cep290 mutant flagella have an abnormal protein composition. (A) Western blots of isolated flagella show an accumulation of IFT complex B proteins (IFT20, IFT46, and IFT81) and BBS4, and a reduction in the IFT complex A protein IFT139 and polycystin-2 (PKD2). The arrowhead marks an anti–FMG-1-immunoreactive band that is absent in the mutant flagella. (B) Silver-stained gel of isolated wild-type (WT) and mutant (cep290) flagella demonstrating that some proteins are abnormally present (arrows) and others are missing or reduced (arrowheads) in the mutant flagella. Proteins identified by mass spectrometry include (1) flagellar adenylate kinase and FAP5, (2) FAP12, (3) EF-3, and (4) EF-1α1. The positions of standard proteins and their molecular masses in kD are indicated. (C) Immunofluorescence indicates that bulges (arrowheads) in the cep290 mutant flagella contain IFT proteins. (D) DIC imaging and kymographs of IFT in living cells. (D, a and b) Still images of the videos (Videos 5 and 6) used to create the kymographs (a′ and b′). Bars, 1 µm. (E) Velocities and frequencies of IFT in wild-type and cep290 mutant flagella. Data are represented as mean ± SD (error bars); P-values were determined using a Student’s t test.