MAP6d1 can multimerize.

<p><i>A</i>, COS-7 cells overexpressing MAP6d1-GFP and MAP6d1-myc were lysed. Proteins were immunoprecipitated with antibodies against either GFP or myc, separated by SDS-PAGE and immunoblotted with each antibody. In the control experiments, COS-7 cells over-expressing either MAP6d1-myc or MAP6d1-GFP were lysed, and protein was immunoprecipitated with anti-GFP or anti-myc antibodies, respectively. <i>B</i>, MAP6d1 multimerization does not require cysteines. COS-7 cells overexpressing MAP6d1-GFP and MAP6d1-GGG-myc were lysed, and the proteins were immunoprecipitated as in <i>A.</i> Both proteins are present in the immunoprecipitate. <i>C,</i> Two-hybrid experiments to test the interactions between MAP6d1 and itself, MAP6-E or lamin. MAP6d1 interacts with itself and with MAP6-E. <i>D,</i> MAP6d1 multimerization requires the Mn3 module. COS-7 cells overexpressing MAP6d1-GFP and MAP6d1-ΔMn3-myc were lysed, and proteins were immunoprecipitated as in <i>A.</i> No co-immunoprecipitation was observed between MAP6d1 and MAP6d1-ΔMn3. <i>E,</i> NIH/3T3 cells were transfected with a plasmid encoding MAP6d1-GGG-ΔMn3-myc, and cryosections were labeled using an anti-myc antibody and immuno-gold. MAP6d1-GGG-ΔMn3 is not concentrated on the Golgi apparatus but is dispersed throughout the cytoplasm. Mitochondria are still heavily labeled, but no protein aggregates were observed. <i>F</i>, NIH/3T3 cells were transfected with a plasmid encoding the mutant MAP6d1-ΔMn3-myc and analysed as in <i>E</i>. MAP6d1-ΔMn3 associates with the Golgi, the plasma membrane (<i>arrowheads</i>) and cytoplasmic vesicles (<i>arrows</i>). <i>G:</i> Golgi apparatus; <i>mit:</i> mitochondria. <i>Bars</i>, 200 nm.