Article Title: Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function
Figure Lengend Snippet: (A) Immunofluorescence staining of HA (green) in WDR60 KO cell lines stably expressing HA-tagged WT and mutant WDR60 constructs. (B and C) IFT88 or IFT140 staining (green) with AcTub labeling (red) of the stable cell lines shown in A as well as WT cells. (Bi) Total intensity quantification of IFT88 labeling across the length of primary cilia in each cell line (n=3; 97 WT, 125 WDR60 KO, 202 WDR60 KO+ HA-WDR60, 119 WDR60 KO+ HA-WDR60 [T749M], 150 WDR60 KO+ HA-WDR60 [Q631*] cells quantified). (Ci) Total intensity quantification of IFT140 labeling across the length of primary cilia in each cell line (n=3; 168 WT, 164 WDR60 KO, 63 WDR60 KO+ HA-WDR60, 56 WDR60 KO+ HA-WDR60 [T749M], 71 WDR60 KO+ HA-WDR60 [Q631*] cells quantified). (D and E) WDR34 KO#1 cells stably expressing mGFP-tagged WT and mutant WDR34. (D) Primary cilia staining with Arl13b (red) and AcTub (blue). (Di) Percentage of ciliated cells (n=3; 357 WT, 430 WDR34 KO, 399 WDR34 KO + mGFP-WDR34, 383 WDR34 KO + mGFP-WDR34 [A22V], 432 WDR34 KO + mGFP-WDR34[C148F] cells quantified). (E) IFT88 staining in WT, WDR34 KO#1 cells, and WDR34 KO#1 cells expressing GFP-tagged WT and mutant WDR34. One-way ANOVA followed by Kruskal-Wallis test was used p-value: ****=<0.0001. Arrows point to the ciliary base. (G-H) Immunoprecipitation of HA-tagged GFP, WT, WDR60 [T749M] and HA-WDR60 [Q631*] mutant proteins followed by immunoblot for (F) HA, NudCD3, and GAPDH; (G) WDR34, LIC3, and TCTEX1and (H) IFT140, IFT88, and IFT57.
Article Snippet: The antibodies used, and their dilutions for western blotting (WB) and immunofluorescence (IF) are as follows: Acetylated tubulin (Sigma (Poole, UK) T6793 1:2000 for IF), rabbit anti-HA (Cell Signaling Technologies (New England Biolabs, Hitchin, UK) 1:2000 WB, 1:1000 IF), rabbit IFT88 (Proteintech (Manchester, UK) 13967-1-AP, 1:200 WB, 1:300 IF), rabbit anti-IFT140 (Proteintech 17460-1-AP, 1:200, WB 1:100 IF), rabbit anti-IFT57 (Proteintech 11083-1,-AP 1:200, WB 1:100 IF), rabbit anti-IFT43 (Proteintech 24338-1-AP, 1:50 IF), anti-IFT20 (Proteintech 13615-1-AP, 1:200 IF), anti-TMEM67 (proteintech13975-1-AP, 1:50 IF), anti-RPGRIP1L (Proteintech 55160-1-AP, 1:100 IF), anti-DYNC2HC1 (Proteintech 55473-1-AP, 1:100 IF), rabbit anti-LIC3 (Proteintech 15949-1-AP, 1:250 WB, 1:100 IF), rabbit anti-TCTEX1 (Santa Cruz Biotechnology (from Insight Biotechnology, Wembley, UK) sc-28537, 1:200 WB, 1:100 IF), rabbit anti-Arl13B (Proteintech 17711-1AP, 1:1000 IF), rabbit anti-TCTN1 (Proteintech 15004-1-AP, 1:100 IF), rabbit anti-Smo (Abcam (Cambridge, UK) ab38686, 1:100 IF), Sheep anti-Myc (( ) kindly provided by Harry Mellor, University of Bristol), rabbit anti-WDR60 (Novus Biologicals (from Bio-Techne Abingdon, UK) NBP1-90437 1:300 WB in ), rabbit anti-WDR60 (Sigma HPA021316, 1:300 WB in Fig. S2 and Fig.S5), rabbit anti-WDR34 (Novus NBP188805, 1:300 WB), mouse anti-GAPDH (Abcam ab9484, 1:1000 WB), p150 glued (BD 6127009, 1:1000 WB), LIS1 (Bethyl A300-409A, 1:1000 WB), dic74 (MAB1618 Millipore (Watford, UK), 1:1000 WB), NUDCD3 (Sigma HPA019136, 1:350 WB).
Techniques: Immunofluorescence, Staining, Stable Transfection, Expressing, Mutagenesis, Construct, Labeling, Immunoprecipitation, Western Blot