Figure 3.

Polypyrimidine-tract-binding protein (PTB) and heterogeneous nuclear ribonucleoprotein L (hnRNPL) are components of Complex II. (a) Competition RNA electrophoretic mobility shift assay (R-EMSA) was performed using an in vitro transcribed, ³²P-CTP-labelled 1518-HaeIII RNA probe in the absence (lane 3) or presence of 50 ng (lanes 4, 8 and 12), 100 ng (lanes 4, 9 and 13), 200 ng (lanes 6, 10 and 14) and 400 ng (lanes 7, 11 and 15) oligo-U (lanes 4–7), oligo-dCT (lanes 8–11) and oligo-dCA (lanes 12–15). To show the presence of PTB in Complex II, 1 μg isotype control immunoglobulin G (lane 16) or 1 μg anti-PTB monoclonal antibody (lane 17) was added to the binding reaction 1 hr before the addition of probe. Lanes 1 and 2 show the E5-HaeIII probe alone and with RNase mix in the absence of extract, respectively. (b) Ultraviolet cross-linking competition assay using an in vitro transcribed, ³²P-CTP-labelled E5-HaeIII RNA probe in the presence of increasing amounts of unlabelled competitor RNA. The assay was conducted in the absence (lane 2) and presence (lanes 3–15) of 20 μg Jurkat/D1.1 cytoplasmic extract and RNase mix. Reactions were incubated with 25 ng (lanes 4, 8 and 12), 50 ng (lanes 5, 9 and 13), 100 ng (lanes 6, 10 and 14) and 200 ng (lanes 7, 11 and 15) unlabelled oligo-U (lanes 4–7), oligo-dCT (lanes 8–11) and oligo-dCA (lanes 12–15). Arrows indicate four specific RNA-binding proteins. Lane 1 shows the E5-HaeIII probe alone in the absence of RNase, extract, and competitor RNA. Asterisks indicate non-specific RNA binding proteins. (c) R-EMSA using an in vitro transcribed 1518-HaeIII probe in the presence of 20 μg cytoplasmic extract from Jurkat/D1.1 (lanes 3–5) and incubated with either no antibody (lane 3), 1 μg control immunoglobulin G (lane 4) or 1 μg anti-hnRNPL antibody (lane 5) before addition of the probe. Lane 2 shows reaction products from probe incubated in the presence of RNase mix in the absence of extract.