Quantitative Immunoblotting of Ciliary Proteins

SDS-PAGE and immunoblotting were essentially as described previously (Cao et al., 2013 (link)) with slight modifications (Belzile et al., 2013 (link)). Cells, cilia, and ciliary ectosome samples were boiled in 1 × SDS sample buffer for 5 min, and then subjected to SDS-PAGE analysis on 4–20% gradient gels (GenScript, USA). The antibodies used for immunoblotting were anti-HA (1:1000; Roche), anti-α-tubulin (1:3000 or 1:200,000; Sigma), anti-GSP1 (1:20,000; Wilson et al., 1999 (link)), anti-FMG1 (1:100,00), anti-IFT139 (1:50,000), anti-IFT81 (1:1000), and anti-BUG22 (1:500,000). Mouse monoclonal antibody against FMG1 was generously provided by Robert Bloodgood (University of Virginia). Monoclonal antibodies against IFT81 and IFT139 were generously provided by Dennis Deiner and Joel Rosenbaum (Yale University). Rabbit polyclonal antibody against BUG22 was generously provided by Dan Meng and Junmin Pan (Tsinghua University). The amount of SAG1-HA released into the medium during adhesion was determined by use of ImageJ analysis of immunoblots of equal proportions of cells and medium. Determinations were made under conditions in which the amount of sample loaded was linear with the signal obtained. Similar methods were used to determine the relative abundance of FMG1 and SAG1-HA in equal protein amounts of ciliary ectosomes compared to cilia.