Figure 7. Ubiquitination of tankyrase (TNKS-1)/ is decreased by PJ34.
Confluent monolayers of MDCK/T1 were maintained in −Dox medium for 2 days to induce tankyrase expression. After treatment with PJ34 (80 μM; 2 h; lane 2) or without (lane 1), cells were lysed in buffer A (1 ml/15cm plate) supplemented with 1% deoxycholate. Extracts were precleared by incubating with GST protein (50 μg/ml extracts) for 30 min, and centrifuged at 13 000 g for 10 min. Soluble extracts were incubated with GST-IRAP78–108 resins [25] (15 μg of protein/ml extracts) to precipitate tankyrase. The precipitates were immunoblotted using anti-ubiquitin (Ub) (upper panel) and anti-tankyrase (middle panel) antibodies. Extracts were immunoblotted using anti-PAR antibodies (lower panel). WB, Western blot.