Figure 2
From: X-Linked thrombocytopenia causing mutations in WASP (L46P and A47D) impair T cell chemotaxis
WASP mutants WASP R L46Pand WASP R A47Dare stably expressed in JurkatWASP-KDT cells. A) JurkatWASP-KD T cells expressing RFP or WASPR-RFP fusions (WT or mutants) were generated by microporating JurkatWASP-KD T cells with plasmids expressing the constructs under the transcriptional regulation of WASP promoter followed by one week of selection with neomycin. The cell lysate were resolved and probed with either anti-WASP, anti-WIP and anti-GAPDH antibodies. B) Total RNA from JurkatWASP-KD T cells expressing WASP-RFP fusions or mutant derivatives were isolated and used to carryout quantitative real time PCR analysis to analyze expression of WASP or its mutant at mRNA level. C) Jurkat T cells expressing the RFP tagged WT or mutants WASP were incubated with either MG132 or calpeptin for 5 hours. Cells were lysed and immunoblotted for WASP and GAPDH antibodies.