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. 2020 Feb 10;10(8):3366–3381. doi: 10.7150/thno.41849

Figure 4.

Figure 4

GRP78 recruits SIAH2 to mediate the degradation of AR-V7. (A) Co-IP assay was performed to identify the interaction between GRP78 and SIAH2, STUB1 or MDM2 in 22Rv1 cells. (B) Truncated mutants of GRP78 fused with FLAG-tag on their C-terminals were engineered and (C) co-transfected with HA-AR-V7 into HEK293T cells. Co-IP assays were performed using FLAG-tag antibodies and immunoblotted for HA and FLAG. (D) Truncated mutants of GRP78 fused with FLAG-tag on their C-terminals were co-transfected into HEK293T cells. Co-IP assays were performed using Flag-tag antibodies and immunoblotted for Myc and FLAG. IgG was blocked using mouse anti-rabbit IgG antibodies (Cell Signaling Technology, #5127) in the following immunoblots. (E) Western blot assays were performed using Myc or HA antibodies in HEK293T cells transfected with HA-AR-V7 plasmids with or without Myc-SIAH2 plasmids for 48 h. (F) Western blot assays were performed using SIAH2 or AR-V7 antibodies in 22Rv1 cells transfected with Myc-SIAH2 plasmids for 48 h. (G) Co-IP assay was performed using AR-V7 antibodies and immunoblotted for K48-Ub and AR-V7 in 22Rv1 cells transfected with Myc-SIAH2 plasmids for 48 h, and exposed to MG132 (10 μM) for 6 h before harvest. (H) Cytoplasmic and nuclear proteins were separated from 22Rv1 cells. Co-IP assays were performed using AR-V7 antibodies or GRP78 antibodies and immunoblotted for GRP78, SIAH2 and AR-V7 in cytoplasmic and nuclear extracts.