Figure 6.

Detection of mRNA of RYs in beta cells by RNase protection assay. Radiolabeled cRNA probes of RY₁ (lanes 1–4), RY₃ (lanes 5–8), and RY₂ (lanes 9–15) were incubated with RNA, digested with RNase, and separated on a 6% polyacrylamide gel containing 7 M urea. RY₁: lane 1, digested probe without sample RNA; lane 2, full-length probe without RNase treatment; lane 3, 5 μg total RNA from skeletal muscle; lane 4, 60 μg total RNA from βTC-3 cells. RY₃: lane 5, digested probe without sample RNA; lane 6, full-length probe without RNase treatment; lane 7, 30 μg total RNA from brain; lane 8, 60 μg total RNA from βTC-3 cells. RY₂: lane 9, digested probe without sample RNA; lane 10, full-length probe without RNase treatment; lanes 11 and 12, 5 and 1 μg total RNA from heart, respectively; lane 13, 15 μg mRNA from islets; lane 14, 60 μg total RNA from islets; lane 15, 15 μg mRNA from βTC-3 cells; lane 16, 60 μg total RNA from βTC-3 cells. Molecular weight (in bases) of protected fragment was determined by pBluescript(SK−) digested with HpaII and labeled by fill-in reaction using Klenow enzyme and [α-³²P]dCTP. The figure represents experiments repeated three times.