Fig. 4

CLK1 phosphorylates SRSF5 on Serine 250 in human pancreatic cancer cells. a Volcano plot of differential expression genes between CLK-KD and CLK1-vector groups. b, c GO and KEGG analysis of the DEGs was performed and results were represented. d Flowchart showing the process of evaluating the involvement of CLK1 in regulating alternative splicing related proteins. e Phosphorylation mass spectrometry was performed, and clusters plots represents differentially phosphorylation expressed sites (DPS) between CLK1-konckdown and control cells. f–h GO analysis of the DPS related proteins was performed and results were represented. i Changes of phosphorylation in the splicing factors (SRSF1, SRSF3, SRSF5, SRSF6) screened by phosphorylation mass spectrometry were confirmed by western blot assays. j Logo pictures represented the 250-Ser of SRSF5. k Relationship between CLK1 expression and phosphorylation level of SRSF5 on Ser250 in different cell lines and l CLK overexpressed or knockdown cell lines was determined by western blot assays. m CLK1 activated the phosphorylation level of SRSF5 on Ser250. n Up, the interaction between CLK1 and SRSF5 was analyzed in Panc-1 cell transfected with plasmids encoding HA-CLK1 and Flag-SRSF5 by co-immunoprecipitation and western blot assays. Bottom, Endogenous interaction between CLK1 and SRSF5 was assayed in PANC-1 cell lysates by co-im0munoprecipitation and western blot assays using an anti-CLK1 or anti-SRSF5 antibody, respectively. Ten percent of the input was loaded, and normal IgG was used as a control. o Immunofluorescence staining of CLK1 (green), SRSF5 (red) and DAPI (blue) in PANC-1 and BxPC3 cells was conducted. Scale bars for the images are 50 μm. p The expressions of phosphorylated SRSF5 and CLK1 in tumor tissues of PDAC patients were evaluated by IHC. q The correlation between P-SRSF5 and CLK1 levels in tumor tissues was analyzed. r–t Kaplan–Meier analyses of the correlations between P-SRSF5 (r), SRSF5 in the condition of High CLK1 expressions (s) CLK1 in the condition of High SRSF5 expressions (t) and overall survival of PDAC patients. Data are shown as mean ± SD from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, between the indicated groups