Fig. 3: The WD40 domain mediates the interaction with the inner scaffold protein POC5. | Nature Communications

Fig. 3: The WD40 domain mediates the interaction with the inner scaffold protein POC5.

From: An interaction network of inner centriole proteins organised by POC1A-POC1B heterodimer crosslinks ensures centriolar integrity

Fig. 3

a, b AlphaFold-Multimer predictions of the WD40 domain of the POC1 proteins and POC5 reveal a potential binding site comprising blades 5 and 6 of the β-propeller and to a lesser extend blade 1. The lower panel of (a, b) focuses on blade 1, showing loss of the interaction when blade 1 is either broken in the case of POC1A or blocked due to an additional β-sheet (named Intra) from the linker region of POC1B. Percentages indicate the occurrence of the predicted ensembles. Confidence scores are shown in Supplementary Fig. 6. c Domain organisation of the POC1 proteins and POC5. d Different HA-tagged constructs of POC5 used for immunoprecipitation (IP) experiments. e Representative FLAG IP from HEK293T cells expressing either POC1A-FLAG or POC1B-FLAG together with HA-tagged subdomains of POC5. Vinculin (Vinc) was used as input control. f Quantification of the FLAG IP shown in (e). Due to differences in the expression levels of the FLAG constructs, the signal intensity of the prey band from the IP sample was normalised to the signal intensity of the bait and used as an indication for the binding efficiency between POC5 and the POC1 proteins. Data are presented as mean ± SD. All statistics were derived from two-tail unpaired t-test of N = 3 biologically independent experiments. g Representative FLAG IP from HEK293T cells expressing FLAG-tagged WD40 domain of either POC1A and POC1B or chimeric versions together with HA-tagged full-length POC5. Vinculin is input control. h, i Quantification of the prey/bait ratio of the IP samples shown in (g). Quantifications show the result from one representative experiment out of N = 3 biologically independent experiments. Although the outcomes of the experiments were identical, there was variation between them. j FLAG IP from HEK293T cells expressing FLAG-tagged POC1 proteins with HA-tagged full-length POC5 or POC5Δ472–532 to verify the predicted interacting site. GAPDH is used as input control. N = 2 biologically independent experiments. f, h, i The prey/bait ratio of each experiment and the corresponding immunoblot is provided as a Source Data file.

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