Figure S1. Characterization of CAS and... | Rockefeller University Press

Figure S1.

$Characterization of CAS and its mutants. (A) Western blot of endoCAS and mCherry-CAS constructs expressed in HEK293T cells. All mCherry-CAS constructs were stably expressed and did not degrade. The epitope of anti-CAS is on the C-terminus of CAS, thus only CAS with non-truncated C-termini are detected. (B) Validation of CAS, CAS∆Cloop, and ∆40NCAS by SDS gel and Western blot. The fractions collected after size exclusion chromotography are indicated by a rectangle. (C) Polydispersity of CAS, CAS∆Cloop, and ∆40NCAS∆Cloop measured by mass photometry. (D) Binding of various CAS mutants with RanGTP in the presence and absence of Kapα measured by microscale thermophoresis (MST). It is worth noting that (i) preloading of Kapα to CAS inhibits subsequent RanGTP binding (left panel, solid red circles); and (ii) binding between ∆40NCAS and Kapα is enhanced. N = 3. Error bars denote standard deviation. Source data are available for this figure: SourceData FS1.$

Characterization of CAS and its mutants. (A) Western blot of endoCAS and mCherry-CAS constructs expressed in HEK293T cells. All mCherry-CAS constructs were stably expressed and did not degrade. The epitope of anti-CAS is on the C-terminus of CAS, thus only CAS with non-truncated C-termini are detected. (B) Validation of CAS, CAS∆Cloop, and ∆40NCAS by SDS gel and Western blot. The fractions collected after size exclusion chromotography are indicated by a rectangle. (C) Polydispersity of CAS, CAS∆Cloop, and ∆40NCAS∆Cloop measured by mass photometry. (D) Binding of various CAS mutants with RanGTP in the presence and absence of Kapα measured by microscale thermophoresis (MST). It is worth noting that (i) preloading of Kapα to CAS inhibits subsequent RanGTP binding (left panel, solid red circles); and (ii) binding between ∆40NCAS and Kapα is enhanced. N = 3. Error bars denote standard deviation. Source data are available for this figure: SourceData FS1.