Figure 7.
Panel A shows fluorescence microscopy images of TGN46, phosphorylated Serine 294, Enhanced Green Fluorescent Protein-G-PKDrep, and merged phosphorylated Serine 294 plus Enhanced Green Fluorescent Protein-G-PKDrep in cells cultured on Fibronectin for 1 hour, 4 hours, and 24 hours, and on Poly-L-lysine for 1 hour, 4 hours, and 24 hours. Panel B shows a superplot of Golgi Protein Kinase D activity (phosphorylated Serine 294/Enhanced Green Fluorescent Protein intensity), with the x-axis labeled Fibronectin and Poly-L-lysine at 1 hour, 4 hours, and 24 hours, and the y-axis labeled Golgi Protein Kinase D activity (phosphorylated Serine 294/Enhanced Green Fluorescent Protein intensity).
Mechanical cues modulate PKD activity at the Golgi membranes. (A) Representative confocal microscopy images of HeLa cells stably expressing the EGFP-tagged Golgi-localized PKD activity reporter (EGFP-G-PKDrep), seeded on FN or PLL for the indicated times, fixed, and immunostained for TGN46 and phosphorylated PKD substrate motif Ser294 (pS294). Scale bar, 10 µm. (B) SuperPlot (N∼10 cells per biological replicate; n = 3 biological replicates) of the quantification of PKD activity, measured as the ratio between pS294 intensity and total EGFP-G-PKD signal at the Golgi. A repeated-measures two-way ANOVA test was performed. P values using Tukey’s post hoc multiple comparison test are reported.