Both AK1 and AK1B contain the GA-binding GM130-interacting domain, but only AK1 binds MTs. (A) GFP-AK1–, GFP-AK1B–, and GFP-AK2–expressing cell extracts were immunoprecipitated with the anti-GM130 antibody, and blots were revealed for GM130 and GFP. Ctl, control; SP, supernatant. (B) Co-IP from flag-AK1B and YFP-GM130 double-transfected cell extracts using an anti-flag antibody. Blots were revealed for flag and GM130. (C) Control (left) or GM130-depleted (right) cells were transfected with the flag-AK1B construct and then NZ treated to fragment the GA into stacks. Labelings were for flag, GM130, and GMAP210 as indicated. Merged images are shown. Enlarged boxes on the right show single labelings or merges corresponding to outlined areas. T, transfected; D, depleted. Arrows indicate GA ministacks. Bars, 5 µm. (D) Cells coexpressing GFP- and flag-tagged versions of AK1 (left) or AK1B (right) were immunoprecipitated with an anti-GFP antibody, and blots were revealed for GFP and flag. (E) GFP-AK1–transfected cell extracts were immunoprecipitated either with anti-p150glued or anti-GFP antibodies (left). IPs were then tested for the presence of GFP-AK1 or p150glued. (right) A similar anti-GFP IP from GFP-AK1B–expressing cells is shown. Black lines separate parts from different gels. (F) MTs were polymerized from RPE-1 cell lysates expressing GFP-AK1 or GFP-AK1B in the presence of exogenous tubulin and taxol. After centrifugation through a sucrose cushion, supernatants (SP) and pellets (MT) were analyzed by WB for GFP, α-tubulin, and p150glued as a positive control. (G) A summary of properties of AKAP450 and the N-terminal AK1 and AK1B fragments. αtub, α-tubulin; γTuSC, γ-tubulin small complex.