Gapdh ap0063

Total protein was obtained from the ventricular myocardial tissues using tissue homogenates, centrifugation and heat denaturation. The protein lysates were electrophoresed and separated by 6–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore Immobion-P; BioSharp, Anhui, China). The membranes were blocked with 5% skim milk at room temperature for 1 h, and then incubated overnight at 4°C with primary antibodies, including rabbit anti-Bcl-2 (BA0412; 1:500), rabbit anti-Bax (BA0315-2; 1:500), rabbit anti-Calr (BM1798; 1:500), rabbit anti-GRP78 (BA2042; 1:500) (all from Boster Biotechnology, Inc., Wuhan, China), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (AP0063; 1:5,000; BioWorld, Inc., Jiangsu, China). The membranes were then incubated with IRDye800-conjugated secondary antibodies (1:20,000; Rockland, Inc., Gilbertsville, PE, USA) at room temperature for 1 h. The Odyssey double color infrared laser imaging system (LI-COR; Lincoln, NE, USA) was used to detect the antigen-antibody complexes in a western blotting detection system (Bio-Rad). The results were expressed as density values normalized to GAPDH.

LPMCs culture supernatant (serum free) were precipitated with methanol and chloroform for secreted proteins, and the cell lysates were collected by direct lysis by 1x Laemmli loading buffer. Samples were boiled to denature at 100 °C for 10 min before the SDS-PAGE gel electrophoresis for protein separation. After electrophoresis, the protein was wet-transferred to a 0.45 μm nitrocellulose membrane (Millipore), and then blotted for indicated proteins using the antibodies as following: mouse caspase-1 p10 (sc-514, dilution 1:400), mouse IL-1β (sc-7884, dilution 1:400), and ASC (sc-22514, dilution 1:400) were from Santa Cruz, NLRP3 (Cryo2, dilution 1:1000) was from Adipogen, and GAPDH (ap0063, dilution 1:10000) was from Bioworld. The uncropped blot images can be found in Supplementary Fig. 8.

Protein from brown adipocytes or brown adipose tissue was extracted using lysing buffer. Protein concentration was determined using BCA Protein Assay kit (Beyotime Institute of Biotechnology, Nanjing, China). Proteins (30 μg) were separated by SDS-PAGE, transferred to PVDF nitrocellulose membrane (Millipore, Boston, MA, USA), blocked with 5% fat-free milk for 2 h at room temperature and then incubated with primary antibodies in 5% milk overnight at 4°C. Sirt1 (ab110304), Sirt2 (ab191383), CHOP (ab179823), GRP78 (ab108615), ATF4 (ab184909), ERDJ4 (ab118282), Bax (ab32503), Apaf-1 (ab32372), UCP1 (ab2384) and PRDM16 (ab106410) antibodies were all purchased from Abcam (Cambridge, UK). Active-Caspase 3 (bs7004), active-Caspase 9 (bs7070), Bcl-2 (bs1511) and GAPDH (ap0063) antibodies were purchased from Bioworld (Nanjing, China). Smad3 (9523S) and IRE1 (3294S) antibodies were purchased from Cell Signaling Technology (CST, Boston, MA, USA). Rabbit HRP-conjugated secondary antibody (Boaoshen, Beijing, China) was added and incubated at room temperature for 2 h. Proteins were visualized using chemiluminescent peroxidase substrate (Millipore, Boston, MA, USA), and then the blots were quantified using ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).

For protein content analysis, skeletal (quadriceps femoris) and cardiac (left ventricle) muscle samples were homogenized and analyzed by Western blotting. The following antibodies were used: BNIP3 (ab109362, rabbit, 1 : 1000), p-AMPK (ab131357, rabbit, 1 : 500) from Abcam (USA); PGC-1α (sc-13067, rabbit, 1 : 200) from Santa Cruz (USA); CS (16131-1-AP, rabbit, 1 : 1000), LC 3 I/II (14600-1-AP, rabbit, 1 : 500), AMPK (10929-2-AP, rabbit, 1 : 500), DRP1 (12957-1-AP, rabbit, 1 : 500), MFN1 (13798-1-AP, rabbit, 1 : 500), and p62 (18420-1-AP, rabbit, 1 : 1000) from Proteintech (USA); and GAPDH (AP0063, rabbit, 1 : 5000) from Bioworld (USA). Membranes were analyzed and quantified using the Quantity One Imaging System (BIO-RAS, USA). Protein expression was normalized to that of GAPDH.

Apigenin (98% in purity) was obtained from Aladdin (Shanghai, China). GAPDH (AP0063) was purchased from Bioworld (Nanjing, China). IL-1 beta (26048-1-AP), IL-6 (66146-1-lg), MMP3 (17873-1-AP), and MMP13 (18165-1-AP) were purchased from Proteintech (Wuhan, China). JNK (ET1601-28), P-JNK (ET1609-42), ERK (ET1601-29), P-ERK (ET1610-13), P38 (ET1602-26), P-P38 (ER2001-52), TRPM7 (ER61334), p-mTOR (HA600094), and mTOR (ET1608-5) were purchased from HUABIO (Hangzhou China). Bax (#40635) and Bcl-2 (#40639) were purchased from SAB (Nanjing, China). Cleaved-caspase3 (TA7022S) was purchased from Abmart (Shanghai, China).