Figure 3.

Supershift assay identifying the subunit composition of the inducible NF-κB complexes in the human testis. Nuclear protein extracts (10 μg) of human seminiferous tubules cultured for 2.5 hours in serum-free conditions were incubated with a ³²P-labeled κB oligonucleotide, after which various antibodies were added to the binding reactions as indicated. The NF-κB complexes A and B were retarded by the anti-p50 and anti-RelA antibodies, indicating that the inducible NF-κB in the human testis is composed of the corresponding proteins. The other antibodies, which did not affect the electrophoretic mobility of the testicular NF-κB complexes, bound to the corresponding DNA-protein complexes in EMSAs of control nuclear extracts described in Materials and Methods (data not shown). The three NF-κB complexes in cultured seminiferous tubules were observed in 11 independent experiments and the result of the supershift assay is representative of two independent experiments.