Fig. 6: SRC-1 and TIF2 are degraded by autophagy, and thus are significantly reduced in Rubicon-ablated adipocytes or aged adipocytes.

a Immunoblotting to detect SRC-1 and TIF2 in Luciferase- or Rubicon-knockdown 3T3-L1 cells treated with or without 125 nM Baf A1 for 8 h. Knockdown was performed for 48 h starting on day 8. n = 3 independent experiments. b Immunoblotting to detect SRC-1 and TIF2 in wild-type eWAT depots explanted in DMEM, treated with or without 20 mM ammonium chloride and 200 μM leupeptin for 2 h. n = 3 independent experiments. c, d Immunoblotting to detect SRC-1 and TIF2 in eWAT depots of 21-week-old mice of the indicated genotypes on an NCD. n = 5 mice. e Immunoblotting to detect the indicated proteins in eWAT depots of 3- or 25-month-old wild-type mice on an NCD. n = 3 mice. f Immunoblotting to detect SRC-1 and TIF2 in the eWAT depots of 3-, 12-, 18-month-old control mice on an NCD. n = 4 mice. g Immunoblotting to detect the indicated proteins in eWAT depots of fed or 48-h-fasted 3-month-old wild-type mice. n = 3 mice. h Immunoblotting to detect the indicated proteins in 3T3-L1 cells. The cells on day 10 were subjected to starvation for the indicated times. n = 3 independent experiments. i Relative mRNA expression of adipogenic genes in 3T3-L1 cells. The cells on day 10 were subjected to starvation for the indicated times. n = 4 independent experiments. Quantification data are shown in the graphs at the right of each blot. Error bars indicate means ± SEM. Data were analysed by two-tailed Student’s t test (b, c, i), one-way ANOVA followed by Tukey’s test (d, f) or Dunnett’s test (a). P value from top to bottom and left to right: 0.0083, 0.0018, 0.0065, 0.0019 (a), 0.0163, 0.0256 (b), <0.0001, <0.0001 (c), <0.0001, 0.1577, <0.0001, 0.1894 (d), 0.0008, 0.0013, 0.0077, 0.0359 (f), <0.0001, 0.5116, 0.0094, 0.0018, <0.0001, <0.0001, <0.0001 (i). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001. N.S. not significant.