Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability
Fig 3
TTBK2 homodimerizes through its C-terminus.
(A) A schematic representing the various TTBK2 truncations used for co-immunoprecipitation. TTBK2FL corresponds to full length mouse TTBK2. TTBK2CTerm is the amino acids C-terminal to the kinase domain: 306–1243. TTBK2SCA11 corresponds to one of the disease-associated mutations, and includes amino acids 1–443. Each is tagged with either V5 or GFP at the N-terminus as indicated in B and C. (B) Construct were expressed together as indicated in HEK 293T cells, which were lysed and subjected to immunoprecipitation using anti-GFP conjugated beads. Full length TTBK2 tagged with V5 (TTBK2FL-V5) was co-transfected with GFP-tagged full-length TTBK2 (TTBK2FL-GFP) or TTBK2Cterm. TTBK2FL-V5 interacted with TTBK2FL-GFP and TTBK2Cterm-GFP. Dashed line indicates that the blot image was cropped to remove bands that were not part of the current analysis. Input protein amount was 10% of total lysate for all conditions. (C) Constructs encoding either TTBK2FL or TTBK2SCA11 tagged with either GFP or V5 as indicated were expressed in HEK293T cells and subjected to immunoprecipitation using anti-GFP conjugated beads. Again, TTBK2FL-V5 and TTBK2FL-GFP co-precipitated, however TTBK2FL did not co-precipitate with TTBK2SCA11-GFP, and TTBK2SCA11-V5 did not co-precipitate with TTBK2SCA11-GFP. Input protein amount was 10% of total lysate for all conditions. Dashed line indicates that the blot image was cropped to remove bands that were not part of the current analysis. Input protein amount was 10% of total lysate for all conditions.