Figure 2.

Effect of SRPK1 inhibitors on VEGF and SR protein phosphorylation. (A) ARPE-19 cells were treated with 10 μM SRPIN340, MVRL09, SPHINX, or left untreated, and subsequently either treated or not with EGF. Extracted protein was run on Western blots for phospho-SR proteins. (A) Bands were observed at molecular weights corresponding to SRSF1/2, SRSF4, and SRSF6, densitometry analysis was undertaken on SRSF1/2. SRPK inhibition significantly reduced the endogenous phosphorylation of SRSF1/2, moreover pre-incubation of cells with either SRPIN340 or SPHINX significantly blocked an EGF-induced increase in SR protein phosphorylation. (B) Serum-starved primary RPE cells and ARPE-19 cells were treated with inhibitors at 5 μM. Twenty-four hours later, RNA was extracted and cDNA was made. PCR was performed with primers spanning VEGF exon 7b and 8b. Treatment with the inhibitors reduced the expression of VEGF₁₆₅ relative to GAPDH control either in primary RPE cells (SRPIN340) or both cell lines (SPHINX and MVRL09). Water (W) and sample without RT were used as negative controls. (C) Forty-eight hours after treatment, protein was extracted and assayed for total VEGF by ELISA. Protein was assessed using a VEGF_xxxb-specific ELISA and the ratio of VEGF_xxxb over total VEGF calculated. The ratio increased in ARPE-19 during treatment with SPHINX and SRPIN340, and in primary RPE during treatment with SPHINX and MVRL09. Total VEGF expression relative to total protein was also calculated. In primary RPE, VEGF expression was reduced following SRPIN340 and MVRL09 treatment, in ARPE-19, VEGF expression was increased following SPHINX and MVRL09 treatment (One-way ANOVA, Bonferroni post hoc. *P < 0.05, **P < 0.01).