Figure 4

Mid1 and Mig12 co-sediment with microtubules. (A) Immunofluorescence analysis in Cos7 cells co-transfected with HA-Mid1 (left panels) and MGFPMig12 (middle panel) proteins. Coincidence of the bundles with microtubules is revealed using monoclonal antibodies against β-tubulin (right panel). These images show the different thickness and distribution of the bundles. (B) Cos7 cells were transfected with either MGFPMid and HA-Mig12 (left panel) or HA-Mig12 alone (right panel). Lysates (L) from cells were supplemented with 40 μM taxol to stabilize polymerized microtubules. After sedimentation on sucrose cushion, supernatant (S) and pellet (P) fractions were assayed for the presence of Mid1, Mig12, and tubulin using appropriate antibodies. In the co-transfection (left panel) both Mid1 and Mig12 were detected in the pellet together with the polymerized microtubules. As expected Mig12 is also present in the soluble fraction where neither Mid1 nor the tubulin are found. Mig12 is found partially associated with the polymerized tubulin fraction also in the single HA-Mig12 transfected cells (right panel). (C) Western blot analysis using the anti-Mig12 antibody reveals a 24 KDa protein in two different cell lines lysates (1, Cos7; 2, HeLa cells). To confirm specificity, incubation with the primary antibody was also performed in the presence of either the fusion protein used to immunize rabbits (GST-Mig12) or an unrelated fusion protein (GST-ur). (D) Detection of endogenous Mig12 in the polymerized microtubule fraction (+ taxol) in HeLa cells and as control in the non-treated sample (-taxol); legend as in (A). (E) Single Mig12 transfected Cos7 cells show partial localization with microtubules, particularly in the MTOC region (upper panels) and at the mitotic spindle poles (lower panels).