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. 2003 Feb 25;100(5):2706–2711. doi: 10.1073/pnas.0436037100

Figure 3.

Figure 3

pVHL specifically interacts with the hyperphosphorylated Rpb1 in nuclear extracts from PC12 and RCC cells. Coimmunoprecipitations (IP) using monoclonal antibodies against HA or pVHL or mouse anti-rabbit IgG (RG-96) in nuclear extracts from PC12 cells overexpressing human HA-tagged pVHL [PC12 VHL (WT)] (A and B), or control PC12 cells stably transfected with an empty vector (A). (C) Dephosphorylation of Rpb1 in PC12 cellular extracts for the indicated times by treating the extracts with alkaline phosphatase (AP) in the absence (lanes 3, 4, 7, and 8) or presence (lanes 6 and 9) of NaF. Treated extracts were subjected to immunoprecipitations using anti-HA antibodies. (D) Anti-HA immunoprecipitations in nuclear extracts from RCC 786-O cells lacking pVHL function (lanes 1 and 2) or from cells stably transfected with HA-pVHL (1) (lanes 3 and 4). PC12 and indicated RCC cells were pretreated with 10 μM CbzLLn for 6 h to increase accumulation of the hyperphosphorylated Rpb1. The immunoprecipitates were washed with high-detergent immunoprecipitation buffer (A, C, and D), or immunoprecipitation buffers containing up to 900 mM NaCl and 0.5% Igepal (B). Blots were probed with the indicated antibodies, human (h)pVHL and rat (r)pVHL, respectively.