Sc 398608

For western blotting, anti-VDR (D-6): sc-13133, anti-Fbxo32/MAFbx (F-9): sc-166806, anti-Trim63/MuRF1 (C-11): sc-398608 (Santa Cruz), and anti-β-Actin: 010-27841 (Wako) antibodies were used.

Angiotensin II, chloroquine (CQ), Bafilomycin A1(Baf A1) and 3-methyladenine (3-MA) were purchased from Sigma Aldrich (Sigma Aldrich, USA). Primary antibodies for detecting Sirt3 (rabbit monoclonal) (D22A3) [5490], FoxO1 (rabbit monoclonal) (C29H4) [2880], LC3 (rabbit polyclonal) [2775], LC3 (rabbit polyclonal) [3868], Beclin-1 (rabbit monoclonal) (D40C5) [3495] were purchased from Cell signalling Technology (CST, UK). Primary antibodies against ac-FoxO1 (rabbit polyclonal) (FKHR D19) [sc49437], MuRF1 (mouse monoclonal) [sc398608], MAFBx (mouse monoclonal) (sc166806) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Primary antibodies against p62 (mouse monoclonal) [ab56416] was obtained from Abcam. Primary antibodies against β-Tubulin (mouse monoclonal) [BM1453], GAPDH (mouse monoclonal) [BM1623], α-SMA (mouse monoclonal) [BM0002] was purchased from Boster.

The molecular weights of the Atrogin-1, MuRF1, and GAPDH (internal reference) target proteins are 42, 40, and 36 kDa, respectively. The total protein contents in rats’ gastrocnemius muscles were extracted and determined according to the BCA method. The BCA working fluid and standards used in protein analyses were prepared based on the instructions of the BCA protein concentration determination kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). Briefly, the samples were mixed with buffer solution (5 times their volume) and boiled for 15 min to denature the protein. SDS-PAGE and membrane transfer were carried out, and the membrane was blocked with 5% skimmed milk for 1 h. Thereafter, the diluted first antibody solution was added [rabbit anti-Atrogin-1 antibody (ab74023, Abcam, 1:1000), mouse anti-MuRF1 (sc-398,608, Santa Cruz, 1:1000)], and the mixtures were incubated at 4 °C overnight. Then, the samples were washed before adding the second antibody (5% BSA solution 1:5000) and incubating at room temperature for another 1 h. Finally, a photosensitive solution was added to each sample, resulting in the formation of a photosensitive film that was fixed and scanned. The scanned images were analyzed by ImageJ software, and the optical density of each band was measured. The OD ratios of the target and GADPH bands were used to assess the relative protein expressions.