Figure 3. FAP54, FAP46, FAP74, and... | Rockefeller University Press

$Figure 3. FAP54, FAP46, FAP74, and FAP221 form a single complex. (A) Western blots of isolated axonemes. FAP74 and FAP221 antibodies recognize proteins of the correct molecular weight and that are missing or reduced from pf18 and pf16, but are present in WT, pf6, and cpc1 axonemes. (B) These proteins are also present in anti-CaM IPs using WT axonemal extracts but not pf18 and pf16 extracts. (C) Silver-stained gel of high calcium anti-FAP74 IPs from WT and pf18 NaCl axonemal extracts. All four proteins coprecipitate from WT extracts. The identities of FAP74 and FAP221 were confirmed by Western blot (not depicted). The identities of FAP54 and FAP46 were confirmed by mass spectrometry. (D) Western blots of WT axonemal extracts fractionated on a 5–20% sucrose gradient. FAP74 and FAP221 cosediment at ∼15S. (E) Silver-stained gel of high calcium anti-FAP74 IPs from pooled sucrose gradient fractions (see F). All four members of the complex coprecipitate from pool B (fractions 7–11), and not from pools A, C, or D. (F) Silver-stained gel of WT axonemal extracts fractionated on a 5–20% sucrose gradient. Odd fractions were pooled together in designated groups A–D and used in high calcium anti-FAP74 IPs shown in E. These data support the conclusion that all four proteins form a single complex.$

Figure 3.

FAP54, FAP46, FAP74, and FAP221 form a single complex. (A) Western blots of isolated axonemes. FAP74 and FAP221 antibodies recognize proteins of the correct molecular weight and that are missing or reduced from pf18 and pf16, but are present in WT, pf6, and cpc1 axonemes. (B) These proteins are also present in anti-CaM IPs using WT axonemal extracts but not pf18 and pf16 extracts. (C) Silver-stained gel of high calcium anti-FAP74 IPs from WT and pf18 NaCl axonemal extracts. All four proteins coprecipitate from WT extracts. The identities of FAP74 and FAP221 were confirmed by Western blot (not depicted). The identities of FAP54 and FAP46 were confirmed by mass spectrometry. (D) Western blots of WT axonemal extracts fractionated on a 5–20% sucrose gradient. FAP74 and FAP221 cosediment at ∼15S. (E) Silver-stained gel of high calcium anti-FAP74 IPs from pooled sucrose gradient fractions (see F). All four members of the complex coprecipitate from pool B (fractions 7–11), and not from pools A, C, or D. (F) Silver-stained gel of WT axonemal extracts fractionated on a 5–20% sucrose gradient. Odd fractions were pooled together in designated groups A–D and used in high calcium anti-FAP74 IPs shown in E. These data support the conclusion that all four proteins form a single complex.

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