Figure 4

Coimmunoprecipitation analysis of TSC2-TSC2 interactions using truncated TSC2 proteins. A series of V5-tagged TSC2 truncation proteins were coexpressed with TSC2, or in combination. The truncated proteins were immunoprecipitated and coimmunoprecipitated proteins detected by immunoblotting. (A) Coimmunoprecipitation of untagged, full-length TSC2 with TSC2 truncation proteins. The V5-tagged truncated TSC2 proteins (A614V5, F904V5, A1153V5, M1028V5 and M1453V5; see Materials and Methods and Suppl. Figure 1. for details) were immunoprecipitated with anti-V5 affinity beads and the washed immunoprecipitates were analysed by immunoblotting. TSC2 was coimmunoprecipitated with all 5 TSC2 truncation proteins, but was not visible in the control transfection (control; no V5-tagged protein expressed). (B) Coimmunoprecipitation of the A1153V5 TSC2 truncation protein with untagged TSC2 and the N-terminal TSC2 truncation proteins, M1028V5 and M1453V5. Full-length TSC2 and the M1028V5 and M1453V5 truncation proteins were immunoprecipitated with a polyclonal antibody recognising epitopes encoded by the last exon of TSC2 [5] and the washed immunoprecipitates were analysed by immunoblotting. In each case, the A1153V5 truncation protein was coimmunoprecipitated. No A1153V5 was visible in the immunoprecipitate of control cells, expressing A1153V5 only (control).