Figure 2

Coimmunoprecipitation of distinct epitope-tagged TSC2 isoforms. Untagged TSC2, GFPTSC2, XpTSC2 and/or TSC2V5 were co-expressed in HEK293T cells with or without co-expressed TSC1. Distinct tagged isoforms were immunoprecipitated and the presence of other coimmunoprecipitated tagged isoforms was investigated by immunoblotting. (A) Coimmunoprecipitation of TSC1 and GFPTSC2 with TSC2V5. TSC2V5 was co-expressed with GFPTSC2 or untagged TSC2. GFPTSC2 was detectable as a 220 kDa band in immunoblots of the cell lysates, distinct from the 200 kDa TSC2 and TSC2V5 bands. TSC2V5 was immunoprecipitated from the lysates with V5 affinity beads. Immunoblotting of the washed beads showed that GFPTSC2 was coimmunoprecipitated with TSC2V5. No GFPTSC2 was immunoprecipitated in the absence of TSC2V5. (B) Coimmunoprecipitation of TSC1 and TSC2V5 with XpTSC2. XpTSC2 was immunoprecipitated from cell lysates with an antibody specific for the Xpress tag. The Xpress- and V5-tagged TSC2 isoforms could be distinguished by immunoblotting using the appropriate specific antibodies. TSC2V5 coimmunoprecipitated with XpTSC2. (C) Coimmunoprecipitation of GFPTSC2 variants with TSC2V5. TSC2V5 was co-expressed with GFPTSC2 or GFPTSC2 containing the R611Q (GFPR611Q), R905Q (GFPR905Q) or N1620S (GFPN1620S) pathogenic substitutions, with or without co-expressed TSC1. As a control, GFPTSC2 was co-expressed with V5-tagged B-lactamase (lacV5). All the GFP-tagged TSC2 variants were coimmunoprecipitated with TSC2V5. In contrast, GFPTSC2 was not detected in the lacV5 immunoprecipitates.