Analysis of Flagellar Proteins by Western Blot

Flagellar proteins were separated by SDS-PAGE and transferred to PVDF membrane (Immobilon; Millipore) using standard protocols. The following primary antibodies were used: mouse anti-acetylated tubulin (clone 6-11B-1; 1:10,000; Sigma-Aldrich), mouse anti–α-tubulin (clone B-5-1-2; 1:10,000; Sigma-Aldrich), mouse anti-IC2 (1:50; King and Witman, 1990 (link)), mouse anti-IFT81 (1:200; Cole et al., 1998 (link)), mouse anti-IFT139 (1:50; Cole et al., 1998 (link)), and mouse anti-IFT172 (1:50; Cole et al., 1998 (link)), mouse GT335 (anti-glutamylated tubulin; 1:2,000; AdipoGen), rabbit anti–β-tubulin (1:2,000; Silflow and Rosenbaum, 1981 (link)), rabbit anti-FAP12 (1:1,000; provided by D. Cole, University of Idaho, Moscow, ID), rabbit anti-GFP (1:500; Invitrogen), and rabbit anti-NAB1 (1:5,000; Agrisera). Western blots were developed using anti–mouse or anti–rabbit secondary antibodies conjugated to horseradish peroxidase (Molecular Probes) and chemiluminescence substrate (SuperSignal West Dura; Thermo Fisher Scientific). A ChemiDoc MP imaging system was used for imaging and Image Lab (both Bio-Rad Laboratories) was used for signal quantification.