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. 2002 Jun 17;195(12):1585–1597. doi: 10.1084/jem.20020047

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Copyright © 2002, The Rockefeller University Press

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Figure 6. — Reduction of the pre-TCR turnover by proteasome inhibition. (A) Immunoprecipitation with TCRβ mAb of SCB.29 cells lysed in 1% Brij 96 immediately after surface labeling with biotin (0) or a 2-h chase in the presence of DMSO (−) or epoxomicin (+). Two-dimensional nonreduced versus reduced gels were revealed in Western blots with avidin-HRP and after stripping with CD3ε antibodies. (B) Immunoprecipitation with 145-2C11 mAb of pre-TCR–associated CD3 chains as described in Materials and Methods. Two-dimensional nonreduced versus reduced gels were revealed as previously described. (C) Immunoprecipitation with G3 mAb of pre-TCR–independent ζ chains. After Western blots with avidin-HRP, membranes were stripped and revealed with anti–ζ chain rabbit serum. IP, immunoprecipitation; WB, Western Blots. (D) Densitometric analyses of the biotinylated pre-TCR chains, pre-TCR–associated CD3ε chains, and pre-TCR–independent ζ chains recovered at the indicated times and in the presence of the indicated treatment (means of at least two experiments are plotted).

Figure 6. — Reduction of the pre-TCR turnover by proteasome inhibition. (A) Immunoprecipitation with TCRβ mAb of SCB.29 cells lysed in 1% Brij 96 immediately after surface labeling with biotin (0) or a 2-h chase in the presence of DMSO (−) or epoxomicin (+). Two-dimensional nonreduced versus reduced gels were revealed in Western blots with avidin-HRP and after stripping with CD3ε antibodies. (B) Immunoprecipitation with 145-2C11 mAb of pre-TCR–associated CD3 chains as described in Materials and Methods. Two-dimensional nonreduced versus reduced gels were revealed as previously described. (C) Immunoprecipitation with G3 mAb of pre-TCR–independent ζ chains. After Western blots with avidin-HRP, membranes were stripped and revealed with anti–ζ chain rabbit serum. IP, immunoprecipitation; WB, Western Blots. (D) Densitometric analyses of the biotinylated pre-TCR chains, pre-TCR–associated CD3ε chains, and pre-TCR–independent ζ chains recovered at the indicated times and in the presence of the indicated treatment (means of at least two experiments are plotted).