A44114

Proteins in the brain tissues and lung tissues were harvested by a RIPA buffer (P0013D, Beyotime, China) and their concentrations were qualified with a BCA Kit (pc0020, Solarbio, China). After denaturation, the protein samples were separated by electrophoresis. Proteins in the gel were transferred to a nitrocellulose membrane (10600023, GE Healthcare Life, USA), which was then sealed a 5% skim-milk. After that, they were reacted with primary antibodies at 4 °C overnight. After washing, they were reacted with anti-rabbit HRP (1:5000, #7074, CST, USA) at 37 °C for 1 h. In the end, the protein signals were developed by the ECL reagent (35055, Pierce, USA) in a gel imaging system (A44114, Invitrogen, USA). The primary antibodies of TNF-α (ab205587, 1:1000), IL-1β (ab254360, 1:1000), p-NF-κB (ab76302, 1:1000), NF-κB (1:5000, ab32536), p-IKBα (1:10,000, ab133462), IKBα (1:5000, ab32518), HIF-1α (1:1000, ab179483), and GAPDH (1:5000, ab199554) were obtained from Abcam (UK).

The acquired gastrocnemius tissues were lysed with the assistance of a lysis buffer (abs9229, absin, China). Subsequently, we selected the BCA kit (BI-WB005, SBJBIO, China) to quantify the lysed protein. Thereafter, the quantified protein was electrophoresed for protein separation, which was then loaded onto the PVDF membrane (PW0034, Leagene, China). After being sealed in 5% bovine serum albumin (BL-082, SBJBIO, China) at 37°C for 60 min, the membrane underwent primary antibodies (4°C, overnight). Afterward, the bound antibodies were then exposed to anti-rabbit secondary antibody (31466, Invitrogen, USA) or anti-mouse secondary antibody (S0002, Affinity, USA) with the condition of 37°C for 60 min. Visualization of protein was conducted by applying an ECL reagent (GK10008, GlpBio, USA) on a gel imaging system (A44114, Invitrogen, USA). The primary antibodies of PGC-1α (1:1,000, ab191838), P62 (1:10,000, ab109012), LC3B (1:2,000, ab48394), AMPKα (1:5,000, ab32047), p-AMPKα (1:10,000, ab133448), Smad3 (1:1,000, ab208182), p-Smad3 (1:2,000, ab52903), and β-actin (1:5,000, ab8227) were provided by Abcam (UK).

The treated cells were routinely harvested and lysed by a cell lysis buffer (abs9225, Absin, China) [17 (link)]. After centrifugation, the protein concentration of the cell supernatant was examined using a BCA kit (abs9232, Absin, China). After denaturation, equal quantities of protein were electrophoresed and then transferred onto a polyvinylidene difluoride membrane (#ISEQ00010, Merck-Millipore, USA). After blocking, they were incubated with primary antibody overnight at 4℃ followed by the addition of secondary antibody (S0001, Affinity, USA) for 1 h at 37℃. Finally, protein expression was examined with a color reagent (abs920, Absin, China) in a chemic luminous instrument gel imaging system (A44114, Invitrogen, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed for the housekeeping gene. The primary antibodies of SFRP1 (1:1,000, 35 kDa, #3534) and GAPDH (1:1,000, 37 kDa, #2118) were obtained from CST (USA).