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. 2018 Dec 11;20(1):e46224. doi: 10.15252/embr.201846224

Figure 6. TIAR retains CDK1 in GMGs and attenuates CDK1 activity.

Figure 6

  1. HeLa cells were treated with 0.4 μM APH for 24 h, fixed, and processed for IF microscopy after staining with anti‐TIAR and anti‐CDK1 antibodies. Intensity profiles along the white line in the merged image are presented on the right side; yellow arrows mark GMGs, red arrows represent CDK1 that does not colocalize with GMGs.
  2. IF microscopy analysis as in (A) using anti‐Cyclin B antibody.
  3. HeLa cells transfected with control or TIAR siRNAs were treated with 0.4 μM APH 24 h prior to fixation. Cells were processed for IF microscopy after staining with anti‐PRP19 and anti‐CDK1 antibodies. Intensity profiles from staining of the same cells with anti‐TIAR and anti‐CDK1 antibodies, shown in Fig EV5D, are depicted on the right side.
  4. CDK1 was immunoprecipitated from HeLa cells transfected with control or TIAR siRNAs, tested for in vitro kinase activity using recombinant histone H1 as substrate, and visualized by Western blot analysis and autoradiography. The mean CDK1 activity ± SEM was quantified from n = 5 experiments.
  5. CDK2 activity was analyzed following immunoprecipitation as in (D) (mean ± SEM, n = 3).
  6. HeLa cells were transfected with control or TIAR siRNAs for 72 h before measuring expression levels of p(S22)‐Lamin A by Western blot analysis (mean ± SEM, n = 3).
  7. p(S22)‐Lamin A levels were analyzed by HTM in siRNA‐transfected HeLa cells (n = 3; 1,000 cells examined per experiment and condition).
Data information: In (D–F), statistical significance was determined by unpaired Student's t‐test. In (G), statistical significance was determined by Wilcoxon rank‐sum test; *P < 0.05; ***P < 0.001.