Figure 1.

Mobility shift of KLF8 is due to phosphorylation in its N-terminal 50 amino acid residues. A. The N-terminal region of 50 residues is required for maintaining the doublet of KLF8 protein on SDS-PAGE gels. HA-KLF8, HA-KLF8-dN50, Myc-KLF8 or Myc-KLF8-dN50 was transfected into HEK293 cells. Whole cell lysates were collected after 48 hours and processed for western blotting with anti-HA (1:3000) or anti-Myc (1:2000) antibody with anti-β-actin as loading control. B. Treatment with alkaline phosphatase abolished the upper band of the KLF8 doublet. HA-KLF8 or Myc-KLF8 was oeverexpressed in HEK293 cells. Lysates prepared from the cells were treated with CIP without or with its inhibitor sodium orthovanadate (Na₃VO₄) as described in the Experimental Procedures and processed for western blotting as described above.