Figure 3.

Quantification and subcellular distribution of CK2 in S‐LAMA84 and R‐LAMA84 cells. (A) The indicated ng of recombinant CK2 holoform (α2β2) and μg of lysate proteins from S‐LAMA84 and R‐LAMA84 cells were analysed by Western blot with anti‐CK2α (left panel) and anti‐CK2β (right panel) antibodies. Anti‐α‐tubulin Western blot is shown as a loading control. Means of densitometric values ± SD, expressed in arbitrary units (a.u.), are reported above the relative subunit bands. Cellular CK2 subunit amounts were calculated by densitometric analysis and extrapolation from the calibration curve built on the signal of recombinant CK2. (B) (Left panel) Cells were disrupted by Dounce homogenization and subcellular fractionation was performed by differential centrifugation as detailed in Materials and methods. Subcellular fractions (N, nuclei; Mt, mitochondria; C, cytosol and Mc, microsomes) were resuspended in an equal volume and the same volume of resulting fractions was immunoblotted with the indicated antibodies including the organelle‐specific antibodies against lamin B (nuclei), lactate dehydrogenase (LDH) (cytosol) and S6 ribosomal protein (microsomes and nucleoli). The Figure is representative of five separate experiments. (Right panel) Bars report the mean values ± SD of the densitometric analysis of the CK2‐subunit bands obtained as in left panel. Densitometric values are expressed in arbitrary units. *p < 0.05. (C) Confocal microscopy of double immunofluorescence staining of R‐LAMA84 cells with anti‐CK2α (red) and anti‐Abl (green) antibodies. Nuclei were stained with Hoechst 33342. Co‐localization of red and green fluorescences is visualized by the yellow fluorescence appearing after merging of both signals. Violet appears from the merging of nuclear staining and red fluorescence.