Journal: The Journal of Cell Biology
Article Title: Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus
Figure Lengend Snippet: The FAP76 N-terminus is located at the interface between C1c and C1e projections. (A) Immunofluorescence light microscopy images of axonemes isolated from fap76-1 (left) and fap76-1;BCCP::FAP76 rescue (right) probed by anti-acetylated-tubulin antibody (red) and fluorescently tagged streptavidin (green). The streptavidin signal indicates that the BCCP-tagged FAP76 was assembled into the axoneme of the rescued strain. (B) SDS–polyacrylamide gel stained by a silver enhancement kit (top) to show that a specific band of appropriate relative mobility could be detected in the fap76-1;BCCP::FAP76 axonemes treated with streptavidin-Au, but not in the control (–Au). Coomassie brilliant blue (CBB) staining (bottom) shows the tubulin bands as loading controls. (C) A classification analysis of the C1c psu1 (black arrows in E and F) in WT and fap76-1 , and fap76-1;BCCP::FAP76 rescue axonemes (white arrows in H, I, K, and L). The particle numbers (n) included in the averages for WT, fap76-1 , fap76-1;BCCP::FAP76 (+Au), and fap76-1;BCCP::FAP76 (–Au) were 664, 927, 1,089, and 935 (see Table S3). (D–L) Comparison of tomographic slices (D, E, G, H, J, and K) and isosurface renderings (F, I, and L) of the averaged CA repeats of WT (D–F) versus fap76-1;BCCP::FAP76 rescue strain either without (G–I) or treated with (J–L) streptavidin gold, viewed in cross-sectional (D, G, and J) and longitudinal (E, F, H, I, K, and L) orientations. The additional density of the streptavidin-gold label at the interface between the C1c and C1e projections in the gold-treated rescue strain (blue arrowheads in J–L) is not observed in WT or control CA (white arrowheads in D–I). Thin blue line in D indicates the location for the tomographic slices shown in E, H, and K. Scale bar in A, 5 µm (valid for all fluorescence images); K, 20 nm (valid for all EM images in D–L).
Article Snippet: On the next day, the slides were washed four times over 1 h with blocking buffer, and then treated for 1 h with blocking buffer containing the secondary antibody (F(ab′)2-goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 568, Invitrogen, A11019, 1:1,000) and Streptavidin (Alexa Fluor 488, Invitrogen, A32354, 1:200).
Techniques: Immunofluorescence, Light Microscopy, Isolation, Staining, Fluorescence