Fig. 3

The PI3K pathway is involved in L-plastin Ser5 phosphorylation through the PI3K/SGK3 axis. a BT-20, SKBR3 or HCC38 cells were treated with Apitolisib (A) or Trametinib (T) or with both inhibitors (A + T), with or without subsequent HGF stimulation. Following treatment, residual L-plastin Ser5 phosphorylation and total L-plastin were determined by immunoblot analysis. The graphs show the ratio between Ser5-P-L-plastin and L-plastin. Three independent experiments were performed for each cell line. Data were scaled to the highest signal obtained (= 1) and results are expressed as means ± SEM. Statistical analysis was performed doing one-way ANOVA, relative to the control (CTRL) condition with or without HGF stimulation, respectively (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). b In vitro kinase assay. A total of 10 μg recombinant full-length L-plastin was incubated with 100 ng recombinant kinase and with 50 μM ATP in a reaction volume of 25 μl. RSK1 was used as a positive control kinase and a negative control (CTRL) was performed by omitting a kinase. L-plastin Ser5 phosphorylation and total L-plastin were determined by immunoblot analysis. c HEK 293 T cells were co-transfected with GFP-fused L-plastinWT and FLAG-tagged SGK3 WT, activated myristoylated SGK3 (Myr SGK3) or the empty vector (Ctrl). Cell extracts were prepared 48 h after transfection and immunoblot analysis was performed to determine L-plastin Ser5 phosphorylation and total L-plastin as well as SGK3 Thr320 phosphorylation and total SGK3