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. 2020 Dec 9;9:e61669. doi: 10.7554/eLife.61669

Figure 2. AKAP6 anchors centrosomal proteins to nesprin-1α through its SR domains.

Figure 2.

(A) Schematic representation of AKAP6, Pcnt, and AKAP9. (B) Immunostaining of Pcnt (red) or AKAP9 (red), cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in siControl and siAKAP6-treated rat P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 suggesting that the SR domains of AKAP6 are sufficient to bind to the nuclear envelope and to anchor centrosomal proteins. Transfected cells are indicated with a yellow arrowhead. (C) Immunoprecipitation to demonstrate the interaction of SR1-3 of AKAP6 with the PACT domain of Pcnt or AKAP9. Lysates from HEK293 cells transfected with GFP-AKAP6-SR1-3 in the absence or presence of FLAG-Pcnt-PACT or FLAG-AKAP9-PACT were immunoprecipitated with an anti-FLAG antibody and analyzed by western blotting with antibodies against GFP and FLAG, as indicated. The experiment was performed three times (n = 3); shown is a representative image. (D) Schematic representation of the results in E. +: interaction; -: no interaction. (E) Lysates from HEK293 cells co-transfected with FLAG-Pcnt-PACT and the indicated GFP-AKAP6-SR domains were immunoprecipitated with an anti-GFP antibody and analyzed by western blotting with antibodies against FLAG and GFP; as indicated. The experiment was performed three times (n = 3); shown is a representative image. (F) Yeast-two-hybrid assay. Interactions were tested by monitoring the growth of yeast cells expressing AKAP6-SR1 fused to the DNA binding domain of GAL4 (BD) and Pcnt-PACT or AKAP9-PACT fused to the GAL4 activation domain (AD) proteins on DBO agar plates (double dropout; SD /-Leu /- Trp) (upper), or on QDO plates (quadruple dropout; SD /-Ade /- His/-Leu/-Trp) (lower). Growth on QDO plates indicate interaction. The experiment was performed twice (n = 2); shown is a representative image. (G–I) Immunostaining of (H) Pcnt (red) or (I) AKAP9, cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1 and subsequent quantification of Pcnt- or AKAP9-positive cardiomyocyte nuclei in transfected cells (G) Transfected cells are labeled with a yellow arrowhead. Data are represented as individual biological replicates, together with mean ± SD. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. ****: p<0.0001. n = 10. Scale bars: 10 µm.

Figure 2—source data 1. Underlying data for graphs in panel 2G.