Figure 3. Conserved Interaction of BIN1 andNesprin in C. elegans (A) Diagram displaying C. elegans (Ce) nesprin2homolog protein (ANC-1) and constructs. Ce ANC-1 contains two conserved CH motifs formingan ABD, a KASH NE-binding domain, and a central coiled-coil region containing numerous SRs (only the five-repeat-rich region is shown). Lysates from HEK293 cells transfected with Ce ABD+KASHFlag and Ce AMPH-1-Myc were immunoprecipitated using anti-Flag or anti-Myc antibodies. Ce ABD+KASH co-immunoprecipitates with Ce AMPH-1 and vice versa. Before developing theblot, the nitrocellulose membranes were cut right below the 50 kDa bands to avoid interference of IgG heavy chain bands. In the blot on the left, avertical line indicates that the IP-Myc lane was not adjacent to the other three lanes in the gel. The IgG lane also contains a molecular weight marker.IP, immunoprecipitation; WB, western blot; IgG, immunoglobulin G.(B) Endogenous Ce ANC-1 and Ce AMPH-1 localization in intestinal cells of wild-type young adult. A mask for the co-localization was created with the ImageJ software (right). (C) The fluorescence profiles of ANC-1 (green) andAMPH-1 (red) across the minor axis of the nucleus are shown. Co-localization of ANC-1 and AMPH-1 signals was estimated using the Pearson coefficient(r). 0 indicates a lack of co-localization, and 1 indicates complete co-localization; r = 0.90. (D) Multichannel 3D projection (z stack 3 um) ofthe nucleus displayed in (B). (E) Endogenous Ce AMPH-1 localization in intestinal cells of anc-1
) young adult animal showing absence of perinuclear AMPH-1 compared to (B) in wild-type animals. DNA was stainedwith Hoechst. Scale bars, 10 um. See also Figure S3.