Figure 5
From: X-Linked thrombocytopenia causing mutations in WASP (L46P and A47D) impair T cell chemotaxis
WASP mutants exist in open conformation even in the presence of WIP. A) S. cerevisiae cells expressing NLS-WASP-Sensor or mutant (L46P, A47D) sensors + NLS-WIP were grown to exponential phase at 24°C and analyzed by fluorescence microscopy. B) Quantification of fluorescence intensity of S. cerevisiae cells expressing NLS-WASP-Sensor or mutants (L46P, A47D) + NLS-WIP. ***p < 0.001 compared to NLS-WASP + NLS-WIP expressing cells. C) Western blot analysis of S. cerevisiae cells expressing of NLS-WASP-Sensor or mutants + NLS-WIP using anti-GFP, anti-WIP and anti-Hexokinase serum. D) HEK293T cells were co-transfected with WASP or mutants (L46P, A47D) sensor with WIP and analyzed by fluorescence microscopy. E) Quantification of fluorescence intensity of WASP or mutants (L46P, A47D) in the presence of WIP. ***p < 0.001 compared to WASP sensor + WIP expressing cells. F) Western blot analysis of HEK293T cells expressing WASP or mutant sensors with WIP using anti-GFP (WASP), anti-His (WIP) and anti-GAPDH. G) His tag pull down assay showing the interaction of WASP or mutants (L46P, A47D) (tagged with GFP) with WIP-His in HEK293T cells.