Supplementary Figure 7: Analysis of protein and transcript levels in DZIP1L-mutant cells.

a,b) Staining with a C-terminal DZIP1L antibody (Sigma; green) reveals no signal in Dzip1l^wpy/wpy MEFs (arrows in b), consistent with the truncated nature of the protein; (c,d,) Staining with an antibody raised to the entire DZIP1L protein (Abnova; green) reveals a residual level of truncated protein in Dzip1l^wpy/wpy MEFs (arrows in d). The cilium is marked with acetylated-α-tubulin and γ-tubulin in (a,b; magenta) and IFT88 in (c,d; magenta). (e) Based on an unpaired two-tailed Student’s t-test, qRT-PCR revealed significantly reduced but not absent Dzip1l transcript levels in Dzip1l^wpy/wpy MEFs relative to Dzip1l^+/+ wild-type MEFs, t(6)=5.905, **p=0.001 (n=4, consisting of 3 cell lines derived from independent embryos, analysed across two experiments (separate RNA preparations from one cell line analysed in both experiments); each data point is the average of 3 separate culture wells). Dzip1 levels were not altered, t(6)=0.385, p=0.7135, ns, not significant, to compensate for reduced Dzip1l. Error bars, s.e.m. (f) DZIP1L (magenta) localizes at the transition zone in control human dermal fibroblasts, but is not detectable in DZIP1L mutant dermal fibroblasts from individual B155 (p.Gln155*) with the C-terminal Sigma antibody (arrows in g). Ciliary axonemes and basal bodies were labelled with anti-acetylated tubulin and γ-tubulin antibodies, respectively (green). (h) Immunoblotting with anti-DZIP1L antibody (Abnova) revealed a band corresponding to full length DZIP1L in control cell lysate (arrow) but not in lysate from DZIP1L mutant human fibroblasts. Scale bars in a-d = 2.5μm; g,h = 1μm.