Megaprime labeling kit

Confluent HaCat cells were serum-deprived overnight and treated (or not) with EGF (500ng/mL). 5μg/mL Actinomycin D (Act.D, Sigma) was then added to replicate flasks, which were subsequently harvested for mRNA extraction at defined time points. mRNA was quantified by spectrophotometry and 10μg of each sample separated on a 1.2% formalin-agarose gel and transferred overnight onto nylon membranes (Hybond N+, Amersham) by capillary blotting in 10× SSC. Blots were UV-crosslinked before hybridization. MICA, MICB and GAPDH probes were prepared by PCR and labeled with α³²P dCTP using Megaprime labeling kit (Amersham) following manufacturer’s instructions. Probes were denatured at 95°C for 5min and hybridization was carried out in Expresshyb solution (Clontech) following manufacturer’s instructions. Blots were incubated with probe in roller bottles for 1h at 68°C, washed with 2× SSC, 0.5% SDS at room temperature, then with 0.1× SSC, 0.1% SDS at 50°C, and exposed to a storage phosphor screen and acquired using the Storm PhosphorImager (Amersham). Bands were quantitated using ImageJ software.