Fig. 5

AGEs promote Caveolin1-mediated LDL transcytosis through the NF-κB-RAGE-NF-κB signaling loop. A–C After pretreating HUVECs with PDTC (10 μmol/ L) for 1 h, HUVECs were exposed to BSA or AGEs for 24 h. Western blot was used to detect the expression of NF-κB related molecules A and summary bar graph showing the expression of the indicated proteins (B). Normalized amount of LDL transcytosis(C); n = 3. D–G 48 h after transfection with control or NF-κB p65 siRNA, HUVECs were treated with BSA or AGEs for 24 h, and the expression of RAGE and Caveolin-1 in HUVECs were detected by Western blot. Representative western blotting analyses of the indicated proteins D and summary bar graph showing the expression of the indicated proteins (E); n = 3. F Dual luciferase reporter assay was performed to measure the activities of the RAGE promoter or Caveolin-1 promoter when PGL3-RAGE or PGL3-Caveolin-1 co-infiltrated with or without pcDNA3.1-NF-κB p65 in HUVECs; n = 3. G–I 48 h after transfection with control or RAGE siRNA, HUVECs were treated with BSA or AGEs for 24 h, the expression of NF-κB related molecules and Caveolin-1 in HUVECs were detected by Western blot G and summary graph showing the expression of the indicated proteins (H). Normalized amount of LDL transcytosis (I); n = 3. J Representative immunofluorescence images of p65 in in HUVECs treated as above. Scale bar, 20 mm