Ab174833

Total protein was extracted from scraped intestinal mucosa as described (38 (link)). Wells with confluent organoids were washed with PBS. Matrigel-embedded organoids were scratched out of the wells into Cell Recovery Solution (Corning) and kept on ice for 45 minutes. Eluted organoids were washed in PBS, resuspended in cell lysis buffer (20 mM Tris pH 8, 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, 5% glycerol, 100 mM NaF, 1 mM Na vanadate, 50 mM β-glycerophosphate 1× protease inhibitors), and disrupted through an 18-gauge needle. Total protein extract was recovered by centrifugation at 25,200g, at 4°C, for 10 minutes. Glass Dounce Tissue homogenizer was used to prepare the nuclear extract (7 (link)). The nuclear pellet was resuspended in 80 μL of buffer B (25 mM Tris pH 7.5, 420 mM NaCl, 1.5 mM MgCl₂, 0.5 mM EDTA, 1 mM DTT, 20% glycerol, 1× protease inhibitor) and incubated in ice for 30 minutes. The suspension was centrifuged at 11,200g for 15 minutes at 4°C to collect the nuclear protein fraction. For Western blotting, 50 μg of total protein and 15 μg of nuclear protein were run on 10% and 12.5% SDS-PAGE gels, transferred to PVDF membranes, and probed with rabbit anti-RBM47 (Abcam ab94638; 1:2,000) and rabbit anti-FNDC5 (Abcam ab174833; 1:1,000) antibodies. Rabbit anti-actin (MilliporeSigma; ab179467; 1:2,000) antibody was used to confirm equal loading of protein.

Immunofluorescent staining was carried out on frozen muscle sections (10 μm). In summary, sections were incubated for 60 min at room temperature with BSA (Bovine Serum Albumine, Sigma Aldrich, Saint-Quentin-Fallavier, France) diluted to 3% in PBS (Phosphate Buffered Saline, Sigma Aldrich). They were then incubated for 60 min with a primary antibody cocktail containing FNDC5 (1:200, ab174833, Abcam) and MHC I or MHC IIA antibodies (1:200, BA-D5 or SC-71, respectively, DSHB) at 37 °C, followed by three rinses. Fluorescence-conjugated secondary antibodies (Alexa Fluor 488 and Alexa fluor 555 (1:250, A11008 and A21422, respectively, Thermofischer, Villebon-sur-Yvette, France) or Alexa fluor 647 (1:250, 1090-31, Southern Biotech, Clinisciences, Nanterre, France)) were applied for 30 min at 37 °C in obscurity, followed by three rinses. Finally, the sections were mounted in ProLong Gold Antifade Mountant (P36934, Invitrogen, Villebon-sur-Yvette, France) and coverslipped. Images were acquired using a Leica DMI8 inverted microscope, fitted with an automated motorized platform for mosaic imaging with the LAS X software (https://imillermicroscopes.com/pages/software-download accessed on 4 February 2024, Leica, Nanterre, France).

Coimmunoprecipitation (CO‐IP) was conducted as described previously.24 Protein A/G PLUS‐Agarose (sc‐2003; Santa) and mouse anti‐integrin alpha V/beta 5 antibody (sc‐81632; Santa) were used for immunoprecipitation. Rabbit anti‐irisin antibody (1:1000, ab174833; Abcam), rabbit anti‐integrin alpha V antibody (1:1000, ab179475; Abcam) and rabbit anti‐integrin beta 5 antibody (1:1000, ab15459; Abcam) were used for WB.

Western blot analysis was performed as described previously.22 PVDF membranes were incubated with primary rabbit anti‐irisin antibody (1:1000 dilution, ab174833; Abcam); anti‐claudin‐1 antibody (1:1000 dilution, 13050‐1‐AP; Proteintech); rabbit anti‐occludin antibody (1:1000 dilution, ab216327; Abcam); rabbit anti‐AMPKα antibody (1:1000 dilution; Cell Signaling Technology); rabbit anti‐PAMPKα antibody (1:1000 dilution; Cell Signaling Technology); rabbit anti‐UCP 2 antibody (1:1000 dilution, ab203244; Abcam); rabbit anti‐UCP1 antibody (1:1000 dilution, 14670S; Cell Signaling Technology); rabbit anti‐IRE1 antibody (1:1000 dilution, ab37073; Abcam); rabbit anti‐CHOP antibody (1:1000 dilution, 5554; Cell Signaling Technology); or mouse anti‐β‐actin monoclonal antibody (1:1000 dilution, HRP‐60008; Proteintech) overnight at 4°C. Then, secondary HRP‐conjugated goat anti‐rabbit IgG (1:2000 dilution, SA00001‐2; Proteintech) was incubated for 1 hour at room temperature. Protein expression was detected by a chemiluminescence system (Bio‐Rad) and quantified by ImageJ2x software.

Western blotting was performed as described previously [31 (link)] Overexpression and knockout cells were washed with PBS and lysed in ice-cold lysis buffer with a protease inhibitor cocktail (Mammalian Protein Extraction Kit, KW Biotechnology, Beijing, China). Protein concentrations were measured using the BCA Protein Assay Kit (23,225, Thermo, USA). Proteins (10 mg) were separated with SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes, and incubated with primary antibodies overnight at 4 °C. Signals were then visualized using the Thermo Scientific Pierce ECL western blotting substrate and the Tanon 5200 (Tanon, Shanghai, China) detection system.
The primary antibodies used were anti-DLGAP1 (bs-12138R, Bioss), anti-UCP1 (Ab10983, Abcam), anti-PRDM16 (63,976, ABclonal), anti-FNDC5 (Ab174833, Abcam), anti-ACC (21923–1-AP, Proteintech), anti-PPARγ (16643–1-AP, Proteintech), anti-ASC1 (Ab70627, Abcam), anti-PSAT1 (10501–1-AP, Proteintech), anti-FABP4 (15872–1-AP, Proteintech), anti-PGC1α (20658–1-AP, Proteintech), anti-FASN (10624–2-AP, Proteintech), anti-LEPTIN (Ab16227, Abcam), anti-GAPDH (Ab9485, Abcam) and anti-α-tubulin (11224–1-AP, Proteintech). The secondary antibody was HRP-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L) (SA00001–2, Proteintech).

The RNA extraction, cDNA synthesis, and qPCR for C2C12 cells were performed similarly to the above descriptions for muscle tissue. All data were normalized against the mRNA expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Determinations were performed in triplicate. The primer sequences in Table 1 were designed and synthesized by GenePharma Biotechnology Co. Ltd, Shanghai city, China.
Total protein extraction, SDS-PAGE gel electrophoresis, and western blotting for cells were performed according to Cai et al. (2017) (link). The FNDC5 protein was separated using 12% SDS electrophoresis, transferred onto a cellulose acetate membrane, and then incubated with recombinant rabbit anti-FNDC5 monoclonal antibodies (ab174833, Abcam, Cambridge, UK), or a monoclonal anti- β-Actin antibody produced in mouse (A5441, Merck Life Science Co., Ltd, Shanghai city, China. Chemiluminesence detection was performed on an FR-1800 Luminescent and Fluorescent Biological Image Analysis System of Furi company, Shanghai city, China. The scanned images were processed and analyzed using Gel-Pro analyzer software from Media Cybernetics, Rockville, MD, USA. The relative content of FNDC5 protein in a sample was calculated through the ratio of the gray value to that of the internal reference actin.

NP cells were lysed with RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (NCM Biotech). The following antibodies were used for western blotting: rabbit monoclonal antibodies: (GAPDH, 1:1000 dilution, #5174, CST; p62, 1:1000 dilution, ab240635, Abcam; FNDC5, 1:1000 dilution, ab174833, Abcam; p-AMPK, 1:1000 dilution, #2535, CST; AMPK, 1:1000 dilution, #5832, CST; p-mTOR, 1:1000 dilution, #5536, CST; mTOR, 1:1000 dilution, #2983, CST); rabbit polyclonal antibodies: (cleaved-caspase3 1:1000 dilution, #9661, CST; p16INK4a 1:1000 dilution, A0262, ABclonal); mouse monoclonal anti-LC3B (1:1000 dilution, #83506; CST).