International Journal of Cell Biology / 2012 / Article / Fig 4

Research Article

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

Figure 4

APF induces serine phosphorylation of CKAP4. (a) APF treatment induces a significant increase in serine phosphorylation of CKAP4 as demonstrated by immunoprecipitation of CKAP4 followed immunoblotting to detect phosphoserine. Whole cell lysates (500 μg) from HeLa cells treated with 20 nM APF or serum-starved controls were immunoprecipitated with CKAP4 antibody (Alexis) overnight at 4°C. Samples were then bound to Protein A, washed (4X with RIPA/Empigen buffer), eluted (4X LDS sample buffer; Invitrogen), boiled at 95°C for 5 min and resolved on a 4–12% Bis-Tris gel and transferred to a nitrocellulose membrane under reducing conditions. Western Blot analysis for pSer detected phospho-serine using primary (Invitrogen and secondary antibodies (goat anti-rabbit HRP-labeled antibody; Pierce)) developed with Enhanced Chemiluminescence reagent (Pierce) and exposed to film. The membrane was stripped with Restore Stripping Buffer (Pierce) and reprobed for CKAP4 (Alexis) to normalize the phosphoserine signal to the amount of immunoprecipitated CKAP4. (b) Densitometric analysis of the immunoreactive bands was done using ImageJ, and the ratios of phosphorylated to nonphosphorylated CKAP4 were determined.

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