Figure 3. Ana1 provides a molecular linkage between Cep135 and Asl.
(a) Recombinant MBP or MBP-Ana1 was immobilized on amylose resin and incubated with 35S-Met-labelled Cep135, Asl or Sas-6 (negative control) synthesized by coupled in vitro transcription-translation (IVTT). Inputs (9%, left panel) and affinity-purified complexes (middle and right panels) were subjected to SDS-PAGE, stained (Coomassie, upper panels), and dried for autoradiography (lower panels). Note: MBP-Ana1 directly and specifically binds to Cep135 and Asl. Uncropped scans are in Supplementary Fig. 6.
(b) GFP or GFP-Ana1 was transiently co-expressed with Flag-Cep135 or Flag-Asl in D.Mel-2 cells for GFP-Trap pull-downs. Inputs and bound proteins were analyzed by Western blotting. GFP-Ana1 specifically co-purifies with Flag-Cep135 and Flag-Asl. Uncropped scans are in Supplementary Fig. 6.
(c) Cep135 or Asl was transiently co-expressed with Ana1. Proteins were tagged either at the N- or C-terminus as indicated (cartoon, upper part). Representative images show both Cep135 (lower part, middle panel) and Asl (lower part, bottom panel) co-localize with Ana1 in cytoplasm, observed by GFP (green) or mRFP (red) signal. Flag-Asl was detected with anti-Flag antibody (green).
(d) Cep135 and Asl were transiently co-expressed in cells, with or without additional Ana1 construct. Cep135 visualized by GFP fluorescence; Asl and Ana1, by mRFP fluorescence or Flag immunostaining as appropriate (cartoon, upper part). Cep135 and Asl form independent assemblies in cytoplasm in the absence of exogenous Ana1 (lower part, upper panel) but colocalize in the presence of exogenous Ana1 (lower part, lower panel). Scale bars in c and d, 5 μm.
(e) Glycerol gradient sedimentation. D.Mel-2 cells were transiently co-transfected with GFP-Cep135 and Asl-mRFP (Control) or Ana1-Flag, GFP-Cep135 and Asl-mRFP (Complex), lysed and sedimented on 10-30% linear glycerol gradients and collected as 34 fractions. In the presence of Ana1-Flag, GFP-Cep135 and Asl-mRFP perfectly co-sediment (lower panel); when Ana1-Flag is absent, they only partially co-migrate with distinct main peaks (upper panel).