Fig. 5

KPNA2 promotes STAT3 phosphorylation and the nuclear accumulation of phosphorylated STAT3. A Western blot analysis of the changes in KPNA2, STAT3, P-STAT3 and ANPTG2 levels in HUVEC under hypoxia for 12 h after overexpression and knockdown of KPNA2. GAPDH was used as an internal reference. B KPNA2 was overexpressed and knocked down in HUVEC under hypoxia for 12 h, and P-STAT3 was detected by Western blotting after separation of the nuclear and cytoplasmic proteins. The nuclear reference was Laminb1, and the cytoplasmic reference was GAPDH. C Immunofluorescence detection of P-STAT3 (red) and DAPI staining of the nuclei (blue) under hypoxia for 12 h after KPNA2 was overexpressed or knocked down in adenovirus-infected HUVEC. Scale bar: 25 μm D Quantitative RT-PCR detection of VEGF and ANGPT2 mRNA expression under hypoxia in HUVEC in which KPNA2 was overexpressed or knocked down. E After adenovirus infection of HUVEC to knock down KPNA2 under hypoxia for 12 h, STAT3 was assessed by Co-IP, and Western blotting was used to detect JAK1, JAK2, SRC, TYK2, and STAT3. F KPNA2 was knocked down in HUVEC under hypoxia for 12 h, and P-JAK1 was detected by Western blotting. G Molecular simulations with ZDOCK showed possible interactions between S220 (KPNA2)-Y657 (STAT3), N228 (KPNA2)-Q643 (STAT3), and Y225 (KPNA2)-Q644 (STAT3). H Constructs from truncated plasmids corresponding to 4 domains of STAT3 with added Flag tags were prepared; the constructs corresponded to amino acids 1–137, 138–319, 320–493, and 494–770. I Four truncated STAT3 plasmids were transfected into 293 T cells, KPNA2 was used for Co-IP, and Flag was detected by Western blotting. Each experiment was repeated three times. NC negative control