Journal: Virus Research

Article Title: The alternative splicing factor hnRNP A1 is up-regulated during virus-infected epithelial cell differentiation and binds the human papillomavirus type 16 late regulatory element

doi: 10.1016/j.virusres.2007.09.006

Figure Lengend Snippet: 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- 32 P] rUTP-labelled LRE RNA probe alone (the LRE RNA is a stem loop structure ; 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 U2AF 65 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- 32 P] 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.

Article Snippet: Using in vitro transcription, riboprobes were synthesised with the Stratagene RNA transcription kit with addition of 25 μCi [α- 32 P]UTP (800 mCi/mmol; NEN) following the manufacturer's protocol.

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Incubation, Western Blot, Fractionation, SDS Page

Journal: Virus Research

Article Title: The alternative splicing factor hnRNP A1 is up-regulated during virus-infected epithelial cell differentiation and binds the human papillomavirus type 16 late regulatory element

doi: 10.1016/j.virusres.2007.09.006

Figure Lengend Snippet: Nuclear hnRNP A1 binds the LRE directly. (A) Electrophoretic mobility shift analysis of binding of GST (500 nM) and GST-hnRNP A1 (45, 90, 180 nM) to [α- 32 P] rUTP-labelled LRE or truncated LRE RNA probes. The probes used are shown in and indicated below the autoradiograph. Arrows indicate free probe. A bracket indicates RNA/protein complexes. (B) Competition electrophoretic mobility shift experiment using a non-specific competitor, unlabelled in vitro -transcribed pBluescript polylinker RNA (70 nts) (pBS) (lanes 1–5) or a specific competitor, unlabelled in vitro transcribed LRE RNA (79 nts) (lanes 6–10). P; [α- 32 P] rUTP-labelled LRE probe alone, N; probe plus nuclear extract. Both competitors were added to reactions at 1, 2, 4, 8 and 16-fold molar excess. Arrows indicate free probe. A bracket indicates protein/RNA complexes.

Article Snippet: Using in vitro transcription, riboprobes were synthesised with the Stratagene RNA transcription kit with addition of 25 μCi [α- 32 P]UTP (800 mCi/mmol; NEN) following the manufacturer's protocol.

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Autoradiography, In Vitro