a. Domain structure of EHD1 and loss of function mutations.
b. Immunoblot analysis of siRNA resistant (Res) GFP-EHD1 wildtype, -K483E and -W485A proteins stably expressed in RPE cells 72h after transfection with siControl or siEHD1#1. Endogenous and GFP-EHD proteins expression levels were detected using anti-EHD1 antibody. Note that the EHD1 antibody also recognizes endogenous EHD4 as indicated. Un-cropped images of blots are shown in Supplementary Fig 6.
c. Quantification of cilia in cells treated with siEHD1#1 as described in (b) and serum starved the last 24h. Means ± SD are pooled data from 3 independent experiments with n=8 areas imaged (total number of cells from all experiments: ResGFP-EHD1, 293; ResGFP-EHD1 K483E, 269; ResGFP-EHD1 W485A, 263).Two tailed t-test analysis compared with WT.
d. Cell lines described in (b) were serum starved for 24h and stained with Actub antibody to mark the cilia. Scale bar: 2 μm.
e. Representative SIM image of RPE cells expressing the siRNA resistant GFP-K483E mutant transfected with EHD1 siRNA as in (c) and stained with CEP164 antibody. 9 out of 15 cells (60%) imaged expressing the K483E showed DAV-like structures. Arrow marks orthogonal view (bottom panels) and corresponds to fluorescence profile plots. Scale bar: 500nm.
f. Quantification of CP110 localization on the mother and daughter centrioles in RPE cells treated as in (c) and stained as described in Fig 6b. Means ± SD are pooled data from 3 independent experiments with n=6 areas imaged (total number of cells from all experiments: siCtrl, 148; siEHD1#1, 114). Two tailed t-tests compared with siCtrl.
*** P<0.0001. Statistics source data for Fig7 c,f can be found in Supplementary Table 2.