Ab8937

Whole-cell protein extracts were obtained by direct lysis in SDS sample buffer heated to 75°C. Proteins resolved by SDS-PAGE were transferred to a polyacrylamide membrane (Bio-rad laboratories, Calfornia, USA.) and probed with antibodies raised against 11βHSD1, 1:5000 (AB83552, Abcam, UK); CRABP2, 1:5000 (HPA004135, SIGMA, Stockholm, Sweden); FABP5, 1:1000 (AB37267, Abcam, UK); RARα, 1:1700, (AB28767, Abcam, UK); RXRα,1:1000 (5388S, Cell Signaling, Danvers, USA); PPARβ/δ, 1:1000 (AB8937, Abcam, UK); Cyp26a1, 1:2000 (SC53618, SantaCruz); DHRS3, 1:200 (15393-1-AP; Proteintech Group Manchester, UK); RBP4,1:1000 (SC46688, SantaCruz); RDH12, 1:1000 (AB87038, Abcam, UK); and β-actin,1:5000 (A2066, SIGMA, UK). After incubation with peroxidase-conjugated secondary antibody, anti-rabbit or anti-mouse IgG diluted 1:1000–1:50,000 (Jackson Immunoresearch Laboratories, West Grobe, USA), detection of chemiluminescence reaction was visualized using the ECL plus chemiluminescence kit (PCC-NCI 32132 Amersham, GE Healthcare, Japan) and normalized to β-actin. The Western blots were quantified using ImageJ software.

Snap frozen mouse retinal and choroid/RPE tissues were homogenized in RIPA buffer containing 1mM dithiothreitol (DTT) (Sigma-Aldrich, St. Louis, MO, USA), 1× protease inhibitor (Roche, Switzerland), 1× phosphatase inhibitor (Sigma-Aldrich, USA), and 1mM phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, USA) before being centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatants were subjected to Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA) for total protein analysis before being separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto an Immobilon-PSQ 0.2 µm Polyvinylidene Fluoride (PVDF) Membrane (Merck Millipore, Burlington, MA, USA). Blots were probed with PPAR delta antibody (rabbit polyclonal, ab8937, Abcam, Cambridge, UK, RRID:AB_306872), GAPDH antibody (rabbit polyclonal, sc-25778., Santa Cruz Biotechnology, Inc., Dallas, TX, USA, RRID:AB_10167668), or RPE65 antibody (mouse monoclonal, ab13826, Abcam, UK, RRID:AB_2181006), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Bethyl Laboratories, Inc., Montgomery, TX, USA).

Cells grown on poly-L-lysine coated culture coverslips (Matsunami Glass, Osaka, Japan) in a 24-well plate were infected with FITC-conjugated M. leprae for 48 h. After discard of the supernatants, cells were washed with PBS 5 times to remove excess extracellular M. leprae, fixed with 10% buffered formalin (Wako Pure Chemical) for 15 min, permeabilized with 0.3% Triton X-100 (Wako Pure Chemical) in PBS for 5 min, and blocked with 0.5% bovine serum albumin (BSA) (Sigma Aldrich) in PBS for 1 h. Immunofluorescence staining was performed by incubating the coverslips with a rabbit anti-PPAR-δ antibody (ab8937, Abcam; 1:500) or a rabbit anti-PPAR-γ antibody (#2435, Cell Signaling Technology; 1:500) in PBS at 4°C overnight. After washing with PBS, coverslips were then incubated with a mixture of Alexa Fluor 594-conjugated chicken anti-rabbit IgG antibody (Life Technologies; 1:1,000) for 1 h at room temperature. The nuclei were counterstained with Hoechst 33258 (Life Technologies; 1:1,000) for 3 min at room temperature. Cover slips were placed on a piece of glass slide with fluorescence mounting medium (Dako, Tokyo, Japan). Immunofluorescence was visualized and the images were captured with an FV10i-LIV laser scanning microscope (Olympus, Tokyo, Japan).