Ab23875

For confocal analysis, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After a 10-min incubation with 3% hydrogen peroxide to block endogenous peroxidase activity, the samples were incubated with primary antibodies overnight at 4 °C. Then, the slides were washed with PBS and incubated with a secondary antibody (1:500, Invitrogen, Carlsbad, CA, USA) at room temperature for 45 min [22] (link). The nuclei were stained using DAPI. The images were acquired via fluorescence microscopy (Olympus BX-61). The following primary antibodies were used in the present study: p-AMPK (1:1000, Abcam, #ab23875), Tom20 (1:1000, Abcam, #ab186735), LAMP1 (1:1000, Abcam, #ab24170), cleaved caspase3 (1:1000, Abcam, #ab49822), caspase9 (1:1000, Cell Signaling Technology, #9504), Parkin (1:1000, Cell Signaling Technology, Inc.) [23] (link).

Proteins from kidney tissues or HK2 cells were extracted with RIPA buffer (Solarbio, China) containing protease inhibitor cocktail, and protein concentrations were determined with a BCA protein assay kit (Thermo, USA). Then, proteins were separated by SDS/PAGE, transferred to nitrocellulose filter membranes, blocked with 5% milk for 1 h at room temperature, and then the membranes were incubated with the following primary antibodies overnight at 4°C: Bax (1:1000, Proteintech, No. 50599-2-IG), Bcl-2 (1:2000, CST, 4223s), Caspase 3/p17/p19 (1:1000, Proteintech, No.19677-1-AP), p-AMPK (1:2000, Abcam, ab23875), AMPK (1:2000, Abcam, ab80039), MCAD (1:10,000, Abcam, ab92461), FOXA2 (1:1000, Proteintech, No.22474-1-AP), H3 (1:10,000, Abcam, ab1791), GAPDH (1:10,000, Bioworld, AP0066). After washing with TBST, the membranes were incubated with secondary antibodies for 1 hour and then visualized with an enhanced chemiluminescence system. Band intensities were analyzed using ImageJ software and normalized to the expression of GAPDH.

The suspended SVFs from adipose depots of 10-wk-old male mice were fixed, blocked, and stained with conjugated antibodies, including anti-CD45, anti-Siglec-5, anti-CD11b, and anti-CD206 (eBioscience and BioLegend), to identify macrophage subsets. To detect ILC2s (CD45⁺Lin⁻CD90.2⁺ST2⁺), SVF cells were incubated with conjugated anti-CD45, anti-Lin (CD3e, CD11b, B220, CD11c, and Gr-1), anti-CD90.2, and anti-ST2 (eBioscience and BioLegend) after fixation unless specified otherwise. In Fig. S1 D, we used additional markers, including anti-RORC and anti-GATA3 (eBioscience), to gate ILC2s (CD45⁺Lin⁻CD90.2⁺RORC⁻ST2⁺GATA3⁺). Anti-AMPK α 1 (phospho-T183) and AMPK α 2 (phospho-T172) antibody (ab23875; Abcam), eFluor 660–conjugated anti-phospho-IκBα (S32/S36; eBioscience), and PE-conjugated phospho-IKKα/β (Ser176/180; Cell Signaling) were used to detect phospho-AMPK, phospho-IκBα and phospho-IKK in ILC2. For the staining of GATA3, RORC and phosphorylated proteins, cells were permeabilized with 0.25% Triton X-100 for 20 min after fixation. FACS analysis was performed on a FACS Calibur (BD PharMingen), and the data were analyzed with FlowJo software as described previously (Dong et al., 2013 (link)). The gating strategy used for ILC2s, eosinophils, and macrophages in adipose tissue is shown in Fig. S3, I–K.