Fig 1: The TBC1D31 signalling circuitry controls ciliogenesis AsiRNA transfected HEK293 cells were serum‐deprived and stained for acetylated‐tubulin, TBC1D31 and DRAQ5.BQuantitative analysis of the experiments shown in (A). A mean value ± SD of four independent experiments is shown. Student’s t test *P < 0.05.CHEK293 cells expressing flag‐OFD1 or flag‐S735A were serum‐deprived and subjected to staining analysis for acetylated α‐tubulin, flag and DRAQ5. Images were collected by super‐resolution microscopy (upper panels). Where indicated, flag‐OFD1 or flag‐S735A was co‐transfected with cilia‐APEX‐GFP vector (middle panels). NIH3T3 expressing flag‐OFD1 or flag‐S735A were stained with anti‐ARL13B and anti‐flag antibodies.D, EStatistical analysis of the experiments shown in (C). A mean value ± SD of three independent experiments is shown. For each experimental group, a minimum of 40 cilia were analysed. Student’s t test ***P < 0.001.FHEK293 cells transiently transfected with flag‐OFD1 or flag‐S735A were serum‐deprived, treated with FSK (6 h) and stained for flag, acetylated‐tubulin and DRAQ5.GStatistical analysis of the experiments shown in (F). A mean value ± SD of three independent experiments is shown. Cells analysed for each experiment: 300 for NT, 80 for flag‐OFD1 and 80 for flag‐S735A. Student’s t test, *P < 0.05, **P < 0.01.HQuantitative RT–PCR analysis showing levels of Gli2 mRNA in NIH3T3 fibroblasts transfected with vectors for flag‐OFD1 and flag‐S735A. An empty vector (CMV) was used as control. Serum‐deprived cells were stimulated with the SHH ligand purmorphamine (1 μM) for 24 h. Where indicated, cells were treated with FSK for 6 h before harvesting. The data represent a mean value ± SD of three independent experiments. Student’s t test, P** < 0.01. Source data are available online for this figure.
Fig 2: TBC1D31 binds and targets praja2 to the centrosome ACo‐immunoprecipitation of flag‐praja2 and GFP‐TBC1D31 from lysates of HEK293 cells. The immunoprecipitation (Ip) was performed using an anti‐flag antibody or control IgG.B, CSame as in (A), with the exception that cells expressing flag‐praja2rm or praja2 deletion mutants (praja21–530, praja21–630 and praja2Δ530–630) were included in the analysis.DLysates expressing flag‐praja2 were subjected to pull down assay with GST and GST‐TBC1D31940–970 polypeptides.ECo‐immunoprecipitation of endogenous TBC1D31/praja2/PKAc complex from cell lysates.FStaining of HEK293 cells with anti‐TBC1D31, anti‐γ‐tubulin and anti‐praja2 antibodies. Nuclei were stained with DRAQ5 (blue). Where indicated, cells were transfected with GFP‐TBC1D31. Arrows indicate the pool of praja2 colocalizing with TBC1D31 staining at the centrosome.GCells transfected with control siRNA (siCNT) or siRNA targeting TBC1D31 (siTBC1D31) were stained for praja2, anti‐γ‐tubulin and DRAQ5.HSchematic picture of TBC1D31/praja2/PKA complex. Source data are available online for this figure.
Fig 3: Modelling TBC1D31/praja2 binding in vitro ALysates from HEK293 cells expressing GFP‐TBC1D31 were subjected to pull down assay with GST and GST‐praja2531–631 polypeptides.BMST signal (normalized fluorescence) of P1 (red curve), P2 (green curve) and P3 (cyan curve) plotted against TBC1D31, at increasing concentrations of peptides. The threading modelled structure of the overlapping binding segment of praja2 (praja2550–570) is shown.CCo‐immunoprecipitation of GFP‐TBC1D31 and flag‐praja2 ring mutant (flag‐praja2rm) or praja2Δ550–570.DThreading modelled structure of TBC1D31, with a zoom of its C‐terminus. Mutated residues are highlighted in stick coloured by atom type.EMD derived binding mode of praja2530–570 (green cartoon) to the C‐terminal region of TBC1D31 (red cartoon).FMST signal of P1 plotted against increasing concentrations of TBC1D31 peptides: wild‐type (red curve), R948A‐R951A (violet curve) and R957‐R959D‐H960A (orange curve). Source data are available online for this figure.
Fig 4: Centrosomal localization of praja2, TBC1D31, OFD1 and PKAc AHEK293 cells transfected with GFP‐TBC1D31 were fixed and immunostained with anti‐OFD1 and anti‐γ‐tubulin antibodies.BHEK293 cells transfected with GFP‐TBC1D31 were fixed and immunostained with anti‐praja2 and anti‐γ‐tubulin antibodies.CHEK293 cells transfected with GFP‐TBC1D31 were fixed and immunostained with anti‐OFD1 and anti‐PKAc antibodies.
Fig 5: Localization of praja2 and TBC1D31 at centrosome AHEK293 cells were fixed and stained with anti‐praja2 and anti‐γ‐tubulin antibodies. Nuclei were stained with DRAQ5.BHEK293 cells transiently transfected with control siRNA or siRNA targeting endogenous praja2 were stained with anti‐praja2 antibody and DRAQ5.CImmunoblot analysis of TBC1D31 and Hsp90 in siRNA‐silenced cells.DCells were transiently transfected with control siRNA or siRNA targeting endogenous TBC1D31. Total RNA was extracted and analysed by quantitative RT–PCR.ECells transiently transfected with control siRNA or siRNA targeting endogenous praja2 were stained for TBC1D31, γ‐tubulin and DRAQ5.FImmunoblot analysis of praja2 and Hsp90 in siRNA‐silenced cells.
Supplier Page from Abcam for Anti-WDR67 antibody