MOLECULAR-CELL-D-20-00113 "Structural insights into pseudokinase domains of receptor tyrosine kinases" Sheetz et al.
Description
Figure 5A: Expression of ROR1 and ROR1∆ICR in BaF3 stable clones by anti-HA Western blot (with beta-tubulin as loading control). Figure 5C: Wnt5a stimulation increases phosphorylation of AKT/ERK and SRC signaling in BaF3-ROR1 cells. BaF3, BaF3-ROR1 and BaF3-ROR1∆ICR cells were serum starved overnight, and then treated with recombinant Wnt5a (50 ng/ml) for the indicated times. Cell lysates were then immunoblotted with the indicated antibodies. ROR1 or ROR1∆ICR protein levels were determined using anti-HA blotting. Figure 5E: Assessment of stable BaF3 clones expressing (HA-tagged) wild-type ROR1, a variant with the YxxxYY motif in the activation loop mutated to FxxxFF (Y641F/Y645F/Y646F), and a K506A-mutated variant by Western blotting with anti-HA and beta-tubulin as loading control. Figure 6D: Cellular thermal shift assay (CETSA) demonstrating stabilization of ROR1 in cells upon addition of ponatinib or GZD824. BaF3-ROR1 cells were treated with 10 uM ponatinib or GZD824, and subjected to the noted temperatures as described in STAR Methods. Cell lysates were blotted with anti-HA to assess ROR1 levels, and with anti-beta-tubulin as a loading control. Figure S5A: BaF3 parental and BaF3-ROR1, BaF3-ROR2 and BaF3-PTK7 stable clones were analyzed for expression of ROR1, ROR2 and PTK7 by Western blotting with the indicated antibodies and beta-tubulin as loading control. Figure S5C: Wnt5a stimulation increases phosphorylation of ERK in BaF3-ROR1, but not BaF3-ROR2 cells. Experiments were performed as described for Fig. 5C, with 50 ng/ml Wnt5a, and determination of ROR1 and ROR2 protein levels using anti-HA blotting. Figure S6A: Representative Western blots showing stabilization of ROR1 expressed in BaF3 cells by increasing doses of ponatinib or GZD824. Cells were incubated at 48˚C as described in STAR Methods prior to immunoblotting to quantitate remaining non-degraded protein. Figure S7D: Adding ponatinib or GZD824 at 1 uM to BaF3 cells expressing full-length ROR1 inhibits the Wnt5a-dependent increases in ERK, AKT, and SRC phosphorylation shown in Fig. 5C. Cells were stimulated with 50 ng/ml Wnt5a for 0, 0.5, 2, or 4 h in the absence or presence of the inhibitors. Cell lysates were then blotted with anti-ROR1 (to assess total ROR1 levels), and with anti-pERK, anti-ERK, anti-pAKT, anti-AKT, and anti-beta-tubulin (as a loading control) as described in STAR Methods.