Figure S2. Defining the interfaces... | Rockefeller University Press

Figure S2.

$Defining the interfaces between Myo2 and Pea2. (A) Upper panel: Ponceau staining of GST (lane 1) or GST fusions to CBD (lane 2) and CBDY1415R (lane 3) used for pull-downs shown in Fig. 1 D. Equal fractions of the elutions from Glutathione Sepharose beads were separated by SDS PAGE and transferred onto nitrocellulose. Lower panel: Anti-myc staining of unbound Pea2-9myc of the pull-down shown in Fig. 1 D. Loading as in upper panel. (B) Upper panel: Anti-His Western blot of the unbound 6His-Pea2221–420 (lane 1), 6His-Pea2221–350 (lane 2), and 6His-Pea2351–420 (lane 3) used in the pull-down of Fig. 1 F. Shown are the supernatants after incubation with immobilized GST-CBD. Lower panels: Ponceau staining of GST-CBD and GST used for the pull-downs shown in Fig. 1 F after elution from beads, SDS-PAGE, and transfer onto nitrocellulose. (C) Extended split-Ub assay of Fig. 1 G with centromeric plasmids containing indicated Nub-Pea2 fragments in haploid yeast cells expressing CBD, Pea2, Spa21–535, and Bud6CRU. (D) Extended split-Ub assay of Fig. 2 B with indicated CBDCRU mutants tested against Nub-Pea2, -Sec4, -Ypt11, -Ypt31, -Ypt32, and -Vac17.$

Defining the interfaces between Myo2 and Pea2. (A) Upper panel: Ponceau staining of GST (lane 1) or GST fusions to CBD (lane 2) and CBD_Y1415R (lane 3) used for pull-downs shown in Fig. 1 D. Equal fractions of the elutions from Glutathione Sepharose beads were separated by SDS PAGE and transferred onto nitrocellulose. Lower panel: Anti-myc staining of unbound Pea2-9myc of the pull-down shown in Fig. 1 D. Loading as in upper panel. (B) Upper panel: Anti-His Western blot of the unbound 6His-Pea2_221–420 (lane 1), 6His-Pea2_221–350 (lane 2), and 6His-Pea2_351–420 (lane 3) used in the pull-down of Fig. 1 F. Shown are the supernatants after incubation with immobilized GST-CBD. Lower panels: Ponceau staining of GST-CBD and GST used for the pull-downs shown in Fig. 1 F after elution from beads, SDS-PAGE, and transfer onto nitrocellulose. (C) Extended split-Ub assay of Fig. 1 G with centromeric plasmids containing indicated N_ub-Pea2 fragments in haploid yeast cells expressing CBD, Pea2, Spa2_1–535, and Bud6CRU. (D) Extended split-Ub assay of Fig. 2 B with indicated CBDCRU mutants tested against N_ub-Pea2, -Sec4, -Ypt11, -Ypt31, -Ypt32, and -Vac17.