2.6. Measurement of NF-kB p65 with ELISA

NF-kB p65, the most abundant member of the NF-kB transcription family, was measured with PhosphoTracer NF-kB p65 assay kit (Abcam, UK), according to the manufacturer’s indications. This kit detects the endogenous level of this molecule in cellular lysates using an immuno-sandwich technique. Briefly, cells were seeded and irradiated as described in sub-chapters 2.1. and 2.2., respectively. After 2, 4, 8 and 24 h from UVB exposure, the cells were lysed with 100 μl lysis solution containing a combination of detergents, phosphatase inhibitors, salts and buffers, available in the ELISA kit. 50 μl/well cell lysates and 50 μl/well antibody mix (capture antibody + detection antibody) were added to PhosphoTracer assay microplate. Fluorescence was recorded at 530–540 nm with an ELISA microplate reader (Tecan Sunrise, Grödig/Salzburg, Austria).