Fig. 7. Substrate drives SCF complex assembly in a Cand1/2- and neddylation-dependent manner.

(A) Steady state assembly of osTir1^9xMyc with ^3xFLAGCul1 in Wt and DKO cells in the absence of auxin. Wt and DKO HEK293^3xFLAG-Cul1 cells stably expressing osTir1^9xMyc and Tet-inducible H2B^AID-YFP were lysed in the presence of rCul1^GSTRbx1 and subjected to IP with anti-Myc. The bound (IP) and unbound (FT) fractions were Western blotted with the indicated antibodies. (B) Degradation of H2B ^AID-YFP substrate is hindered in the absence of Cand1/2. The cell lines from (A) were treated with tetracycline for 24 hrs to activate H2B^AID-YFP expression, treated with 500 µM auxin, and at the indicated time points after auxin addition samples were lysed and analyzed by Western blot. The half-life of H2B^AID-YFP was calculated from 2 biological replicates. (C) Auxin-induced osTir1 ^AID-YFP assembly with Cul1 is rapid. Wt cells from (A) were treated with 500 µM auxin for the indicated time, lysed in the presence of rCul1^GSTRbx1, and subjected to IP followed by Western blot. (D) Auxin-driven SCF^Tir1 assembly peaks in 30’ and depends on Cand1/2. Cells from (A) were treated as described in (C) for the indicated amount of time. Fold change in osTir1^9xMyc association with ^3xFLAGCul1 is displayed below. (E) Inhibition of neddylation blocks substrate driven SCF complex assembly. Same as (D) except cells were treated ± MLN4924 and auxin, as indicated, for 30’ prior to cell lysis. (F) Substrate increases the association of neddylated Cul1 interacting with osTir1^9xMyc. Cells were treated and processed as described above except that auxin treatment was for 30’ or 60’ and IP was for osTir1^9xMyc. Percent neddylated Cul1 (N8-Cul1) is indicated. S.E., L.E.: short and long exposures.