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Fig. 2. RNAi of VCP, p47, Ufd1 and ß5 subunit of the proteasome in HeLa cells. HeLa cells were subjected to RNAi of the indicated proteins for 4 days or treated with 5 µM MG132 for 24 hours. (A) Equal amounts of whole cell lysate protein were subjected to western blotting with the indicated antibodies. The anti-BIP antibody detects both BIP/Grp78 (lower band) and Grp94 (upper band); (B) Ubiquitin levels were quantified by densitometry of western blots. The ubiquitin level from control lysates was assigned a value of 100% and the levels from experimental lysates are expressed as a percentage of the control. Results represent mean values (±s.e.m.) from three independent experiments. The value for each experimental group was compared to the control by Student's t test. The asterisk indicates P<0.05. (C) Lysates were assayed for the chymotrypsin-like activity of the proteasome by hydrolysis of Suc-LLVY-AMC. Activities from control lysates were assigned a value of 100% and activities from experimental samples are expressed as a percentage of the control. Results represent mean values (±s.e.m.) of three independent experiments. Each experimental group was compared to the control by Student's t test. The asterisk indicates P<0.05.
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