Fig. 4.

Hypoxia promoted the internalization of claudin-5 in a CAV-1 upregulation-dependent manner. A C57BL/6 mice injected with 300 mg/kg MβCD were exposed to HH for 24 h and then reinjected with MβCD, which cycled for 1 h. Brain slices were colabelled with claudin-5 and CAV-1 antibodies to observe the colocalization of CAV-1 and claudin-5 (n = 10). B to E bEnd.3 cells were exposed to 1% O₂ for 24 h. Claudin-5 and ZO-1 antibodies were labelled by immunofluorescence (B) to quantify the fluorescence intensity of claudin-5 on the cell membrane and in the cytoplasm (C). Claudin-5 and CAV-1 antibodies were labelled by immunofluorescence (D) to calculate the colocalization of CAV-1 and claudin-5 (E) (** P < 0.01 and *** P < 0.001). F to J After transfection with siCAV1 for 48 h, bEnd.3 cells were exposed to 1% O₂ for 24 h. The CAV-1 and claudin-5 protein levels were detected by Western blot (F) and quantified (G). CAV-1 and claudin-5 antibodies were labelled by immunofluorescence assay (H) to quantify the fluorescence intensity of claudin-5 on the cell membrane (I) or in the cytoplasm (J) (*P < 0.05, **P < 0.01 and ***P < 0.001)