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. 2020 Oct 7;23(10):101604. doi: 10.1016/j.isci.2020.101604

Figure 1.

Figure 1

SMYD3 Directly Interacts with ATM, CHK2, and BRCA2 In Vitro and In Cellulo

(A) Upper panel: Diagram showing the nine overlapping GST-BRCA2 (B2-1 to B2-9) fusion proteins used in this study. Lower panel: HIS-SMYD3 bound to histidine beads was incubated with GST-BRCA2 fusion proteins and washed. Bound proteins were visualized by immunoblotting using anti-GST and anti-HIS antibodies.

(B) Upper panel: Diagram showing the eight overlapping GST-ATM (A-1 to A-8) fusion proteins used in this study. Lower panel: HIS-SMYD3 bound to histidine beads was incubated with GST-ATM fusion proteins and washed. Bound proteins were visualized by immunoblotting using anti-GST and anti-HIS antibodies.

(C) Competition assay: HIS-SMYD3 bound to histidine beads was incubated with GST-BRCA2 B2-4 and GST-ATM A-8 fusion proteins in the presence of escalating doses of the purified P1 and P10 tripeptides, respectively. Bound proteins were visualized by immunoblotting using anti-GST and anti-HIS antibodies.

(D) Co-immunoprecipitation assay in MYC-SMYD3- and BRCA2-overexpressing HEK-293 cells using anti-MYC and anti-BRCA2 antibodies.

(E) Co-immunoprecipitation assay in MYC-SMYD3- and FLAG-ATM-overexpressing HEK-293 cells using anti-MYC and anti-FLAG antibodies.

(F) Co-immunoprecipitation of endogenous SMYD3 using anti-SMYD3 antibodies in MDA-MB-231 nuclei treated with MNase to limit indirect interaction through polynucleosomes.

(G) Upper panel: Domain structure of CHK2 protein. Lower panel: HIS-SMYD3 bound to histidine beads was incubated with recombinant CHK2 protein and washed. Bound proteins were visualized by immunoblotting using anti-GST and anti-CHK2 antibodies.

(H) Co-immunoprecipitation assay in MYC-SMYD3- and FLAG-CHK2-overexpressing HEK-293 cells using anti-MYC and anti-FLAG antibodies.

(A, B, C, and G) A 10% input of the purified fusion proteins was used as a loading control. (A, B, and G) GST-HSP90 C-terminal (616–736) was used as a positive control. (D, E, F, and H) Anti-IgGs were used as negative controls. P, P-tripeptide. Results are representative of at least three independent experiments. See also Figures S1 and S2.