Co-localization of RRM2B with PYCR1 and PYCR2 in Mitochondria.
(A,B) Mitochondrial fraction (M) and cytosolic (C) fraction were isolated from 293 T-REx-Flag-RRM2B cells, (C) HFF-hTERT cells transduced with MSCV retroviruses expressing either empty control or RRM2B and (D) HFF-hTERT cells treated with Adriamycin (Adr) (+) or left untreated (−) for 24 hours. (A, C and D) Equal amount of proteins from each fraction separated electrophoretically on gels was transferred to membranes and blotted with antibodies. TOM20 was used as mitochondrial marker and α-TUBULIN (TUBA) was used as cytosolic maker to validate purity of each fraction. (B) Proteins from mitochondrial fraction were subjected to immunoprecipitation with normal mouse IgG or anti-Flag antibodies. Western blot analysis was performed to detect Flag-RRM2B, RRM2B, PYCR1 and PYCR2.