Fig. 1: Identification of ZNF185 as a PKA substrate in HUVECs.

a Several PKA substrates are phosphorylated using forskolin in HUVECs. Forskolin (10 µM) was added to HUVECs for 1 h. pPKA substrates are indicated by arrows. Representative blots are shown (n = 3). b Representative silver-staining of the immunoprecipitated PKA substrates in HUVECs. Forskolin (10 µM) was added to the HUVECs for 1 h. pPKA substrates indicated by asterisks were immunoprecipitated by a pPKA substrate antibody (n = 3). The band indicated by a red asterisk was ZNF185 (Supplementary Fig. 1). c Overexpressed ZNF185 was phosphorylated by forskolin in HUVECs. Stable cell lines of HUVECs overexpressing Myc-ZNF185-HA were generated. One hour after the administration of forskolin (10 µM), anti-Myc beads were used for immunoprecipitation. Densitometric analysis of pZNF185. The black arrows indicate overexpressed ZNF185 and the arrowhead indicates endogenous ZNF185. The red arrows indicate phosphorylated ZNF185. *p < 0.05 (n = 3). d Overexpressed ZNF185 was phosphorylated by forskolin within 5 min. Time course of ZNF185 phosphorylation after the administration of forskolin (10 µM) is shown. Anti-Myc beads were used for immunoprecipitation. Densitometric analysis of pZNF185. The arrows indicate overexpressed ZNF185. **p  < 0.01 (n = 3). e Overexpressed pZNF185 is localized to the membrane region. Immunofluorescence staining of pPKA (green) and Myc (magenta) in HUVECs overexpressing Myc-ZNF185-HA is shown. pZNF185 accumulates at the membrane region after the administration of forskolin (10 µM) for 1 h. Arrowheads indicate the cell boundaries. Arrows indicate pZNF185 localized to the membrane region. VE-cadherin was used to stain the cell membrane. Representative images are shown (n = 3). Scale bars, 10 µm. Data are presented as the mean ± standard error.