CC and MIT domains are required for stable association of TMF with the Golgi.

<p>(A) Diagram of the TMF aa sequence with the marked MIT and CC motifs (top). Constructs 1–5 transfected into NIH3T3 cells are depicted below. The TMF aa included in each construct are shown, and the presence of the CC or MIT motifs in each construct are marked in the brackets. (B-F) Confocal microscopy analysis of EGFP-fused TMF segments, ectopically expressed (green) in NIH3T3 cells. (G) Confocal microscopy analysis of TMF segment containing the MIT and c-terminus CC domain fused to EGFP (EGFP-TMF C5) (green) overlapping the immuno-staining of GM130 (Golgi marker, red.). Arrows represent co-localization (yellow color). The different fluorescence channels are presented in the right panels. Bar represents 20μm. Nuclei were visualized with Hoechst solution (blue). (H) As in <b>G</b>, only that these cells were treated for 20 min with colchicine. Arrow indicates the Golgi apparatus. Images represent typical fluorescence and staining profiles obtained for all transfected cells in four independent transfection experiments. (I) Confocal microscope analysis of the full TMF protein fused to EGFP (green) overlapping the immuno-stained Golgi (golgin 97- red.) (J) As in <b>I</b>, only that cells were treated for 20 min with colchicine. Arrow indicates the Golgi apparatus. Nuclei were visualized with Hoechst solution (blue).Images represent typical fluorescence and staining profiles obtained for all transfected cells in four independent transfection experiments. (K) IP of GFP-TMF protein fragments from lysates of NIH3T3 cells expressing either GFP-TMF containing the MIT domain (TMF-MIT) or GFP-TMF fragment containing the CC domains without the MIT motif (TMF-CC).Whole cell lysate (WCL) represents the input of the IP. IgG beads served as a negative control. The IP was followed by a WB analysis to assess the co-IP of the TMF fragment and tubulin. Images represent one out of five independent typical WB analyses which gave similar results.