Figure 7
BECN1 interacts with the ISG15 E3 ligase HERC5.A PAMs were co-transfected with the plasmids Flag-BECN1 and Myc-HERC5 or Flag-RFP and Myc-HERC5 as a control. Cells were lysed and subjected to an IP assay with Anti-Flag M2 Affinity Gel. The precipitated proteins were detected by Western blot with an anti-Myc antibody. B PAMs were co-transfected with the plasmids Flag-BECN1 and Myc-HERC5 or Flag-RFP and Myc-HERC5 as a control. Cells were lysed and subjected to an IP assay with an anti-c-Myc immunoprecipitation kit. The precipitated proteins were analyzed by Western blot with an anti-Flag antibody. C GST and GST-BECN1 fusion proteins expressed in E. coli BL21 cells were conjugated with glutathione-Sepharose resin and incubated with lysate of HEK-293T cells overexpressing the Myc-HERC5 protein. The bound proteins were detected by Western blot with an anti-Myc antibody. D GST and GST-HERC5 fusion proteins expressed in E. coli were conjugated with glutathione-Sepharose resin and incubated with lysate of HEK-293T cells overexpressing the Flag-BECN1 protein. The bound proteins were detected by Western blot with an anti-Flag antibody