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. 2002 Jan;160(1):205–218. doi: 10.1016/s0002-9440(10)64364-7

Figure 2.

Figure 2.

Time course of activation of the transcription factor NF-κB during human testicular apoptosis. Segments of human seminiferous tubules were cultured under serum-free conditions for 0 to 10 hours to induce apoptotic cell death, after which apoptotic DNA fragmentation and activation of NF-κB were studied at different time points. A: Southern blot analysis of apoptotic low molecular weight DNA fragmentation (185 bp multiples). a: DNA was extracted from the seminiferous tubules cultured for various periods of time, after which 1 μg of the total DNA in each sample was 3′-end labeled with Dig-dd-UTP and subjected to electrophoresis. The labeled DNA was detected with chemiluminescence as described in Materials and Methods. b: Low molecular weight DNA (<1.3 kb) fragmentation was quantified from the radiograph presented in a, as described in Materials and Methods, and plotted as a function of time. The digitized quantification of the low molecular weight DNA fragments in the sample cultured for 10 hours was taken as 100% apoptosis, and the amounts of low molecular weight DNA fragments in the other samples were expressed in relation to this. B: EMSA demonstrating the increase in NF-κB DNA-binding activity during in vitro induced testicular apoptosis. a: Nuclear protein extracts (10 μg) from the seminiferous tubules were incubated with a 32P-labeled κB oligonucleotide, and the DNA-protein complexes formed were resolved on polyacrylamide gel electrophoresis. Competition experiments using an unlabeled κβ oligonucleotide (cold) or unlabeled mutated κB oligonucleotide (mut.) confirmed the specificity of binding to the κB oligonucleotide. b: NF-κB DNA binding was quantified from the radiograph presented in a, as described in Materials and Methods, and plotted as a function of time. The digitized quantification of the specific NF-κB bands in the sample cultured for 5 hours was set as 100%, and the intensities of the bands in other samples were expressed in relation to this. All of the results shown are representative of two independent experiments.