Figure 5
From: Retrotransposition and mutation events yield Rap1 GTPases with differential signalling capacity
Determination of the GTP/GDP ratio bound to mRap1A-retro1, -retro2 and hRap1B-retro. A. Comparison of GTP binding between Rap1 retrogenes and Rap1V12. Cos-7 cells were transfected with 2 μg different Rap1 constructs as indicated, starved overnight, labelled with [32P] orthophosphate for 5 h. HA-Rap1 was immunoprecipitated with the 12CA5 anti-HA monoclonal antibody, and bound nucleotides were separated using thin layer chromatography, followed by PhosphorImager detection. SD, standard deviation. B. Rap retrogenes are sensitive to Rap1Gap. Cos7 cells were transfected with HA-Rap1Gap in combination with mRap1A-retro1, mRap1A-retro2 and hRap1B-retro. GDP and GTP, bound to transfected Rap1, was measured as indicated above. The bottom panels indicate the expression of HA-Rap1Gap and HA-Rap as determined by Western blotting using anti-HA as a probe.