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. 2020 Jul 17;11:3586. doi: 10.1038/s41467-020-17415-x

Fig. 7. PEAK1 regulates FA turnover by PXN phosphorylation downstream of AXL.

Fig. 7

a PXN phosphorylation is PEAK1 dependent in TNBC cells. Hs578T and MDA-MB-231 cells were transfected with 100 nM siCTRL or siPEAK1 and their lysates were used for anti-PXN immunoprecipitation. Phosphorylation levels of PXN was analyzed via western blotting. b PEAK1 expression increases PXN phosphorylation. Lysates of 293T cells expressing the indicated plasmids were used for anti-GFP immunoprecipitation. Levels of PXN phosphorylation is detected via western blotting. c PEAK1 interacts with CSK in an AXL-dependent manner. Lysates of MDA-MB-231 cells treated or not with 1 μM R428 were used for anti-CSK immunoprecipitation. PEAK1 levels in the IP were analyzed via western blotting. d Lysates of 293T cells transfected with the indicated plasmids were subjected to anti-GFP immunoprecipitation. GFP-PXN phosphorylation levels were detected via western blotting. e Lysates of 293T cells transfected with the indicated plasmids were subjected to anti-GFP immunoprecipitation. GFP-PXN phosphorylation levels were detected via western blotting. f MDA-MB-231 GFP-PXN-expressing cells transfected with 100 nM of siCTRL or siPEAK1 and treated with or without GAS6 were imaged live by spinning disk microscopy for a period of 30 min to assess the dynamics of FA turnover. Magenta arrow heads point toward an FA that was followed for its assembly and disassembly times. Scale bar, 5 μm. g, h AXL modulation of FA turnover is PEAK1 mediated. Quantification of assembly and disassembly time (g) and lifetime (h) of the FAs of GFP-PXN expressing MDA-MB-231 cells. ****P < 0.000001; *P = 0.0196 (n = 3 experiments with 90 FAs followed per condition). Data are represented as boxplots where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value, and the lower whisker extends from the hinge to the smallest value. Two-tailed Mann–Whitney test was used to calculate P value. Source data are provided as a Source data file.