FIG. 7.

Sucrose velocity gradients of Far3 and Far7 to Far11. (A) Cosedimentation of the Far proteins on sucrose velocity gradients. Lysates from strains containing epitope-tagged Far3 and Far7 to Far11 were separated on 15 to 40% continuous sucrose velocity gradients. Fractions were collected from the top and analyzed by Western blotting to detect each of the tagged Far proteins. Markers of known size were also separated on sucrose gradients. The fraction in which a particular size marker peaked is indicated below the corresponding lane. (B) Coprecipitation of Far3 with Far7 to Far11 in IPs from the sucrose gradient fraction peak. Far3-HA was immunoprecipitated from sucrose gradient fractions in which it was the most abundant; the presence of Far proteins in the IP was detected by Western blotting as described in Materials and Methods. Pregradient lysates and the gradient fractions used for IP were also analyzed by Western blotting to detect epitope-tagged Far proteins or the loading control, Dpm1. Lane letters indicate strain details and experimental conditions, as follows: B, FAR3 〈pSL2771〉 + IP α-HA; C, far3 〈pSL2784〉 no IP α-HA; D, far3 〈pSL2784〉 + IP α-HA. For both panels A and B, antibodies and strains are the same as those used in the original coIP experiments (Fig. 5). The asterisk (∗) next to the Far11-myc lanes in both panels A and B indicates that the 130-kDa cleavage product is shown, as opposed to the 160-kDa full-length protein.