tra12132-sup-0001-MovieS1.movapplication/unknown773KMovie S1: The cells were processed as described in Figure 5. The movie shows imaging-induced photodamage of lysosomes. GAK KO cells have unstable lysosomal membranes and therefore suffer the damage earlier than the control MEFs. Number of frames corresponds to the dose of 488 nm light the cells absorbed.
tra12132-sup-0002-FigureS1.docWord document610KFigure S1: GAK depletion causes increase in p21 level resulting in decreased CDK2 activity. A) CDK2 was immunoprecipitated from control and GAK KO MEF lysates. Western blot analysis of the immunoprecipitates showed an increase in the amount of p21 bound to CDK2 in GAK KO cells. IN: lysate prior to IP, FT: post-IP (immunodepleted) supernatant. B) The activity of CDK2 immunoprecipitated from control and GAK KO lysates was analyzed using on-beads in vitro kinase assay with histone H10 and [32P]ATP as substrates showing that CDK2 kinase activity is decreased in GAK KO cells.
tra12132-sup-0003-FigureS2.docWord document1534KFigure S2: Plk4 in GAK KO cells resides on centrosomes in mitogen-dependent manner. AdCre-infected MEF cells grown on coverslips, beginning on day 4 post-infection were cultured for 48 h in medium containing 10% FBS (A), 0.1% FBS (B) or 0.1% FBS supplemented with 100 ng/mL EGF (C). The cells were then stained for Plk4, α-tubulin and γ-tubulin. In GAK KO cells cultured in 10% FBS Plk4 resides at centrosomes. Low FBS concentration (0.1%) causes loss of Plk4 from centrosomes, while supplementing low FBS with EGF restores centrosomal Plk4. Scale bars represent 10 µm in low magnification images and 1 µm in high magnification inserts.

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