Fig. 7

RhoA phosphorylation and activity are affected by PFTK1. Neuronal cultures from E13–14 embryos from HET × HET crosses were examined after 18–20 h in vitro. Total RNA (A) or protein (B) was extracted followed by RT-PCR or western blot. Representative images from RT-PCR (A) and western blot (B) assays are shown. C Bar graph represents the average band intensity from 3 KO and 6 WT for mRNA and 7 WT and KO each for protein. Non-parametric Mann-Whitney test. D Assessment of RhoA activity using an IP-based assay. Neuronal extracts were incubated with Rhotekin PBD beads to pull down active RhoA. WT extract preincubated with GTP_γS (positive) or GDP (negative) was used as control. IP was followed by western blotting for RhoA. Two representative assays of four independent assays are shown. E In vitro kinase assay. IPed protein (either with IgG control or PFTK1) from WT cortical neuronal culture extracts was used as source of kinase and incubated with human recombinant RhoA (His-tagged) with or without GDP. Results were analyzed for phospho-serine signal. F RhoA activity assay following in vitro kinase assay. Kinase reactions were analyzed by G-LISA to test for RhoA activity. Bar graph represents mean values ± SEM. Two-way ANOVA analysis of grouped data and Sidak test for multiple comparisons are indicated on bar graph. RHO1, His-RhoA human recombinant protein