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. 2019 Dec 20;10:5828. doi: 10.1038/s41467-019-13782-2

Fig. 6. Tension increase is translated into an inhibitory phosphorylation in the FBP17 F-BAR domain.

Fig. 6

a, b c-Abl inhibits FBP17 tubulation activity. c-Abl or empty vector were expressed with GFP-FBP17 in HeLa cells and c-Abl was stained and examined with confocal microscopy. N = 64 (−) and n = 47 (+) biologically independent cells, representative of 3 independent experiments. Statistical analysis with a two-tailed unpaired t test. ***P < 0.005. Scale bar 10 μm, inset 5 μm. c c-Abl expression does not affect the expression level of GFP-FBP17. Immunoblot showing the levels of GFP-FBP17 and c-Abl and loading control. The relative amount of GFP-FBP17 in each condition is indicated in arbitrary units. d, e GFP-FBP17 and GFP-FBP17 3YF were expressed in HeLa cells and the amount of phosphorylated FBP17 in response to osmotic swelling was determined. N = 3 biologically independent samples from 3 independent experiments. Statistical analysis with a two-tailed unpaired t test. **P < 0.01; ***P < 0.005. f, g GFP-FBP17 and GFP-FBP17 3YE were expressed in HeLa cells and the amount of tubules per cell was calculated. N = 18 and n = 24 biologically independent cells, representative of 3 independent experiments. Statistical analysis with a two-tailed unpaired t test. ***P < 0.005. Scale bar 10 μm, inset 5 μm. h, i Pure GST-FBP17, GST-FBP17 3YE, and control GST were incubated with liposomes and the amount of tubulated liposomes were scored. N = 3 biologically independent samples, from 3 independent experiments. Statistical analysis with a two-tailed unpaired t test. ***P < 0.005. Scale bar 2.5 μm. j, k FBP17 3YE is unable to interact with FBP17 wild type. GFP-tagged FBP17 wild type of 3YE mutant were expressed in cells together with c-Myc-FBP17. GFP versions were immunoprecipitated and their binding to wild-type c-Myc-FBP17 was quantified. Numbers in the right margin indicate kDa. N = 7 and n = 2 biologically independent samples from the corresponding independent experiments. Statistical analysis with a two-tailed unpaired t test. ***P < 0.005. l, m HeLa cells were transfected with GFP-FBP17 or GFP-FBP17 3YF and exposed to osmotic swelling (60 mOsm) for 10 min. The amount of FBP17-labeled tubules was scored. N = 6 (l) and n = 8 (m) biologically independent cells from the corresponding independent experiments. Statistical analysis with a two-tailed unpaired t test. *P < 0.05; ns non-significant. Data represent mean ± S.E.M.