FIG. 9.

Role of P1465 hydroxylation in formation of renal cancer tumors. (A) (i) RT-PCR of mRNA levels in tumorigenic cells showing exogenous wild-type (WT) Rpb1 and P1465A Rpb1 as well as endogenous human Rpb1. (ii) Western blot analysis, using the indicated antibody, of total chromatin-enriched nuclear extracts from pools of 786-O VHL⁺ cells transfected with an empty vector (lane 1), Rpb1 WT (lane 2), or Rpb1 P1465 (lane 3). (B) (i) Frequency (left) and size (right) of tumors resulting from orthotopic xenografts involving injections of 786-O VHL⁺ cells stably expressing Rpb1 WT, P1465A, or empty vector (E). Formation of tumors by VHL⁻ cells transfected with an empty vector is shown as a positive control for tumor formation. In each group a total of 15 mice were injected in three independent series of five; **, P < 0.01; ***, P < 0.001. In each case, tumor size includes the injected kidney. NK, average size of normal mouse kidney based on weight of 20 noninjected kidneys. (ii) Photomicrograph (magnification, ×200) of immunostaining for exogenous histidine-tagged Rpb1 (WT, left; P1465A, right; negative control without primary antibody [Ab], middle) using H1029 antibody. (C) Western blot analysis, using indicated antibodies, of combined chromatin fractions from 14 RCC tumors (T) or adjacent normal kidney (K). G number describes the grade of each tumor.