Gapdh

Total proteins were extracted from cultured cells with RIPA buffer, and protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of proteins were separated using SDS/10% polyacrylamide gel electrophoresis (SDS/PAGE), transferred to 0.22 µm polyvinylidene difluoride (PVDF) membranes, and incubated with specific antibodies. Proteins were detected using chemiluminescence (ECL). The intensity of the bands was quantified by ImageJ. Primary antibodies, ASC (ab227502, ABCAM), caspase-1 (ab62698, ABCAM), p-caspase-1, IL-1β (BA3711, BosterBio), p-IL-1β, IL-18 (PB0057, BosterBio), p-IL-18, and GAPDH (A00227-1, BosterBio) were obtained from Cell Signaling Co. (Cell Signaling Technology, U.S.A.).

Western blotting with proteins of the cortex and myocardium was performed as described previously (Mlyniec et al. 2014a , b (link)). To confirm equal loading of the samples on the gel, the membranes were re-probed with an antibody specific to GAPDH as an internal control. The specific primary antibodies used included rabbit polyclonal antibodies against CAIII (1:1000; abcam), Beclin-1 (1:200; Santa Cruz), LC3 (1:1000; abway), P53 (1:1000; abcam), IL-17 (1:1000; abcam), NF-κB (1:200; Santa Cruz), GAPDH (1:2000; Boster). Finally, the X-ray films were developed and fixed in a dark room.

Whole cell extracts of MC3TE-E1 for western blotting were prepared after the indicated treatment for 3 days as previously described [19 (link)]. 20 μg protein from total cell lysate was prepared using PRO-PREPTM protein extraction solution (Boca Scientific Inc., Boca Raton, FL) and carried out subsequent electrophoresis, according to the manufacturer’s instruction. The primary antibodies against the following proteins: FoxO1 (Abcam, ab52857, 1:1000), SIRT1 (Abcam, ab189494, 1:1000), human catalase (CAT, Abcam, ab130029, 1:1000), glutathione peroxidase (GPX1, Abcam, ab108427, 1:1000), superoxide dismutase 2 (SOD2, Abcam, ab252426, 1:1000). Expression levels of the target protein were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Boster, Wuhan, China, 1:2000) levels in each sample. The following day, HRP-conjugated goat anti-rabbit, which used as secondary antibodies, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the results were detection and analysis by using an iBrightCL1000 (Invitrogen, Carlsbad, CA).

Mice (n = 6 in each group) were decapitated, the hippocampus were immediately dissected, frozen and stored at −80 °C. Tissue samples were lysed and western blots were performed as previously described [11 (link)]. Primary antibodies include GFP (1:1000, Beyotime, cat # AG281, Shanghai, China), Glial fibrillary acidic protein (GFAP) (1:1000, Wanleibio, cat # WL0836, Shenyang, China), Flag (1:1000, abm, cat # G188, Zhenjiang, China), Oligo2 (1:1000, Wanleibio cat # WL04942, Shenyang, China), NeuN (1:1000, Wanleibio, cat # WL03099, Shenyang, China), doublecortin (Dcx) (1:1000, Proteintech, cat # 13925-1-AP, Rosemont, USA), GAPDH (1:5000, Boster, cat # BM1623, Wuhan, China) and β-Tubulin (1:5000, Beidibio, Nanjing, China). HRP-conjugated secondary antibodies purchased from Immunoway Biotechnology Company (goat anti mouse cat # RS0001 and goat anti rabbit cat # RS0002, Plano, TX, USA) were used to probe these blots. Protein visualization was carried out using an enhanced chemiluminescen (ECL, Meilunbio, Dalian, China). The signal intensity was obtained by densitometric scanning.

Proteins from all the skeletal muscles and cells were extracted using the Protein Extraction Reagent (KeyGENBioTECH, Nanjing, China), and the concentrations were determined with the BCA Assay Kit (KeyGENBioTECH, Nanjing, China). Next, equal amounts of protein were separated on 10% SDS-PAGE gels followed by electrotransfer to polyvinylidenedifluoride membranes (PVDF). After blocking with 5% skim milk powder in 0.05% TBST, the membranes were incubated with the following primary antibodies: FTO, phospho-AMPKα2 (Abcam), AMPKα2 (GeneTex), METTL3 (Proteintech), METTL14 (Sigma), FAS, C/ebpα, ATGL, PGC1α (Santa Cruz), GAPDH (Boster) and β-actin (Hua An, Hangzhou, China). The secondary antibodies were HRP-conjugated anti-mouse and anti-rabbitIgG (Hua An, Hangzhou, China). Finally, the immunocomplexes were detected using the ECL Western Blotting Detection System (Amersham, Biosciences, Piscataway, NJ, USA).

Total protein was extracted from the indicated cells or tissues using RIPA lysis buffer (Beyotime, China), and the protein concentration was measured using a BCA protein assay kit (Beyotime, China). The protein was separated by 10% SDS-PAGE and subjected to western blotting, as described previously [43 (link)]. The primary antibodies used in western blotting were as follows: LIMK1(1:1000, ab81046, Abcam), p-cofilin (1:1000, #3313, CST), cofilin (1:1000, #5175, CST), p-CREB(Ser133) (1:1000, #9198, CST), CREB (1:1000, #9197, CST), MMP2(1:1000, ab97779, Abcam), ITGB1(1:1000, ab134179, Abcam) and COL1A1(1:1000, #72026, CST); GAPDH (1:1000, Boster, Wuhan, China) was used as a loading control. Horseradish peroxidase–conjugated anti-mouse or anti-rabbit IgG antibodies (1:3000, Boster, Wuhan, China) were used as secondary antibodies, and the protein expression levels were visualized using an enhanced chemiluminescence system (Bio-Rad, Hercules, EDAUSA). Band intensities from three biological experiments were quantified by densitometry using ImageJ software.