Fig 1: CD146 interacts with CD36. (A) Co-IP assay of CD36 and CD146 in BMDMs. CD146 and CD36 from cell lysates were immunoprecipitated with anti-CD146 mAb and anti-CD36, respectively. (B) Direct interaction between CD36 and CD146. CD146-ECD was first incubated with CD36-ECD. CD146-ECD was pull down by anti-CD146 ME-9F1, and then precipitated by protein G beads. Bound proteins were subsequently analyzed by western blotting. (C) Co-IP assay showed that the CD36 and CD146 interaction was enhanced in the presence of oxLDL in BMDMs. Immunoblot analysis of CD146-precipitated proteins from BMDMs treated with oxLDL (50 μg/ml) for the indicated times. Right panel: quantification of CD36 level relative to CD146. (D) Co-IP assay showed that the CD36 and CD146 interaction was decreased by anti-CD146 antibody AA98. Right panel: quantification of CD36 level relative to CD146. (E) Direct interaction of CD36/CD146 or CD36/CD146D4-5. CD36-ECD was first incubated with CD146-ECD and CD146D4-5, respectively. CD36-ECD was pull down by anti-CD36 antibody, and then precipitated by protein G beads. (F) AA98 blocked CD36 and CD146 interaction. His-CD146-ECD or His-CD146D4-5 was first incubated with BMDM cell lysates in the presence of mIgG or AA98 (50 μg/ml). CD36 proteins bound to His-CD146-ECD or His-CD146D4-5 were detected by immunoblot with anti-CD36 antibody. *P < 0.05, **P < 0.01. The data represent three independent experiments.
Fig 2: CD146 facilitates CD36 internalization. Western blot (A, B) and FACS analysis (C, D) of membrane CD146 and CD36 in oxLDL-stimulated BMDMs isolated from CD146WT or CD146M-KO mice (A, C) or BMDMs with or without pretreatment with anti-CD146 AA98 (50 μg/ml) (B, D). Membrane fractions were immunoblotted with the indicated antibodies. SP (sodium pump) served as a loading control for membrane fractions. Right panel: quantification of CD36 or CD146 levels relative to SP (A, B). Bottom panel: quantification of the MFI of CD36 or CD146 (C, D). (E, F) Western blots of CD146 and CD36 in endosomal fractions of BMDMs isolated from CD146WT or CD146M-KO mice (E) or BMDMs with or without pretreatment with anti-CD146 AA98 (50 μg/ml) (F). Endosomal fractions were immunoblotted with the indicated antibodies. Right panel: quantification of CD36 or CD146. (G, H) BMDMs from CD146WT or CD146M-KO mice or BMDMs with or without pretreatment with AA98 (50 μg/ml) were labeled with a CD36-cross-linking antibody. Then, the cells were incubated at 37 °C to cross-link CD36. The cells were then washed with cold acid wash buffer to deplete surface CD36. Confocal microscopy was used to detect and quantify CD36 internalization. Bottom panel: quantification of the MFI of CD36. *P < 0.05, **P< 0.01, ***P< 0.001. The data represent three independent experiments.
Fig 3: CD146 controls the expression of macrophage migratory factors in response to oxLDL. (A, B) Quantitative real-time (RT) PCR analysis of mRNA levels of the macrophage migratory factors Cd36, Netrin-1, Sema 3E and Ccr7 in BMDMs that were incubated with oxLDL (50 μg/ml) for 24 h. (A) BMDMs were isolated from WT, CD146M-KO or CD36KO mice and stimulated as indicated; (B) WT BDMDs were stimulated as indicated in the presence of control mIgG or anti-CD146 AA98 (50 μg/ml). (C, D) Western blot analysis of the macrophage migratory factors CD36, Netrin-1 and CCR7 in BMDMs that were treated as indicated. GAPDH was used as a loading control. (C) BMDMs were isolated from WT, CD146M-KO or CD36KO mice and stimulated as indicated; (D) WT BDMDs were stimulated as indicated in the presence of control mIgG or anti-CD146 AA98 (50 μg/ml). Right panel: quantification of protein expression level relative to GAPDH. *P < 0.05, **P < 0.01. The data represent three independent experiments.
Supplier Page from Sino Biological, Inc. for Mouse CD36 / SCARB3 Protein (His Tag)