FIGURE 7.

Deletion of insulin‐like growth factor‐binding protein 5 (IGFBP5) increases HIF1α expression by suppressing ubiquitination via ubiquitin ligase Von Hippel‐Lindau (VHL). (A) Western blotting images and quantification of HIF1α in sh‐NC‐ and sh‐IGFBP5‐infected human umbilical vein endothelial cells (HUVECs) (n = 6 in each group). (B) Western blotting images and quantification of HIF1α in NC‐overexpression (OE‐NC)‐ and IGFBP5‐overexpression (OE‐IGFBP5)‐transfected HUVECs (n = 6 in each group). (C) The stability of HIF1α expression in sh‐NC‐ and sh‐IGFBP5‐transfected HUVECs in the presence of 20 μg/mL cycloheximide (CHX) determined by Western blot (n = 5 in each group). (D) The stability of HIF1α expression in OE‐NC‐ and OE‐IGFBP5‐transfected HUVECs in the presence of MG132 (MG) determined by Western blot. (E) Western blotting images of total ubiquitin, K48 ubiquitin and K63 ubiquitin of HIF1α in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs (n = 3). (F) Western blotting images and quantification of VHL in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs (n = 5 in each group). (G) Co‐immunoprecipitation of the interaction between VHL and HIF1α detected by Western blot in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs (n = 3). (H) Western blot images and quantification of HIF1α expression in sh‐NC‐ and sh‐IGFBP5‐infected HUVECs in the presence or absence of IGF1R inhibitor picropodophyllin (PPP) (n = 5 in each group).