Figure 3. RAC1 activity is reduced upon overexpression of SRGAP1 mutants in cultured HEK293T cells.
(A) Active GTP-bound RAC1 precipitated by GST-PAK1 pulldown (PD) from HEK293T cells transfected with wild-type (WT) versus mutant SRGAP1 constructs. Note that HEK293T cells transfected with human SRGAP1 mutants C269Y and P665T exhibit an enhanced decrease in RAC1 activity compared to wild-type SRGAP1. The efficiency of SRGAP1 transfections and 5% input control were confirmed by immunoblotting with anti-SRGAP1 and anti-actin antibodies, respectively.
(B) Relative RAC1 activities (PAK1-bound RAC1/total RAC1) based on anti-RAC1 immunoblot signal intensities in (A). Ratios are normalized to mock transfection. A and B represent 5 experiments each.
*, P< 0.05 (Student’s t-test); Error bars indicate one standard deviation (n=3).