P1250

The protein suspension of liver tissues for Western blotting was obtained using a total protein extraction kit (APPLYGEN P1250, Beijing, China) and protein concentrations were determined by BCA protein assay kit following the manufacturer’s instructions. CD68 (ab283654, Abcam), HSP90 (ab59459, Abcam), HSF1 (ab242138, Abcam), SGT1 (11019-2, Proteintech), IL-1β (ab234437, Abcam), Caspase 1 (ab179515, Abcam), cleaved-IL-1β (#63124, CST), cleaved-caspase-1 (#89332, CST), NLRP3 (ab263899, Abcam), ASC (#67824, CST), GAPDH (#5174, CST), and β-actin (sc-69879, Santa Cruze) anti-bodies were used and blots were quantified using Image J Software (NIH, Bethesda, MD, USA). Each group was tested using three samples that were created by mixing the liver samples from mice numbered 1–3, 4–6, and 7–8, respectively. Western blot results were normalized to the GAPDH band or the β-actin band.

The concentration of glucose was detected using the glucose oxidase method (Li et al., 2015 (link)). The assay kit (E1010; Applygen Technologies, Beijing, China) was purchased from Applygen Technologies (Beijing, China). After treatment, the PC12 cells were collected, and the concentration of total protein was extracted using a total protein extraction kit (P1250; Applygen, Beijing, China) and detected using a BCA protein assay kit (P0012; Beyotime, Beijing, China). Then, according to the instructions, the absorbance was measured at 570 nm with an automatic microplate reader and calculated against a glucose standard curve.

The total protein was extracted from 3 T3-L1 adipocytes and mouse epididymal adipose tissues with a total protein extraction kit (P1250, Applygen). Protein expression levels of AQP7 and PPARγ were detected. Samples were separated by 10% SDS-PAGE and transferred onto 0.45 μm PVDF membranes (Bio-Rad). The membranes were blocked in 5% nonfat dry milk followed by incubation with primary antibodies against AQP7 (AB15568, Merck-Millipore, Germany), PPARγ (ab45036, Abcam, UK), phosphorylated peroxisome proliferator-activated receptor γ (pPPARγ, Ser112) (abs130911a, Absin, China) and Tubulin-α (AF7010, Affinity, US) overnight at 4 °C. The secondary antibody (ZSGB-BIO, China) was goat anti-rabbit immunoglobulin G antibody. After washing, the immunocomplex was incubated with secondary antibody (1:10,000) for 2 h. Bands were visualized by chemiluminescence (Millipore Corporation, Billerica, MA, USA).