International Journal of Cell Biology / 2012 / Article / Fig 2

Research Article

Antiproliferative Factor-Induced Changes in Phosphorylation and Palmitoylation of Cytoskeleton-Associated Protein-4 Regulate Its Nuclear Translocation and DNA Binding

Figure 2

Surface-labeled CKAP4 translocates from the plasma membrane into the nucleus following APF exposure. (a) HeLa cell-surface proteins were labeled with Sulfo NHS-biotin as described in Section 2. Following exposure to 20 nM APF for 24 hours (or no treatment), the cells were harvested and the nuclear protein fraction was isolated (Pierce NE-PER), separated by SDS-PAGE, and transferred to nitrocellulose. The membrane was then probed with streptavidin-HRP (1 : 5000; Pierce) to bind biotinylated proteins, and the signal was detected by ECL (Pierce). Following detection of the biotinylated proteins from the nucleus, the (streptavidin) HRP on the membrane was inactivated by incubating the blot in PBS containing 3% H2O2 and 1% sodium azide. The same membrane was then reprobed with antibodies to CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals) and fibrillarin (a nuclear marker and loading control; Abcam; diluted 1 : 1000). (b) HeLa cells were treated with APF (20 nM) for 24 hours, which resulted in a significant increase in the abundance of CKAP4 in the nucleus compared to control samples. Treated cells were harvested and the nuclear and cytosolic fractions were isolated and separated by SDS-PAGE as described in Section 2. Protein expression was analyzed by Western Blotting with antibodies for β-tubulin (diluted 1 : 1000, Abcam; loading control for the nonnuclear fraction), CKAP4 (“anti-CLIMP-63”, diluted 1 : 1000, Alexis Biochemicals), and fibrillarin (diluted 1 : 1000, Abcam; loading control and specific marker for the nuclear fraction), and then with an HRP-conjugated anti-mouse secondary antibody (1 : 20000; ThermoFisher Scientific). The proteins were detected by ECL (Pierce) with multiple exposures to film. The integrated density of the bands on the film was measured using ImageJ. Exposure times were controlled to ensure that the signals on film were not saturated. (c) The nuclear/cytosolic ratio represents the relative distribution of CKAP4 in the nuclear versus cytosolic fractions extracted from cells treated with or without APF. CKAP4 abundance in the APF-treated and control samples were normalized for loading to β-tubulin for the nonnuclear fractions and to fibrillarin for the nuclear fractions. The nuclear/cytosolic ratio for CKAP4 in the APF and control samples was determined from these normalized values. The standard deviation describes the variability among the normalized, nuclear, and cytosolic ratios from three independent experiments. A two tailed, paired -test of the two data arrays (plus APF and control) indicate that the difference between these ratios is significant ( ; ). Cells treated with APF stop dividing, so the 10 cm dishes containing control and APF treated cells contained fewer cells (and protein) at the end of the experiment, normalizing the CKAP4 signals to loading controls corrected for this disparity. Fibrillarin is a well-characterized nuclear marker that is also known to localize to nucleoli. The data shown are representative of four independent experiments.

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