Figure 2.

RNA binding of Ku antigen in nuclear extract. (A) Gel filtration chromatography of HeLa nuclear extracts (2 ml) on Sepharose 6B. Elution of Ku antigen and NDH II after digestion of nuclear extracts by MNase (150 U/ml) plus RNase A (0.1 mg/ml) or by MNase (150 U) only for 15 min at room temperature was shown by western blotting (Aa). The multiple elution peaks of Ku86 from MNase plus RNase A treated nuclear extracts are indicated. The Sepharose 6B column was calibrated with the indicated molecular weight standards. Nucleic acids from the nuclease-treated extracts are shown by agarose gel electrophoresis (Ab). (B) RNAs were immunoprecipitated from 0.35 M NaCl nuclear extracts by the antibody against Ku86 and labeled with T4 RNA ligase and [5′-³²P]pCp after protein extraction with phenol–chloroform and RNA precipitation with ethanol. After labeling, the solutions were divided for digestion with RNase A as indicated. DNase I (0.1 mg/ml) was always present to remove possible contaminating DNA fragments. The labeling mixtures were then separated by electrophoresis through a 7 M urea–8% polyacrylamide gel. The RNA length standard was obtained from Roche; the positions of small nuclear RNAs (U1, U2, U4, U5 and U6) are indicated.