The human phosphatase CDC14A modulates primary cilium length by regulating centrosomal actin nucleation (2018)
Figure 4

Figure 4. The phosphoprotein DBN1 is a substrate for hCDC14A

hCDC14A, not the inactive hCDC14AC278S, dephosphorylates the serine residue 142 of DBN1 in vivo. RPE1 cells expressing GFP, hCDC14A-GFP, and hCDC14AC278S-GFP under TetON promoter were serum starved in presence or absence of Dox for 48 h prior to fixation for immunofluorescence. DBN1pS142 was detected with a phospho-specific antibody. DNA was stained with DAPI. Scale Bar = 10 μm.

Immunoblot analysis of samples from (A). The DBN1pS142/DBN1 ratio was densitometrically measured from the Dox treated samples and represented in the graph next to the immunoblot. In this experiment two GAPDH blots were used to normalize protein levels because DBN1 and DBN1pS142 were analyzed in separated blots. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001.

DBN1S142 was hyper-phosphorylated in hCDC14APD cell lines during ciliogenesis compared to the WT control. In this experiment we sequentially analyzed DBN1pS142 and DBN1 on the same blot. The specificity of the DBN1pS142 antibody was validated by siRNA mediated knock-down of DBN1 in both hCDC14APD cells and GFP/hCDC14A-GFP/hCDC14AC278S-GFP stable cell lines. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001.

In vitro dephosphorylation of DBN1 by purified hCDC14A. DBN1-GFP was transfected into HEK293T cells and expressed for 48 h. DBN1-GFP was immunoprecipitated from cell lysates with GFP binder. The immunoprecipitated phospho-DBN1 was then incubated with and without recombinant hCDC14A in vitro. The DBN1pS142/DBN1 ratio was densitometrically measured. Three independent experiments. Mean ± SD. **** P≤ 0.0001.

hCDC14A, not the inactive hCDC14AC278S, dephosphorylates the serine residue 142 of DBN1 in vivo. RPE1 cells expressing GFP, hCDC14A-GFP, and hCDC14AC278S-GFP under TetON promoter were serum starved in presence or absence of Dox for 48 h prior to fixation for immunofluorescence. DBN1pS142 was detected with a phospho-specific antibody. DNA was stained with DAPI. Scale Bar = 10 μm.

Immunoblot analysis of samples from (A). The DBN1pS142/DBN1 ratio was densitometrically measured from the Dox treated samples and represented in the graph next to the immunoblot. In this experiment two GAPDH blots were used to normalize protein levels because DBN1 and DBN1pS142 were analyzed in separated blots. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001.

DBN1S142 was hyper-phosphorylated in hCDC14APD cell lines during ciliogenesis compared to the WT control. In this experiment we sequentially analyzed DBN1pS142 and DBN1 on the same blot. The specificity of the DBN1pS142 antibody was validated by siRNA mediated knock-down of DBN1 in both hCDC14APD cells and GFP/hCDC14A-GFP/hCDC14AC278S-GFP stable cell lines. Three independent experiments. Mean ± SD. ** P≤ 0.01, *** P≤ 0.001.

In vitro dephosphorylation of DBN1 by purified hCDC14A. DBN1-GFP was transfected into HEK293T cells and expressed for 48 h. DBN1-GFP was immunoprecipitated from cell lysates with GFP binder. The immunoprecipitated phospho-DBN1 was then incubated with and without recombinant hCDC14A in vitro. The DBN1pS142/DBN1 ratio was densitometrically measured. Three independent experiments. Mean ± SD. **** P≤ 0.0001.

Data information: (B, C) One-way ANOVA. (D) Unpaired Student's t-test.