Fig. 5

PAK5 interacts and phosphorylates p65. a MDA-MB-231 cells were transfected with pcDNA3.1-Myc-control plasmids and pcDNA3.1-Myc-PAK5 plasmids. Lysates were immunoprecipitated with anti-p65 antibody and immunoblotted with anti-Myc and anti-p65 antibody. b MDA-MB-231 cells were transfected with pcDNA3.1-Myc-control plasmids, pcDNA3.1-p65 plasmids and pcDNA3.1-Myc-PAK5 plasmids. Lysates were immunoprecipitated with anti-p65 antibody and immunoblotted with anti-Myc, and anti-p65 antibody. c MDA-MB-231 cells transfected with pcDNA3.1-Myc-control plasmids and pcDNA3.1-Myc-PAK5 plasmids. Then concentrated protein was used for WB. The top p-p65 represents the mobility shift of phosphorylated p65 proteins in SDS-PAGE with polyacrylamide-bound Mn²⁺-Phos-tag. The bottom p65 represents non-phosphorylated counterpart in SDS-PAGE with polyacrylamide-bound Mn²⁺-Phos-tag. d, e Luciferase reporter assay was carried out in BT549 and MDA-MB-231 cells. The relative luciferase activities of pcDNA3.1 + pGL3-basic, pcDNA3.1-p65 + pGL3-basic and pcDNA3.1-p65 + pGL3-Cyclin D1 groups normalized against pcDNA3.1 + pGL3-Cyclin D1 group. Data are shown as mean ± SD for three independent experiments. **, P < 0.01