Fig. 6. In vitro binding assays of hGAR17 and hGAR22 proteins. hGAR17 (A) or hGAR22 (B) proteins labeled with ³⁵[S]-methionine were incubated with preassembled MFs (+MF) or MTs (+MT), the reaction mixtures centrifuged at high speed and the resulting supernatant (S) and pellet (P) fractions run on an SDS-PAGE gel. The gels were dried and the radioactively labeled proteins visualized by autoradiography. The presence of a protein in a pellet fraction indicates binding to the corresponding filaments. Reactions without filaments (–MF, –MT) were run to control for non-specific protein aggregation. BSA was used as a non-binding negative control; in this case the protein was visualized by Coomassie staining. The results of the assays are indicated to the right of the gels (+, binding; –, absence of binding). Since hCt22 sedimented in the absence of MFs, its sedimentation with MFs (+/–) is most probably due to non-specific aggregation rather than specific filament binding.