Chemiluminescent detection reagent

Western blot analysis of the distal colonic mucosal samples was performed by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to membrane. For the detection, primary antibodies against HSP70 (ADI-SPA-812), HSP25 (ADI-SPA-801), and HSC70 (ADI-SPA-815, Enzo) and the corresponding secondary antibodies (goat anti-rabbit IgG HRP, sc-2004; or goat anti-rat IgG HRP, dv-2006, Santa Cruz Biotechnology) were used. Proteins were visualized using chemiluminescent detection reagent (Thermo Fisher Scientific, MA, USA) and analyzed using the FluorChem FC3 Chemiluminescent system (ProteinSimple Ltd., USA).

Mice ears were injected with chemically-modified FFAR2 and control siRNAs followed by application with 2% PA and UVB exposure for 3 days. On third day mice ears were cut, homogenized and then lysed with RIPA buffer (Thermo Fisher Scientific). Cell lysates (30 µg) were subjected to 10% SDS-PAGE gel, which were then transferred to a poly(vinylidene fluoride) (PVDF) membrane (Sigma) and blocked with 5% (w/v) nonfat milk before incubation overnight with primary antibodies to FFAR2 Rabbit PolyAb (Proteintech, Rosemont, IL, USA) at 4 °C or β-actin (1:1,000; Cusabio Technology, Houston, TX, USA). This was followed by treatment with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000) (Thermo Fisher Scientific) for 1 h. Protein bands were detected with a chemiluminescent detection reagent (Thermo Fisher Scientific) and Omega Lum C Imaging System (Gel Co., San Francisco, CA, USA). Protein bands were conducted using ImageJ software (https://imagej.nih.gov/ij/; Version 1.53e).

Protein was extracted and homogenized from heart tissue samples in RIPA buffer (Sigma-Aldrich, R0278) containing proteinase/phosphatase inhibitor cocktail (Roche). Samples in SDS sample buffer were run on SDS-PAGE and immunoblotted with anti-CACT (Sigma, 1:1000) and anti-α-tubulin (Cell Signaling, 1:2000). Secondary HRP-conjugated antibodies were from Cell Signaling and chemiluminescent detection reagent was from Thermo Scientific. Bio-Rad Quantity One software was used for quantitation. Two-tailed Student's t-test for unpaired data was used

The 3T3-L1 cells were grown in DMEM or differentiation media for six days. Differentiated 3T3-L1 cells were treated with or without 0.1 µM of GLPG-0974 in the presence of media collected from culture of 100 µL/mL L. mesenteroides EH-1 bacteria (10⁷ CFU/mL) and 2% glucose for 30 min on Day 0, 2, 4, and 6. Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific). Mouse abdominal fat depots were ground and lysed with tissue lysis buffer containing protease inhibitors. Protein (30 µg) was loaded to a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, then transferred into a polyvinylidene difluoride (PVDF) membrane (Sigma) and blocked with 5% (w/v) nonfat milk before overnight incubation with primary antibody to PPAR-γ (1:1000), or β-actin (1:5000). Thereafter, the blotting was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-mouse (1:5000) Thermo Fisher Scientific) for 1 h. Chemiluminescent detection reagent (Thermo Fisher Scientific) and Omega Lum C Imaging System (Gel Co., San Francisco, CA, USA) were used to detect protein bands, and then analyzed with ImageJ software 1.50b (National Institutes of Health, Bethesda, MD, USA).

Cytoplasmic as well as nucleus-specific proteins in the PVN and SON were isolated by using the Nucleus and Cytoplasmic extraction kit (Thermo Scientific, Catalogue No. 78833), according to the manufacturer’s instruction. Briefly, protein concentration was measured by the Bradford dye-binding assay (Bio-Rad, Hercules, CA, USA, Catalog No. 5000006), and then, equal amount of proteins from each sample were separated by SDS-PAGE, transferred onto the PVDF membranes by electrophoretic transfer. The membrane was blocked with 5% non-fat skim milk in TBS-tween, and then incubated with anti-TonEBP (Santa Cruz, Catalog No. sc-398171; 1:1000 dilution). The membrane was incubated with HRP-conjugated mouse secondary antibody (Cell signaling, Danver, MA, USA, Catalog No. 7076; 1:3000 dilution), and the immunoreactive signals were detected by Chemiluminescent detection reagent (Thermo Scientific, Catalog No. 34095). Protein density was normalized using an anti-Hsc70 antibody (Rockland, Limerick, PA, USA, Catalog No. 200-301-A28; 1:1000 dilution), and ImageJ software was utilized to analyze data.