1(bs-1402R, Bioss, Beijing, China), anti-Pax7 (ab61067, Abcam, Cambridge, UK), anti-MYOD (bs-2442R, Bioss, Beijing, China), and anti-MYOG (bs-3550R, Bioss, Beijing, China). Thereafter, the membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (bs-0295G; Bioss, Beijing, China) for 1 h. β-actin (ab8226, Abcam, Cambridge, UK) was used as an endogenous control. A grayscale intensity analysis was performed using ImageJ software (NIH).