Figure 2
Figure 2. Tyrosine motifs are important for degradation of CD33 following cross-linking. (A) CD33WT and CD33Y340F/Y358F stable Ba/F3 cells were incubated with α-CD33 (IC7/1) for 20 minutes and cross-linked with GAM for 30 minutes. The cells were pretreated with and without MG132 (0.5 μM) and LLNL (0.5 μM) for 30 minutes. Lysates were immunoprecipitated with α-Flag and immunoblotted with α-Flag (Ai). WCL was immunoblotted with α-STAT5B as a loading control (Aii). (B) CD33WT and CD33Y340F/Y358F stable Ba/F3 cells were incubated with α-CD33 (IC7/1) for 20 minutes and cross-linked with goat antimouse for 5 minutes. Samples were incubated with PE-conjugated α-CD33 or isotype control for 15 minutes and analyzed by FACS. In the bottom panel, about 70% of the Ba/F3 cells are stably infected with the expression construct for CD33Y340F/Y358F. As a result, the peak in the lower-left corner represents the cells negative for CD33Y340F/Y358F expression.

Tyrosine motifs are important for degradation of CD33 following cross-linking. (A) CD33WT and CD33Y340F/Y358F stable Ba/F3 cells were incubated with α-CD33 (IC7/1) for 20 minutes and cross-linked with GAM for 30 minutes. The cells were pretreated with and without MG132 (0.5 μM) and LLNL (0.5 μM) for 30 minutes. Lysates were immunoprecipitated with α-Flag and immunoblotted with α-Flag (Ai). WCL was immunoblotted with α-STAT5B as a loading control (Aii). (B) CD33WT and CD33Y340F/Y358F stable Ba/F3 cells were incubated with α-CD33 (IC7/1) for 20 minutes and cross-linked with goat antimouse for 5 minutes. Samples were incubated with PE-conjugated α-CD33 or isotype control for 15 minutes and analyzed by FACS. In the bottom panel, about 70% of the Ba/F3 cells are stably infected with the expression construct for CD33Y340F/Y358F. As a result, the peak in the lower-left corner represents the cells negative for CD33Y340F/Y358F expression.

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