SOCS3 accelerates proteasomal degradation of CD33. (A) 293T cells were transiently transfected with 2 μg EV, CD33WT, and CD33Y340F in the presence and absence of 2 μg SOCS3 and treated with pervanadate (1.4 mM) for 15 minutes. Lysates were immunoprecipitated with α-CD33 (My9) and immunoblotted with either (Ai) α-Flag, (Aii) α-phospho-Tyr (PY20), or (Aiv) α-SHP-2. (Aiii) Lysates were also immunoprecipitated with α-SOCS3 (008) and immunoblotted with α-Flag. (Av) WCL was immunoblotted with α-STAT5B as a loading control. (B-C) 293T cells were transiently transfected with 2 μg CD33WT or CD33Y340F/Y358F in the presence and absence of SOCS3 and treated with cycloheximide for 30 minutes prior to pervanadate treatment for 10, 30, and 60 minutes. (B-C) Lysates were immunoprecipitated with α-CD33 and immunoblotted with (Bi,Ci) α-Flag or (Bii,Cii) α-Phospho-Tyr. (Biii,Ciii) WCL was immunoblotted with α-Flag. (D) Stable CD33WT Tet-regulated SOCS3 Ba/F3 cells were incubated with α-CD33 (IC7/1) for 20 minutes and cross-linked with GAM for 5, 10, and 15 minutes. Lysates were immunoprecipitated with α-Flag and immunoblotted with (Di-ii) α-Flag. (Diii) WCL was immunoblotted with α-STAT5B as a loading control.