Gpr43

Cells or muscles were cracked by the RIPA lysis buffer containing 1 mM PMSF. For the nuclear or cytoplasmic protein extraction, the procedure of protein extraction was followed by the nuclear extraction kit (BB3112, Bestbio). Protein concentration was detected by a BCA protein assays kit. After sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis gels, total protein lysates (20 μg) were immunoblotted with primary antibody (P-Foxo3: AP0684, ABclonal; Foxo3: A0102, ABclonal; P-mTOR: 5536S, CST; mTOR: ab185696, Abcam; P-AKT, 4071S, CST; AKT: 9272S, CST; Ubiquitin: A2129, ABclonal; Puromycin: MABE343, EMD Millipore Corporation; P-JAK2: BS-2485R, Bioss; JAK2: 3230S, CST; P-STAT3: 9145S, CST; STAT3: 12640S, CST; MyoD: sc-377460, Santa Cruz Biotechnology; MyoG: 382257, CST; MyHC: MAB4470, CST; Histone H3: 4499S, CST; Pan-acetylation: 66289-1-lg, Proteintech; GPR43: sc-293202, Santa Cruz Biotechnology; followed by incubating with goat anti-rabbit or goat anti-mouse HRP-conjugated secondary antibody (1:50 000). The levels of GAPDH, VDAC and β-actin served as the loading control. Protein expression levels were determined using MetaMorph software ImageJ (National Institutes of Health, USA).

Total protein was extracted from 40 mg frozen gastrocnemius muscle samples as previously described [41 (link)]. Protein concentration was measured with a Pierce BCA Protein Assay kit (23225; Thermo Scientific). Antibodies and their sources are: Adiponectin (BS6961, Bioworld), AdipoR1 (BS6797, Bioworld), AdipoR2 (14361-1-AP, Proteintech), AMPKα112 (BS1009, Bioworld), p-AMPKα1/2 (sc-33524, Santa Cruz), PGC-1α (sc-13067, Santa Cruz), UCP3 (BS2849, Bioworld), UCP2 (BS2917, Bioworld), GPR43 (sc-32906, Santa Cruz), GPR41 (sc-98332, Santa Cruz), CPT-1b (sc-20670, Santa Cruz), COX4 (MB0102, Bioworld), HDAC1 (BS6485, Bioworld), GAPDH (AP0063, Bioworld), β-actin (AP0063, Bioworld). The phosphorylated AMPK was normalized by the total AMPK.

The distal ileum, colon, spleen, and adipose tissue were immersed in 4% paraformaldehyde. After fixation, the tissues were washed in 75% alcohol, dehydrated in a graded ethyl alcohol series (85%, 95% I, 95% II, 100% I, and 100% II), cleared in xylene, and embedded in paraffin. The tissues were serially sectioned into 4 µm-thicknesses on a rotary microtome. The paraffin sections were stained using an SABC kit (Boster, Wuhan, China), following the manufacturer′s instructions, and incubated with GPR41 (1∶50 diluted) and GPR43 (1∶50 diluted) antibody (Santa Cruz Biotechnology, Texas) at 4°C overnight. After immunoreaction, the images were captured on each slide at 400× magnification under a spot camera (Olympus, Tokyo). To check the specificity of the secondary antibody, the sections incubated without the primary antibody were stained by the secondary antibody as a negative control.