T-cell activation up-regulates the level of the mRNA for LAT.
(A) Jurkat T cells were incubated as described in the legend for Figure1 for 8 hours at 37°C with RPMI-5% FCS (lane 1), RPMI-5% FCS containing 1 ng/mL of PMA (lane 2), or RPMI-5% FCS containing 1 μg/mL of anti-CD3 mAb (α-CD3; lane 3). After washing, total RNA was extracted, reverse transcribed to generate first-strand cDNA, and the resultant cDNA amplified by PCR using LAT- or GAPDH-specific primers, as described in “Materials and methods.” GAPDH gene expression was analyzed to control for mRNA integrity and loading. PCR products were visualized by ethidium bromide staining of the gel. This experiment was repeated 3 times with similar results. (B) A total of 2 × 107 Jurkat T cells were incubated for 8 hours at 37°C in RPMI-5% FCS or in RPMI-5% FCS containing the indicated concentrations of PMA. After incubation, the cells were washed in PBS, and poly A+ RNA was extracted using the Oligotex mRNA mini kit according to the manufacturer's recommendations. The resultant mRNA was subjected to 1% agarose electrophoresis and then transferred to positively charged nylon membranes. The membranes were then prehybridized for 4 hours at 50°C using the reagents provided in the Northern blotting (Max-Gly) kit. After prehybridization, the membranes were incubated for 16 hours at 50°C with a mixture of 5′-biotinylated LAT antisense oligonucleotides (upper panel). After incubation, the membranes were extensively washed, and the membrane-bound biotinylated probe was visualized as described in “Materials and methods.” To ensure equal loading, the membranes were stripped of LAT oligonucleotides by boiling for 30 minutes in 0.1% SDS and were then reprobed with a 5′-biotinylated β-actin antisense oligonucleotide. This experiment was repeated twice with similar results.
