Fig. 3. Western analysis reveals CrPRF in cytoplasmic and flagellar compartments. (A,B) Coomassie Blue staining (A) and anti-CrPRF immunoblot (B) comparing cytoplasmic and flagellar fractions. (A) Each lane contained 20 µg protein from cell body extract (C.B.ext), isolated flagella from wild-type cells (WTfla), isolated axonemes from wild-type flagella (WTaxo) and isolated flagella from pf14 cells (pf14fla). Molecular weight standard positions are shown on the left. (B) Lanes 3-6 are the corresponding western blots using the affinity-purified CrPRF antibody for detection. Lanes 1 and 2 were loaded with 5 ng and 1 ng purified, recombinant CrPRF. Notably, isolated flagellae contained a significant fraction of profilin (lanes 4 and 6). By contrast, axonemes contained very little profilin (lane 5). (C) Western blot comparing CrPRF in flagellar fractions. Notably, little CrPRF was found in the axoneme (Axo; lane 2). By contrast, nearly all of the flagellar CrPRF was detergent soluble and found in the membrane-matrix fraction (Memb./Matr.; lane 3).