Anti red fluorescent protein

Msmeg cultures were growth-synchronized before ATc was added to a final concentration of 100 ng ml⁻¹. For each time point, 20 OD₆₀₀ units of culture were harvested by centrifugation (4000g, 10 min) and resuspended in 600 μl of lysis buffer [50 mM tris and 150 mM NaCl (pH 7.4)] containing a protease inhibitor cocktail (Sigma-Aldrich, #11873580001) before lysis by bead beating in Lysis B Matrix tubes (MP Biomedicals, #116911050) using a Precellys Evolution homogenizer (Bertin Instruments, #P000062-PEVO0-A; 3 × 10,000 rpm, 30-s intervals). The cell lysates were cleared by centrifugation (20,000g, 10 min), and a 20-μl aliquot was incubated with 4× lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, #NP0007) supplemented with dithiothreitol at 70°C for 10 min. Samples were separated on a 4 to 12% bis-tris polyacrylamide gel (Invitrogen, #NP0323BOX) in Mops or MES running buffer, transferred to a nitrocellulose membrane using the TransBlot Turbo Transfer System (Bio-Rad, #1704150), and incubated for 1 hour in blocking buffer (LI-COR, #927-60001). Proteins were probed with anti-RpoB (BioLegend, #663905), anti–red fluorescent protein (RFP; Rockland, #600-401-379), or anti-FLAG (Sigma-Aldrich, #F3165) primary antibodies overnight at 4°C and subsequently detected with fluorescent goat anti-mouse and anti-rabbit secondary antibodies (Bio-Rad, #12004159 and #12004162).

Mouse tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin according to standard protocols. Immunohistochemical staining of paraffin sections was carried out with 1:200 anti-UCP1 (Abcam). For Immunofluorescence, slides were fixed with 4% PFA and permeabilized in ice cold methanol. Heat-mediated antigen retrieval with a 0.01 M citric acid (pH 6.0) was performed for 5 min in a microwave. After blocking with 4% BSA, the sections were blocked with unconjugated AffiniPure Fab Fragment (1:100; Jackson), then incubated with primary antibodies anti-UCP1 (rabbit; 1:200; Abcam), anti-red fluorescent protein (rabbit; 1:500; Rockland), anti-PDGFRα (goat; 1:200; R&D) or anti-DPP4 (goat; 1:200; R&D) followed by detection with secondary antibodies Alexa Fluor^® 594 conjugated goat antibody to rabbit IgG (1:1,000; Invitrogen) or Alexa Fluor^® 647 conjugated donkey antibody to goat IgG (1:1,000; Invitrogen). Finally, the sections were incubated with DAPI or Hoechst. The adipocytes were stained with BODIPY^® 493/503 (Invitrogen). EdU incorporation assay was performed following the instruction of Click-iT^® Plus EdU Alexa Fluor^® 488 Imaging Kit (Invitrogen).