Fig. 4.

hnRNP A1 binds the HPV16 LRE. (A) Electrophoretic mobility shift assay showing interaction of hnRNP A1 and LRE RNA in HeLa and W12 cells. Free probe and the RNA/protein complexes formed are indicated. Asterisks and an arrowhead indicate RNA/protein complexes that have been supershifted due to binding of the anti-hnRNP A1 antibody in tracks 3 and 5. Track 1, [α-³²P] rUTP-labelled LRE RNA probe alone (the LRE RNA is a stem loop structure (Cumming et al., 2003); the upper band is due to secondary structure formation); track 2, LRE RNA probe incubated with HeLa nuclear extract; track 3, as in track 2 but nuclear extract preincubated with anti-hnRNP A1 antibody; track 4, LRE RNA probe incubated with W12E nuclear extract; track 5, as in track 4 but nuclear extract preincubated with anti-hnRNP A1 antibody. (B) Western blot of nuclear (tracks 2 and 4) and cytoplasmic (tracks 1 and 3) extracts from undifferentiated (tracks 1 and 2) and differentiated (tracks 3 and 4) W12E cells showing fractionation of U2AF⁶⁵ and hnRNP A1 mainly with the nuclear fraction and involucrin mainly with the cytoplasmic fraction as expected. The increase in levels of involucrin in tracks 3 and 4 confirms that the W12 cells have differentiated. (C) Autoradiogram of an SDS-PAGE fractionation of UV crosslinking of [α-³²P] rUTP-labelled LRE RNA probe with nuclear (NE) and cytoplasmic (CE) fractions of HeLa and undifferentiated (U) and differentiated (D) W12E cells. (D) Western blot of the SDS-PAGE in (C) probed with anti-hnRNP A1 antibody.