Quickblock western

A total of 2 × 10⁶ cells were used to extract protein samples with a mixture of 100μlRIPA buffer (Beyotime, # P0013B) and 2 μl PMSF (Sigma, #329-98-6). The concentrations of protein were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, #NCI3225CH). The total protein (50 μg/sample) added in the loading buffer was boiled at 100 °C for 5 min, separated in a 10% gel at 80 V for 90 min, transferred to a membrane at 4°C using 300 mA for 85 min, and then incubated with QuickBlock™ Western (Beyotime, #P0252) for 10min. The membranes were probed with antihuman TET1 antibody (Abcam #ab191698), anti-OASL (Abcam #ab229136), anti-IRF1 (Abcam #ab191032), and anti-β-actin (Abcam #ab8227)) overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (Abcam#) served as a secondary antibody

For immunoprecipitation experiments, total homogenates from adipose tissue and cultured cells were treated with RIPA lysis buffer (P0013B, Beyotime Biotechnology, China), vortexed for the 30s, and centrifuged for 15 min at 12000 r/min. The tissue or cell extracts was subjected to immunoprecipitation with HIF1α primary antibody at 4°C overnight. The antibody-bound proteins were precipitated with 20 μL protein A/G PLUS-Agarose (Santa Cruz Biotechnology, sc-2003) and rotated for 1 h, then incubated overnight at 4°C. The beads were then gently centrifuged at 1000 r/min for 5 minutes at 4°C. After four RIPA buffer washes, the immunoprecipitates were diluted with 40 μL of 1 × SDS loading buffer (CW0027, Cowin Biotech, China) and boiled at 100°C for 2-3 min to separate complexes from the protein A/G PLUS-Agarose. The samples were then subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). After blocking with QuickBlock™ Western (P0252, Beyotime Biotechnology, China), the membranes were incubated with an anti-acetylated-lysine antibody (Cell Signaling Technology, #9441) overnight at 4°C, washed in PBST three times, and incubated with a secondary goat anti-rabbit polyclonal antibody (SA00001-2, Proteintech Group) at room temperature for 1h. Finally, the signals were tested by WesternBright™ Sirius ECL kit (K-12043-D20, Advansta, USA).

Immunoblotting analysis and qPCR were performed according to previous articles (29 (link)). In brief, Protein-extracts of snap-frozen eWAT and whole-cell lysates of SVF were prepared using standard procedures. Protein concentrations in the supernatants were measured using Bicinchoninic acid (BCA) assay (ASPEN, USA). Proteins were separated on SDS-polyacrylamide gels and transferred to PVDF membranes. After blocking with QuickBlock™ Western (P0252, Beyotime Biotechnology, China), the membranes were incubated with the primary antibodies overnight at 4°C, washed in PBST three times, and incubated with a secondary goat anti-rabbit polyclonal antibody (SA00001-2, Proteintech Group) at room temperature for 1h. Finally, the signals were tested by WesternBright™ Sirius ECL kit (K-12043-D20, Advansta, USA). Protein expression levels were normalized to β-actin.
Total mRNA was extracted from eWAT or SVF cells with GeneJET RNA Purification Kit (K0731, Thermo Fisher Scientific, USA). Reverse transcribed into cDNA using RevertAid First strand cDNA Synthesis kit (K1622, Thermo Fisher Scientific, USA). The StepOne Real-Time PCR (Life tech, Alameda, CA) was used for real-time qPCR analysis. The primers used are described in Table S1. β-actin was used as an internal control. The relative expression quantity 2^-ΔΔCt value was calculated to compare the differences among groups.

The samples were mixed with 5× SDS loading buffer and separated by 12% SDS–polyacrylamide gel, and then transferred onto a nitrocellulose membrane (Millipore, USA). The membrane was blocked with QuickBlock Western (Beyotime, China) and further incubated with appropriate primary antibodies at 4°C overnight. After being washed three times with TBST (Tris-buffered saline plus Tween 20), the membrane was incubated with a secondary antibody for subsequent detection by ECL enhanced chemiluminescence substrate (Thermo Scientific, USA). Flag, His, HA, and histone H3 antibodies, Cy3-labeled goat anti-mouse IgG, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG secondary antibodies, and mouse control IgG were purchased from Beyotime. Goat anti-rabbit IgG-horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, and antiubiquitin (anti-Ub) were purchased from Abcam and Sigma, respectively. Tubulin, p53, HUWE1, and TRAF6 antibodies were prepared in our lab.

Snap-frozen terminal ileum tissues were homogenized and centrifuged and the supernatant was collected. The protein content was calculated by using the bicinchoninic acid approach. Equal amounts of proteins (40 mg) were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes (IPFL00010, Millipore, Burlington, MA), and probed with primary antibodies. Membranes were blocked using QuickBlock Western (Beyotime) and subsequently incubated overnight at 4°C with primary antibodies, including β-actin (20536-1-AP, Proteintech, Rosemont, IL), cleaved caspase-3 (Santa Cruz Biotechnology, Dallas, TX), IκBα (Cell Signaling Technology, Danvers, MA), Grx1 (ab45953, Abcam, Cambridge, UK), IKKβ (ab124957, Abcam), RelA (ab32536, Abcam), p-RelA (ab76302, Abcam), and iNOS (ab178945, Abcam). Following primary incubation, membranes were treated with appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature to visualize immunoreactivity bands. The optical density of these bands was analyzed using ImageJ software (NIH, Bethesda, MD), and band intensities were captured with the Kodak Scientific Imaging System (Kodak, Rochester, NY).

For the analysis of phospho-EGFR inhibition, 5 μM afatinib was added into the culture medium, and organoids were collected after incubation for 2 h. The organoid samples were collected and lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Beyotime). The samples were quantified by BCA Protein Assay Kit (Beyotime), separated by 10% SDS-PAGE (Beyotime), and transferred to a PVDF membrane (Millipore). The membrane was blocked with QuickBlock Western (Beyotime) and incubated with primary antibody (Phospho-EGF Receptor (Tyr1068) (D7A5), Cell Signaling Technology, CST-3777, 1:1000 dilution) at 4 °C overnight followed by secondary antibody incubation. The protein bands were visualized using ChemistarTM ECL Western Blotting Substrate (Tanon).