Figure 3
Effect of MIB on p27 protein stability and ubiquitylation (A) LNCaP cells were maintained in the presence of 1 ng/ml MIB and/or 750 ng/ml CA for 72 h. Total cellular RNA was isolated and p27 and SKP2 RNA levels were determined by Northern blotting as described in Materials and methods. Glycerol aldehyde phosphate dehydrogenase (GAPDH) RNA levels are shown as loading controls. Ribosomal RNA is indicated as size markers. The ethidium bromide (EtBr) stained gel before transfer is show to demonstrate the integrity of the RNA. Blots were quantitated using a phosphoimager and RNA levels normalized to the GAPDH reference are shown in a block diagram (right). (B) LNCaP cells maintained in the absence or presence of 10 ng/ml MIB for 72 h were treated with cycloheximide (CHX, 100 ug/ml) to inhibit protein synthesis. Samples were taken after the indicated times, and p27 abundance was determined by immunoblotting. Blots were scanned and data normalized to the signal of tubulin were blotted in the diagram. (C) In vitro ubiquitylation of p27. Wildtype p27 or a point mutant in which threonine 187 was replaced by alanine (p27-T187A) was radiolabeled with 35S-methionine by coupled in vitro transcription/translation. The labeled substrate was incubated with total protein lysate prepared from LNCaP cells maintained in the absence or presence of 10 ng/ml MIB for 72 hours. The reaction also contained ATP and ubiquitin as described in Materials and methods. Polyubiquitylated p27 species generated in the reaction are indicated (p27-Ubn).