Rhea ecl

For western‐blotting, HeLa cells were plated in 12‐well plates and transfected with 1 µg plasmid. After 48 h transfection, cells were washed with PBS and lysed in 50 µL 1 × SDS loading buffer (50 mm Tris‐HCl pH 6.8, 10% glycerol, 2% SDS, 0.1% bromophenol blue, 1% beta‐mercaptoethanol) at room temperature for 10 min. The cell lysate was collected and then boiled at 95 °C for 10 min. The appropriate amount of protein was loaded onto SDS PAGE gels. The separated proteins were transferred onto a PVDF membrane (Millipore) in an ice‐bath for 2 h. Then the PVDF membrane was blocked in 5% (w/v) BSA (Beijing Dingguo changsheng Biotechnology Co.,Ltd, FA016) in TBST (Tris‐buffered saline, 0.1% Tween 20) at room temperature for 1 h. The blot of protein was stained as indicate for at least 12 h at 4 °C. The blot was washed four times with TBST at room temperature for 5 min each, then stained with 1:5000 HRP‐conjugated Affinipure Goat Anti‐Rabbit IgG(H+L) (Proteintech, SA00001‐2) or HRP‐conjugated Affinipure Goat Anti‐Mouse IgG(H+L) (Proteintech, SA00001‐1) in 5% BSA (w/v) in TBST for 1 h at room temperature. The blots were washed four times with TBST at room temperature for 5 min each time and imaged on Molecular Imager ChemiDocTM XRS+ Imaging System (Bio‐Rad) after incubation with Rhea ECL (US Everbright, Inc).

Total protein was extracted with RIPA lysate (KeyGEN BioTECH, Nanjing, China) and phenylmethylsulfonyl fluoride (PMSF; KeyGEN BioTECH). Samples (60 μg, 5–10 μL) were run on an SDS-PAGE gel followed by blotting to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk, the membranes were incubated with the following primary antibodies: β-actin (1:5000 Abcam, Branford, CT, USA), PGC-1a (1:1000; Abcam), NRF-1 (1:1000; Abcam), and Tfam (1:2000; Abcam), fusion protein mitofusin 1 (Mfn-1) (1:2000; Abcam), optic atrophy 1 (OPA-1) (1:1000; Abcam) and dynamin-related protein 1 (DRP-1) (1:500; Abcam), followed by incubation with appropriate secondary antibodies. Rhea ECL (US Everbright Inc., Suzhou, China) was used as the developer reagent, and the band intensity was assessed by using Image lab software and referenced to β-actin.