Wbluf0500

The cells and mouse tissues were lysed using RIPA buffer (#89901, Thermo fisher Scientific) supplemented with PhosSTOP and complete protease inhibitors (Merck, Beijing, China). The protein samples were subjected to 12.5% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (162-0177, BioRad, Shanghai, China). Blots were probed with appropriate primary antibodies against VCAM-1 (ab134047, Abcam, Cambridge, UK), E-selectin (20894-1-AP, Proteintech, Shanghai, China; sc-137054, Santa Cruz, California, USA), Phosphorylated ACLY (Ser455, 4331, CST, Danvers, MA), ACLY (13390, CST), Raptor (48648, CST), Rictor (9476, CST), phosphorylated FoxO1 (Ser256, 84192, CST), FoxO1 (2880, CST), acetylated FoxO1 (Lys294, AF2305, Affinity, PA, USA), MYC (9402, CST), FASN (3180, CST), ACSS2 (3658, CST), ACC1 (4190, CST) (MA, USA), and pan-acetyl-lysine (PTM-105RM, PTM Biolabs, Shanghai, China) at 4 °C overnight. The blots were then incubated with the appropriate secondary antibodies (1:5000, proteintech) and detected using a horseradish peroxidase substrate (WBLUF0500, Millipore, USA). For internal controls of equal loading, the blots were also stripped with stripping buffer (100 mmol/l 2-mercaptoethanol, 2% SDS, 62.5 mmol/l, Tris pH 6.8) and reprobed with ACLY, FoxO1, or GAPDH antibodies.

BMDM protein lysates were subjected to SDS‐PAGE 10% polyacrylamide electrophoresis (NuPAGE, Thermofisher) and transferred onto a nitrocellulose membrane (iBlot, Thermofisher) according to manufacturer's protocol. Membranes were blocked for 1 hour in 5% milk (Marvel) and probed with the following antibodies overnight at 4°C: anti‐CREB (48H2, Cell signaling), anti‐P‐Ser133 CREB (87G3, Cell signaling), and anti‐beta‐actin (ab8227, Abcam), then detected using a peroxidase‐coupled secondary anti‐rabbit antibody (7074, Cell signaling) and enhanced chemiluminescence (WBLUF0500, Millipore).

The tissues were collected in liquid nitrogen, ground into fine powders, and extracted with UEB buffer as above mentioned. Proteins of 50 μg were loaded onto either 10% or 12% SDS-PAGE gel. Fractionated proteins were transferred to PVDF membranes (GE Healthcare). The membrane was incubated with both primary and secondary antibodies. Immobilon forte western hrp substrate (WBLUF0500, Millipore) was used for membrane exposure and signal detection. The secondary antibodies were anti-biotin-HRP polyclonal antibodies (7075, Cell Signaling Technology, 1:2000), while the useful primary antibodies were the anti-TurboID polyclonal antibodies (AS20 4440, Agrisera, Sweden, 1:2000), anti-actin monoclonal antibody (a0480, Sigma Aldrich,1:5000) and anti-pPATL3 polyclonal antibodies (GL Biochem, 1:1000) targeted to the sequence of MIPQNLGpSFKEESSC.

Protein lysates were diluted in NuPAGE LDS sample buffer (NP0007, Thermofisher Scientific) containing 2.5% 2-mercaptoethanol and boiled at 95°C for 5 min. 10 μg of protein was then separated by electrophoresis using NuPAGE SDS-polyacrylamide gels (Thermofisher Scientific) and transferred to nitrocellulose membranes using the iBlot Dry Blotting System (Thermofisher Scientific). Membranes were blocked for 1 hr in 5% fat-free milk (Marvel) or 5% BSA in Tris-buffered saline containing 0.05% Tween (TBST) at room temperature and incubated overnight at 4°C with the appropriate primary antibody. Bound primary antibodies were detected using peroxidase-coupled secondary anti-rabbit antibody (7074, Cell signalling) and enhanced chemiluminescence (WBLUF0500, Millipore). Blots were exposed digitally using the ChemiDoc MP System (Bio-Rad), and bands were quantified using Image Lab software (Bio-Rad). The expression of proteins was normalised to a housekeeping protein (β-actin), and the phosphorylation status was determined by normalising to a respective total protein. All protein quantification data is expressed as arbitrary units.

Kidney and liver tissue were homogenized in RIPA and were analyzed by SDS‐Page and Immuno‐blotting on PVDF membranes as by the manufacturer protocol (Trans‐Blot Turbo Transfer System, Bio‐Rad). Incubation with primary antibodies (Table S1) was performed at 4°C overnight at a 1:1000 dilution in 5% nonfat dry milk (Bio‐Rad No. 1706404). Incubation of HRP conjugated secondary antibody (different species, Dianova) was performed for 1 h at room temperature. Chemiluminescence (Millipore WBLUF0500) intensity detection was performed with the Azure^® c400 imaging system (Azure^® Biosystems) and quantified using ImageJ 1.53 (Schneider et al., 2012 (link)).

Total protein was extracted using RIPA lysis buffer (20-188, Millipore) supplemented with protease inhibitor (ab201120, Abcam) and quantified using the bicinchoninic acid kit (23225, ThermoFisher). Equivalent amount of proteins was separated using 10% SDS–PAGE and transferred onto PVDF membranes (IPVH00010, Millipore). After blocking with 5% BSA (A3059, Sigma), the membranes were incubated overnight at 4 °C with primary antibodies. The primary antibodies used were PCNA (A0264, Abclonal, Massachusetts, USA), FGF21 (A3908, Abclonal), FGFR1 (A21219, Abclonal), FGFR2 (A19051, Abclonal), FGFR3 (A19052, Abclonal), FGFR4 (A9197, Abclonal), KLB (A15629, Abclonal), p-FGFR (3471S, Cell Signaling Technology), STAT3 (4904, Cell Signaling Technology), p-STAT3 (AP0705, Abclonal), FoXO1 (A2934, Abclonal), p-FoXO1 (AP0172, Abclonal), Akt (ab179463, Abcam), p-Akt (ab192623, Abcam), Bax (A0207, Abclonal), Bcl-2 (A19693, Abclonal), Bcl-xl (A0209, Abclonal), α-Tubulin (AC007, Abclonal), β-Actin (ab8226, Abcam) and GAPDH (AC002, Abclonal). The membranes were then incubated with secondary antibodies (7074, 7076, Cell Signaling Technology, Massachusetts, USA) for 1 h at room temperature. Protein bands were visualized using an HRP substrate (WBLUF0500, Millipore) on a gel imaging system (ChemiDoc XRS+, Bio-Rad, California, USA). The images were analyzed using Image Lab software (version 6.1, Bio-Rad).

Protein analysis by western blot was performed on human preadipocytes and Wt and miR‐424(322)/503‐null MEFs following adipogenesis. Cells were washed with cold PBS and lysed with protein lysis buffer (Cell Signaling Technology, 9803). Protein concentrations were determined using a protein assay kit (Life Technologies, 22660/22663) and equal amounts of proteins were subjected to SDS‐PAGE and transferred to nitrocellulose membranes (Whatman, 10600001). Non‐specific binding was blocked by incubation with TBST (20 mm Tris‐Hcl pH7.4, 150 mm NaCl, 0.1% Tween‐20) plus 5% of non‐fat milk (Bio‐Rad, 170–6404). Membranes were incubated overnight at 4 °C with a 3% BSA (Fisher BioReagents, BP9703‐100)‐TBST solution containing the primary antibody. Thereafter, membranes were incubated for 1 h with secondary HRP‐conjugated antibodies at room temperature (Jackson Immunoresearch, 115‐035‐003 and 111‐035‐003). Signal was detected with a western blot HRP substrate (Millipore, WBLUF0500). The antibodies used in this study include anti‐SNCG (Sigma Aldrich, HPA014404), Anti‐TNFAIP8 (Sigma Aldrich, HPA057089), anti‐UNG (GeneTex, GTX113860), anti‐CHRDL1 (GeneTex, GTX49225), anti‐CEBP‐α (Santacruz, sc‐166258) anti‐AdipoQ (Life Technologies, PA1‐24411), anti‐PPARγ (Santacruz, sc‐7273),anti‐FABP4 (R&D Systems, MAB3150) and anti‐Actin (Sigma Aldrich, A5441).