FIG 5.

PKP2 knockdown impairs EGF-induced responses. (A) Efficiency of PKP2 knockdown using siRNA and shRNA in 293T cells. The mRNA levels of PKP2 were visualized using RT-PCR. To assess the protein levels of PKP2 to test the efficiency of siRNAs and shRNAs, plasmids encoding PKP1 and PKP2 with siRNAs or shRNAs were transfected to 293T cells. At 48 h after transfection, the cell lysates were subjected to Western blot analysis. si-Control, control siRNA; sh-Control, control shRNA. (B) A431 cells were treated with control siRNA or si-PKP2-2 (twice, at 0 and 48 h). At 72 h after the first siRNA transfection, the media were changed to 0.5% FBS-containing DMEM. EGF was added at 98 h after the first siRNA transfection and was harvested at the indicated times. (C) A431 cells were transfected with plasmids encoding control shRNA, shPKP2-2, or shPKP2-4, followed by selection by puromycin. Selected cells were serum starved with 0.5% FBS-containing DMEM for 24 h, and EGF was added and harvested at the indicated times. (D) The ratios of tyrosine 1068-phosphorylated EGFR to total EGFR presented in panel C were quantified. (E) HCT116 cells were transfected with plasmids encoding control shRNA or shPKP2-4, followed by selection with puromycin. Selected cells were serum starved in DMEM with 1% FBS for 24 h, and then EGF was added and harvested at the indicated times. (F) MDA-MB-468 cells were transfected with plasmids encoding control shRNA or shPKP2-4, followed by selection with puromycin. Selected cells were serum starved with 0.5% FBS DMEM for 24 h, and EGF was added and harvested at the indicated times. Cell lysates were immunoprecipitated with anti-EGFR antibody and immunoblotted with antibodies as indicated. (G) A431 cells were transfected with plasmids encoding control shRNA or sh-PKP2-4, followed by selection with puromycin. Selected cells were serum starved with 0.5% FBS DMEM for 24 h, and EGF was added and harvested at the indicated times. Cell lysates were immunoprecipitated with anti-EGFR antibody and immunoblotted with antibodies as indicated. Whole-cell lysates were also immunoblotted as indicated. (H) Expression of PKP2 increased the interactions between EGFR and adopter molecules. Sh-PKP2-4-expressing A431 cells were transfected with plasmids encoding empty PKP2 or increasing amounts of Sh-PKP2-4-resistant PKP2 as indicated. Cell lysates were immunoprecipitated with IgG or anti-EGFR antibody and immunoblotted with antibodies as indicated. Whole-cell lysates were also immunoblotted as indicated. (I) The sh-PKP2-4-expressing A431 cells were transfected with plasmids encoding empty vector (EV) or sh-PKP2-4-resistant PKP2 (PKP2shR). At 24 h after starvation (0.5% FBS-containing DMEM), EGF was added and harvested at the indicated times. (J) Suppression of PKP2 expression reduces the EGFR dimerization rate. The short hairpin control or sh-PKP2-4-expressing A431 cells were transiently transfected with EGFR-GFP and empty vector or EGFR-FLAG as indicated. At 24 h after transfection, cell lysates were immunoprecipitated with FLAG and immunoblotted with antibodies as indicated. Whole-cell lysates were also immunoblotted as indicated. Molecular weights are in thousands.