Figure 4
Figure 4. E3 ligase complex is required for proteasomal degradation of CD33 and SOCS3. (A) 293T cells were transiently transfected with 2 μg EV and CD33WT in the presence and absence of SOCS3. Cells were incubated with α-CD33 (IC7/1) for 20 minutes and cross-linked with GAM for 30 minutes. Lysates were immunoprecipitated with (Ai-ii) α-Flag and immunoblotted with α-Flag. (B) 293T cells were transiently transfected with 2 μg EV and CD33WT with E3 ligase complex (Elongin B/C, Cul5 and Rbx1) in the presence and absence of SOCS3. Cells were incubated with α-CD33 (IC7/1) for 20 minutes and cross-linked with GAM for 30 minutes. The cells were pretreated for 30 minutes with and without MG132 and LLNL. Lysates were immunoprecipitated with α-Flag and immunoblotted with (Bi-iii) α-Flag and with (Biv) α-Myc (9E10). (Bv) WCL was immunoblotted with α-His.

E3 ligase complex is required for proteasomal degradation of CD33 and SOCS3. (A) 293T cells were transiently transfected with 2 μg EV and CD33WT in the presence and absence of SOCS3. Cells were incubated with α-CD33 (IC7/1) for 20 minutes and cross-linked with GAM for 30 minutes. Lysates were immunoprecipitated with (Ai-ii) α-Flag and immunoblotted with α-Flag. (B) 293T cells were transiently transfected with 2 μg EV and CD33WT with E3 ligase complex (Elongin B/C, Cul5 and Rbx1) in the presence and absence of SOCS3. Cells were incubated with α-CD33 (IC7/1) for 20 minutes and cross-linked with GAM for 30 minutes. The cells were pretreated for 30 minutes with and without MG132 and LLNL. Lysates were immunoprecipitated with α-Flag and immunoblotted with (Bi-iii) α-Flag and with (Biv) α-Myc (9E10). (Bv) WCL was immunoblotted with α-His.

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