|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 5. Physical interaction of VRK1 with CREB in a cell-cycle-regulated manner. (A) Protein extracts from HeLa cells transfected with pFlag-VRK1 were analyzed by immunoprecipitation with an anti-Flag antibody. Coprecipitated CREB proteins were detected by immunoblotting using the anti-CREB antibody. Mouse immunoglobulin-G1 (mIg-G1) was used as an immunoprecipitation control. (B) Total cell lysates from HeLa cells were incubated with the GST-CREB protein and pulled down with glutathione-conjugated beads. Bound proteins were evaluated by immunoblotting using anti-VRK isoform-specific antibodies. (C) Recombinant full-length or fragments of CREB (F1-F4) were used for binding analyses with VRK1. Bound VRK1 protein was detected by immunoblotting using the anti-VRK1 antibody. Recombinant proteins were detected using the anti-GST antibody. bZIP, basic leucine zipper; CAD, constitutive activation domain; KID, kinase-inducible domain; Q, glutamine-rich domain. (D,E) The cell cycle was synchronized in S phase using a double thymidine block (D) or in M phase by treatment with nocodazole (E), and then released at the indicated time by removing the cell cycle inhibitors. Expression of VRK1 and CREB was assessed by immunoblotting and interactions were investigated by immunoprecipitation. P-H3 S10 (phosphorylated histone 3) was used as a mitotic marker.
|
