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. 2000 Sep 5;97(19):10430–10435. doi: 10.1073/pnas.190332597

Figure 3.

Figure 3

VHL mutants that do not bind stably to Elongins B and C do not assemble into the VHL complex and do not detectably activate HIF1α ubiquitination. (A) Total cell lysates from Sf21 cells infected with the baculoviruses indicated in the figure were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (B) The cell lysates of A were subjected to immunoprecipitations with anti-VHL antibodies as described in Materials and Methods. The immunoprecipitates were subjected to 8% or 13% SDS/PAGE, and proteins were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using the antibodies indicated in the figure. (C) The cell lysates of A were subjected to immunoprecipitations with anti-VHL antibodies as described in Materials and Methods. The immunoprecipitates were assayed for the ability to activate HIF1α ubiquitination as described in Materials and Methods. Reaction products were subjected to 8% SDS/PAGE, and GST-ubiquitin-HIF1α conjugates (GST-Ub-HIF1α) were transferred to Hybond P membranes and visualized by Western blotting as described in Materials and Methods, using anti-HIF1α antibodies. EloB, Elongin C; EloC, Elongin C.