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. 2000 Jul 18;97(15):8507–8512. doi: 10.1073/pnas.97.15.8507

Figure 4.

Figure 4

Both elongins B and C are needed for pVHL stabilization. VHL expression vectors were transiently transfected into 293T cells, either with no cotransfection (− el BC) or with cotransfection of elongin B (+ el B), elongin C (+ el C), elongin B and elongin C (+ el BC), or elongin B, elongin C, and cul-2 vectors (+ el BC/cul-2). Transfected cells were treated with cycloheximide (CHX) for various time points (indicated above each blot). Whole cell lysates were normalized for equal protein loading (10 μg) in each lane. VHL Western blotting (Upper panels) was performed using either an anti-Flag mAb (A) or mAb 11E12 (B). Elongin B-HA, elongin C-Flag, and cul-2-Flag, detected with antibodies to the appropriate epitope-tag, are indicated by arrowheads (Lower panels). (A) Position of Flag-VHLp24(MPR) is indicated by an arrowhead to the left of the blot (Upper). (B) Positions of VHLp24(MPR) products are indicated by a bracket to the right of the blot (Upper).