(A) p97-Myc was immunoprecipitated from HeLa cells treated or not with 20 µM MG132 for 2 h prior to harvesting the cells. Cells expressing no Myc-tagged protein were used as negative control. The indicated proteins were detected in the immunoprecipitates (top) and in the input cell extracts (bottom) using specific antibodies.
(B) p97-Myc was immunoprecipitated from HeLa cells treated for 48 h with 5 nM of the indicated siRNAs and incubated with MG132 as above. The indicated proteins were detected using specific antibodies, in the immunoprecipitates (left) and in the input cell extracts (right). Luc – luciferase
(C) HeLa cell extracts were fractionated on a Superdex 200 gel filtration column. Individual fractions were concentrated by TCA precipitation and subjected to western blotting using specific antibodies. All proteins were endogenously expressed.