Skip to main content
. Author manuscript; available in PMC: 2019 Mar 1.
Published in final edited form as: Mol Cell. 2018 Mar 1;69(5):773–786.e6. doi: 10.1016/j.molcel.2018.01.038

Figure 5. Experimental concordance with mathematical predictions.

Figure 5

(A) 3xFLAGCul1 overexpression rescues the IκBα degradation defect of DKO cells. IκBα levels were monitored by western blot (WB) at indicated time points after TNFα treatment. Overexpression of 3xFLAGCul1 was induced by tetracycline. Average relative t1/2 of IκBα are shown in the graph. Error bars: range of values, n = 2.

(B) 3xFLAGCul1 overexpression impedes cullin neddylation. WB analysis of cullins in cell lysates from (A). Fold increase in total Cul1 levels and percent neddylation of overexpressed 3xFLAGCul1 and endogenous Cul4a are indicated. A representative result of two replicates is shown.

(C) β-TrCP overexpression does not rescue the IκBα degradation defect of DKO cells. IκBα levels were monitored by western blot (WB) at indicated time points after TNFα treatment. Overexpression of β-TrCP was induced by tetracycline. Average relative t1/2 of IκBα are shown in the graph. Error bars: range of values, n = 2. (See Fig S5E for WB of β-TrCP)

(D–E) Overexpression of Cul1 significantly depletes free β-TrCP in the DKO cells. As illustrated in (D), cells with/without tetracycline induced 3xFLAGCul1 were lysed in buffer containing Cul1•GSTRbx1 sponge protein and subjected to GST pulldown, which probes changes in levels of unbound cellular proteins capable of binding to sponge in cell lysate (see Fig S5F for WB images). Average changes in protein levels compared to non-tetracycline induced control are shown in the graph. Overexpression of 3xFLAGCul1 depleted the pool of free β-TrCP in DKO cells by 80%. Error bars: range of values, n = 2.

(F–G) Overproduction of β-TrCP modestly reduces the efficiency of its assembly with Cul1. As illustrated in (F), cells containing endogenous 3xFLAGCul1 were lysed in buffer containing Cul1•GSTRbx1 sponge protein. β-TrCP bound to endogenous 3xFLAGCul1 was probed by anti-FLAG beads, and free cellular β-TrCP capable of binding to sponge in cell lysate was probed by GST beads (see Fig S5G for WB images). Percentage of β-TrCP bound to endogenous 3xFLAGCul1 in WT and DKO cells with or without tetracycline-induced overexpression of β-TrCP is graphed. Error bars: range of values, n = 2.