Figure 2.

Determination of the transcription start site of murine SOCS-3 mRNA by RNase protection assay. As described in Materials and Methods, a ³²P-labeled antisense probe spanning nucleotides +160 to −273 of the murine SOCS-3 gene was hybridized at 42°C with 10 μg yeast RNA (lanes 2 and 3) or 10 μg total RNA derived from LIF-stimulated AtT-20 cells (lane 4). Lane 2 shows the undigested full-length probe. RNase A/RNase T1 mix was added for digestion of unprotected fragments to samples of lane 3 (negative control) and lane 4. Lanes 5 and 6 show ³³P-sequencing of A and C with antisense primer (+160 to +143). Arrows indicate a protected band in lane 4 and the corresponding nucleotide sequence.