Fig. 2. Localization of FAP133 to flagella and the peri-basal body region. (A) Purified Chlamydomonas flagella were separated in a 5-15% polyacrylamide gel and stained with Coomassie Blue (left) or blotted to nitrocellulose membrane for immunodetection (right). Rabbit polyclonal antibody (CT248) raised against FAP133 specifically recognized a single band of M_r66,000. (B) Equivalent amounts of flagella matrix proteins obtained by freeze-thaw and extracted flagella were separated in a 5-15% polyacrylamide gel and stained with Coomassie Blue (upper panel) or transferred to nitrocellulose and probed with CT248 to detect FAP133 (lower panel). The majority of FAP133 was found in the flagellar matrix fraction. (C) Membrane and matrix proteins were initially extracted from isolated flagella with detergent (M&M). The resulting axonemes were incubated three times with a buffer that contained 10 mM ATP (1^st, 2^nd and 3^rd ATP respectively). Finally ATP-treated axonemes were extracted with a high-salt buffer (0.6 M NaCl). Equivalent amounts of these fractions and the axonemal remnants (Extr. Axon.), were separated in a 5-15% polyacrylamide gel and stained with Coomassie Blue (lower panel) or transferred to nitrocellulose membrane and probed with antibodies against FAP133, D1bLIC and LC2 (upper panels). (D) Chlamydomonas cells were prepared for indirect immunofluorescence microscopy using the CT248 antibody against FAP133. Images were acquired using differential interference contrast optics (left panels) to show the location of the two flagella and also under fluorescence (right panels) to detect the FAP133-specific signal. FAP133 localized primarily to the peri-basal body region as well as in punctate structures along the flagella. Images of the cell in the bottom panel were acquired while focusing at the basal body region and insets show enlargements of this area. Bar, 10 µm.