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Subsequently, the cells were fixed with 4% paraformaldehyde at 16 C for 15 min, permeabilized in phosphate buffered saline (PBS) with 0.1% Triton X-100 for 5 min at 16 C, and incubated with first antibodies at 4 C overnight, followed by incubation with FITC-conjugated secondary antibodies at 16 C for 1 h

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For each phenotype, we divided the significantly associated proteins into those that were upregulated and those that were downregulated

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We also found that in elderly testes, nuclear-FOXO4 + Leydig cells expressed less 3-hydroxysteroid dehydrogenase (3-HSD), the rate-limiting enzyme in testosterone synthesis, than nuclear-FOXO4 - Leydig cells (Figure 2D)