β tubulin

The protocol of western blot was followed according to a previous study [10 (link)]. Briefly, adipocytes were split by radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) by adding protease inhibitor (Pierce, WA, USA). The total protein sample was separated in the SDS-polyacrylamide gel. Then, it was transferred into a PVDF membrane (Millipore, Bedford, MA, USA). Next, the membrane was blocked in 5% defatted milk for 2 h. After that, the membrane was incubated with primary antibodies at 4 °C overnight followed by a secondary antibody at room temperature for 1.5 h. The antibodies Cyclin B, Cyclin D, Cyclin E, PPARγ, and AP2 were purchased from Santa Cruz (CA, USA); C/EBPα and SREBP-1 were purchased from Abcam; p62, LC, ATGL, and HSL were purchased from CST (Boston, MA, USA); and β-tubulin was purchased from Sungene Biotech (Shanghai, China).

The 3T3-L1 cells were harvested and washed twice with PBS, then radio immunoprecipitation assay (RIPA) (Beyotime, China) buffer add with protease inhibitor mix (Pierce, USA) were added to 3T3-L1 cells under low temperature (4°C). Then, we used a cell scraper to separate the cell lysate from the culture plate as much as possible, which was then subjected to 4°C centrifugation (13,000g) for 10 min. Next, one-fourth volume of 5× loading buffer was added to the supernate and the mixture boiled for 10 min in water. A volume of 15–20 µg of protein samples was used for electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel and then the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane was blocked at 4°C for 2 h with 5% milk, incubated with antibodies (1:500) against Cyclin D (Santa Cruz, USA), p27 (Santa Cruz), p38 (Cell Signaling, USA), phosphorylation of p38 (Cell Signaling), G3BP1 (Bosterbio, USA) and β-tubulin (Sungene Biotech, Tianjin, China) overnight at 4°C, followed by incubation with secondary antibody for 1.5–2 h at 4°C. Chemiluminescence reagents (Millipore, USA) were used to visualize the protein bands and the Image Lab software was used to quantify them.

Briefly, 15 μg samples of total lysates from tissues or cells were run on a 10% SDS-PAGE gel and immunoblotted with the primary antibodies (1:1000) to BAMBI (ThermoFisher, PA5-38027), β-Tubulin (Sungene Biotech, KM9003), PPARγ (Abcam, ab3442), aP2 (Santa, Sc-271529), ATGL (CST, 2138), HSL (CST, 4107), Akt (CST, 4691), p-Akt (ser473,CST, 4060), UCP1 (Abcam, ab10983), PGC1α (CST, 2178), C/EBPβ (Santa, sc-7962), oxidative phosphorylation (OXPHOS) (Abcam, ab110413), and Nox4 (ABclonal, A11274). The intensity of bands was measured using ImageJ. All experiments were repeated at least three times and mean values were derived. All the uncropped blots are included in the Source Data file.

The cells or tissues were homogenized in lysis buffer solution (1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, 0.1 mM Na₃VO4) supplemented with complete protease inhibitor cocktail (Sigma-Aldrich). The lysates were separated by SDS-PAGE, and transferred onto PVDF membrane. The membranes were blotted with antibodies against IFI204 (Lifespan), phospho-IRF3 (Santa Cruz), phospho-IκBα (Cell Signaling Technology), IκBα (Cell Signaling), phospho-NF-κB P65 (Cell Signaling), IRF3 (Abcam), IFI204 (Lifespan), STING (Proteintech), GAPDH (Proteintech), or β-Tubulin (Sungene Biotech).

BMDMs infected with bacteria were dissolved by lysis buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1 mM Na₃VO₄, 0.1 mM NaF, 1 mM PMSF, 5 mg/mL Aprotinin, 5 mg/mL Leupeptin, and 5 mg/mL Pepstatin A. Lysate and supernatant was harvested and used for immunoblotting analysis. The lysate (30 mg) were subjected to 12% sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking with 5% milk, the membrane was blotted with antibodies against PARP (Cell Signalling), pMLKL (Abcam), RIPK3 (Abgent), Caspase-1 p20 (Adipogen), Cleaved caspase-3 (Cell Signalling), AIM2 (ebioscience), and β-Tubulin (Sungene Biotech, Tianjin, China).