Fig. 2: Autophagy activities showed gender difference in visceral but not subcutaneous fat.

a, b The steady-state protein levels of LC3 in subcutaneous WAT, analyzed by Western blotting (a) and densitometry (b). c, d The steady-state protein levels of LC3 in visceral WAT, analyzed by Western blotting (c) and densitometry (d). e–h Measurement of autophagy flux in subcutaneous and visceral WAT. The WAT explant cultures were incubated with and without autophagy inhibitor bafilomycin A1 (0.1 μM) and leupeptin (10 μg/ml) for 4 h, and the turnovers of LC3-II and p62 were examined by Western blotting (e, g) and densitometry (f, h). In densitometric analyses, the band densities of investigated proteins were normalized against that of GAPDH or β-actin, and the fold changes were calculated by taking the normalized density of female group as “1”. For autophagy flux, we first normalized the band densities of LC3-II and p62 against that of GAPDH, then calculated the differences of normalized densities in the presence vs. the absence of autophagy inhibitor; lastly, the differences were shown as fold changes by taking the female group as “1”. BL bafilomycin A1 and leupeptin, M male, F female; *p < 0.05; **p < 0.01; n.s. not significant; n = 3–4