Rabbit anti cd36

Equal amounts of mice spinal cord homogenates protein (40 μg) were resolved separately for soluble and membrane fractions on SDS-PAGE, transferred to nitrocellulose membrane, and blocked overnight with 5 % skim milk in TBS-T (0.3 % Tween 20). Blots of the soluble fraction were probed with the following primary antibodies: mouse anti-actin (1:10,000 Sigma-Aldrich, USA), rabbit anti-MCP-1 (1:1000 Peprotech, USA), rabbit anti-IL-6 (1:1000 Peprotech, USA), and mouse anti-Iba-1 (1:1000 Millipore, Germany). Blots of the membrane fraction were probed with the following primary antibodies: mouse anti-actin (1:10,000 Sigma-Aldrich, USA), rabbit anti-occludin (1:1000 Abcam, UK), rabbit anti-claudin 5 (1:500, Sigma- Aldrich, USA), rabbit anti-ZO-1 (1:1000 Sigma-Aldrich, USA), and rabbit anti-cd36 (1:1000 Abcam, UK). Blots were incubated with corresponding secondary antibodies conjugated peroxidase (Sigma- Aldrich, USA) and developed with the EZ-ECL detection kit (Biological Industries, Israel). Quantitative densitometric analysis was performed using the densitometric software EZQuant-Gel (version 2.12).

Radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (BestBio Cat No. BB-3101) was used to extract proteins. The protein concentration was assessed using the Rapid Gold BCA Protein Assay Kit (Thermo Fisher). Western blotting analysis was performed by loading 15 µg of lysate onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferring the gels to polyvinylidene difluoride (PVDF) membranes (Millipore), and incubated with rabbit anti-GDF5 (1:1000, #A13167; ABclonal), rabbit anti-PPARγ (1:1000, #2443; CST), rabbit anti-FASN (1:1000, #D262701; Sangon Biotech), rabbit anti-C/EBPα (1:1000, #2295; CST), rabbit anti-FABP4 (1:1000, #2120; CST), rabbit anti-CD36 (1:1000, #ab1336-25; Abcam), rabbit anti-GAPDH (1:5000, #BS65529; Bioworld), rabbit anti-CD9 (1:1000, #AP68-965-100; Abcepta), rabbit anti-CD63 (1:2000, #D160973; Sangon Biotech), rabbit anti-TSG101 (1:2000, #381538; ZEN BIO), rabbit anti-Alix (1:1000, #D262028; Sangon Biotech) or rabbit anti-Calnexin (1:1000, #D262986; Sangon Biotech). Afterward, goat anti-rabbit secondary antibody (1:50000, # BS13278, Bioworld) conjugated with HRP was used. GAPDH levels served as the loading control. The amount of protein was measured using ImageJ software.

Calcium and magnesium free PBS (PBS⁻) was purchased from Gibco (#10010-023). Triton X-100 was obtained from Sigma-Aldrich (#T8787). Hoechst 33258 (#H3569) and AlexaFluor 568-conjugated Phalloidin (#A12380) were from Invitrogen. Antibodies used were as follows: Mouse anti-RPE65 (#ab78036 [clone 401.8B11.3D9]; Abcam, Cambridge, MA), mouse anti-Cytokeratin (#M0821 [clone MNF116]; Dako A/S, Glostrup, Denmark), rabbit anti-ZO1 (mid) (#40-2200; ThermoFisher Scientific, Waltham, MA), mouse anti-Syntenin-1 (#ab131190 [clone 3D9-G9-H4]; Abcam), mouse anti-TSG101 (#612696; BD Biosciences, San Jose, CA), rabbit anti-SLC39A12 (#ab106570; Abcam), rabbit anti-Calreticulin (#12238 [clone D3E6]; Cell Signaling Technologies, Danvers, MA), mouse anti-BEST1 (#NB300-164, [clone E6-6]; Novus Biologicals, Littleton, CO), mouse anti-Na⁺/K⁺-ATPase alpha (#sc-58628 [clone M7-PB-E9]; Santa Cruz Biotechnology, Dallas, TX), rabbit anti-CD36 (#ab78054; Abcam), AlexaFluor 488-conjugated donkey-anti-rabbit IgG (#A21206, Invitrogen), AlexaFluor 488-conjugated donkey-anti-mouse IgG (#A21202, Invitrogen), AlexaFluor 568-conjugated donkey-anti-rabbit IgG (#A10042; Invitrogen), HRP-conjugated donkey-anti-mouse IgG (#715-035-150, Jackson ImmunoResearch Laboratories, West Grove, PA), and HRP-conjugated donkey-anti-rabbit IgG (#711-035-152, Jackson ImmunoResearch Laboratories).

DC pellets were lysed in RIPA lysis buffer (Sigma-Aldrich) with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). Proteins were then separated with SDS-PAGE 4–20% gel (20 μg protein/slot; Precast Gels, Genscript, Piscataway, NJ) and transferred onto 8.5 × 6 cm PVDF membranes (GE Healthcare, Freiburg, Germany) and blocked with 5% w/v BSA (0.5 h). Membranes were incubated overnight with primary Abs: rabbit anti-p-4E-BP1 (Thr37/46), rabbit anti-p-p70S6K (Thr389/412), rabbit anti-Pparγ, rabbit anti-p-NFκβ p65 (Cell Signaling Technology; 1:1000), rabbit anti-CD36 (Abcam, Cambridge, MA, 1:1000) or mouse anti-β-actin (Sigma-Aldrich; 1:1:2000). HRP-conjugated goat anti-rabbit IgG (H±L) secondary Ab (Cell Signaling Technology; 1:5000) was used (1 h). Signals were detected by Western HRP Substrate on ChemiDoc™MP Imaging System (Sigma-Aldrich; Bio-Rad, Hercules). Densitometric quantification of Western blot signals was performed using Image Lab software (open source: http://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z). All proteins were subsequently normalized to β-actin.