Fig. 2. Primer extension analysis for determination of the 4-1BB mRNA start site. 5 μg total RNA of splenocytes (lane 1), CTLL-R8 (lane 2), and EL4 (lane 3) was used for extension analysis. Total mRNA was annealed with 2 × 10⁴ cpm of the end-labeled oligonucleotide using [α-³²P]dATP at 30℃, overnight, in a hybridization buffer. Synthesis of cDNA using AMV reverse transcriptase was then performed. The mixture was extracted with phenol-chloroform, and precipitated with ethanol. The precipitate was analyzed by gel sequencing. The nucleotide sequence ladder of the pGEM-T easy vector shown on the left is a size marker for the extension products. The specific extension products for 4-1BB mRNA are indicated by arrows with numbers.