Fig. 2: CFAP45 interacts with axonemal dynein complex components. | Nature Communications

Fig. 2: CFAP45 interacts with axonemal dynein complex components.

From: CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module

Fig. 2

a CFAP45 immunoprecipitates from isolated porcine respiratory cells (PRC) identify 113 unique associations by LC/MS–MS including axonemal dynein complex proteins; CFAP45 was identified in all three independent experiments. b AK8 (red) is detectable in human respiratory cilia by IFM. Ciliary axonemes (green) are detected using anti-acetylated α tubulin (AcTub) antibody. Merge images include Hoechst stain (blue) to indicate nuclei. White scale bar equals 10 µm. n = 18 images over three experiments. Polyclonal anti-DNALI1 antibody immunoprecipitates myc-tagged DNALI1 (c) and FLAG-tagged CFAP45 (d), using monoclonal anti-myc and anti-FLAG antibodies by IB, respectively. Polyclonal anti-CFAP45 antibody immunoprecipitates myc-tagged CFAP45 (e) and FLAG-tagged DNALI1 (f), using monoclonal anti-myc and anti-FLAG antibodies by IB, respectively. Recombinant DNALI1 (orange arrowhead) and CFAP45 (blue arrowhead) are ~37 and 74 kilodaltons, respectively. c–f, n = 1 blot from two experiments for each IP combination. g CFAP45 directly interacts with DNALI1, as indicated by growth on LWH + 3-AT media. CFAP45 also shows binary interactions to components associated with the basal body/centrosome and intraflagellar transport (see also Supplementary Fig. 3 and Supplementary Table 3). Polyclonal anti-CFAP45 antibody immunoprecipitates native DNAH11 (h) and CFAP45 (i) from PRC lysates, using polyclonal anti-DNAH11 and anti-CFAP45 antibodies by IB. For reference, polyclonal anti-DNAH11 antibody immunoprecipitates native DNAH11 (j) but not full-length CFAP45 (k) from PRC lysates. Porcine full-length CFAP45 (blue arrowhead) and DNAH11 (green arrowhead) are ~66 and 517 kilodaltons, respectively. (h′, j′) indicate longer exposure times of (h, j) cropped above gray dashed lines. A DNAH11-immunoreactive band of ~85 kDa (red arrowhead) is detectable in both CFAP45 and DNAH11 immunoprecipitates. Species-matched (rabbit) polyclonal normal IgG is used as control. Marker (M) indicates relative molecular weight (blue numerals) in kilodaltons. h–k, n = 1 blot from two experiments for each IP combination.

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