High K8 levels in the PKP3 knockdown clones are due to a decrease in phosphorylation.

<p><b>A.</b> Protein extracts from the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) were resolved on SDSPAGE gels and Western blots performed with the indicated antibodies. <b>B.</b> S9 cells were transfected with either dsRed or the shRNA resistant dsRed PKP3 3.7R cDNA. 48 hours post transfection cells were stained with antibodies to K8 (green) and visualized by confocal microscopy. Note that dsRed PKP3 3.7R localizes to the border as previously described (indicated by arrow) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038561#pone.0038561-Gosavi1" target="_blank">[28]</a>. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. <b>C.</b> A real time PCR analysis to determine the mRNA levels of K8 and K18 was performed on RNA isolated from the vector control and PKP3 knockdown clones. GAPDH was used as an internal control for normalization. The Ct values for all samples are shown on the Y-axis. <b>D.</b> Protein extracts from the PKP3 knockdown clones or the vector control were subjected to 2-dimensional gel electrophoresis and Western blots performed with antibodies to K8. <b>E.</b> Protein extracts from the vector control cells or the PKP3 knockdown clones were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 or phosphospecific antibodies against S73 (α-S73), S431 (α-S431) and actin. <b>F.</b> HCT116 cells transfected with GFPK8 or GFPS73A or GFP S431A were resolved on two dimensional gels followed by Western blots with antibodies to K8. MW markers are indicated. Protein extracts from the transfected cells were resolved on SDS-PAGE gels followed by Western blotting with antibodies to actin to serve as loading controls.