Figure 2.

Nek7 suppression disrupts RGS2 recruitment to the spindle. (A) and (B) In vivo endogenous localization of RGS2 and Nek7 in HeLa cells during mitosis. Endogenous proteins were detected with specific primary antibodies, revealed with Alexa Fluor® 488 or Alexa Fluor® 546 secondary antibodies, and visualized by confocal fluorescence microscopy using Operetta High Content Imaging System (Perkin Elmer). The protein pericentrin was used to stain the centrosome and α-tubulin the cytoskeleton. The nucleus was stained by Hoechst. At least 20 cells were analyzed in each mitotic stage from 3 independent experiments and all cells showed the localization pattern represented in the images. The magnitude is indicated in the figures scale bars. White arrow denotes spindle pole (centrosome), arrowhead indicates mitotic spindle and red arrow indicates astral MTs. (C) Western Blotting (WB) analysis of lysates from HeLa cells treated with control shRNA or Nek7 shRNA, demonstrates the decreased levels of Nek7.WB of 6xHis-Nek7FL recombinant protein shows the antibodies specificity. GAPDH was used as a protein loading control. (D) Immunofluorescence images show loss of RGS2 at the spindle of Nek7-depleted cells. Bottom, graphs show the phenotype quantification. n = 35 counted cells/ triplicated experiments. *P < 0.05.