Journal: Nature Communications

Article Title: Age-dependent loss of adipose Rubicon promotes metabolic disorders via excess autophagy

doi: 10.1038/s41467-020-17985-w

Figure Lengend Snippet: 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.

Article Snippet: The following antibodies were used for western blotting at the indicated dilutions: rabbit monoclonal anti-Rubicon (CST, #8465, 1:1000), mouse monoclonal anti-ULK1 (Santa Cruz Biotechnology, sc-33182, 1:2000), mouse monoclonal anti-ATG13 (Sigma-Aldrich, SAB4200100, 1:2000), rabbit polyclonal anti-ATG12 (CST, #2011, 1:2000), rabbit monoclonal anti-ATG16L1 (CST, #8089, 1:2000), mouse monoclonal anti-Beclin 1 (BD, 612112, 1:2000), rabbit polyclonal anti-LC3 (MBL, PM036, 1:2000), mouse monoclonal anti-p62 (MBL, M162-3, 1:2000 for IP samples), rabbit polyclonal anti-p62 (MBL, PM045, 1:5000 for other samples), rat monoclonal anti-Adiponectin (R&D Systems, MAB1119, 1:2000), mouse monoclonal anti-FABP4 (Santa Cruz Biotechnology, sc-271529, 1:2000), mouse monoclonal anti-PPARγ (Santa Cruz Biotechnology, sc-7273, 1:2000), rabbit monoclonal anti-SRC-1 (CST, #2191, 1:2000), rabbit polyclonal anti-TIF2 (Bethyl Laboratories, A300-346A, 1:2000), rabbit monoclonal anti-Perilipin1 (CST, #9349, 1:2000), rabbit polyclonal anti-Perilipin2 (Abcam, ab52356, 1:2000), mouse monoclonal anti-Complex III subunit Core 1 (Invitrogen, #459140, 1:2000), rabbit polyclonal anti-Tomm20 (Santa Cruz, sc-11415, 1:2000), rabbit polyclonal anti-Parkin (CST, #2132, 1:2000), rabbit polyclonal anti-PINK1 (Abcam, ab23707, 1:2000), rabbit polyclonal anti-Cleaved Caspase-3 (CST, #9661, 1:2000), rabbit polyclonal anti-FLAG (CST, #2368, 1:2000 for IP samples), mouse monoclonal anti-FLAG (Sigma-Aldrich, F1804, 1:2000 for other samples), mouse monoclonal anti-α-tubulin (Sigma-Aldrich, T5168, 1:25,000), mouse monoclonal anti-β-actin (MBL, M177-3, 1:25,000), HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, 111-035-003, 1:2000), HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch, 112-035-003, 1:2000) and HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, 115-035-003, 1:2000).

Techniques: Western Blot, Luciferase, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Age-dependent loss of adipose Rubicon promotes metabolic disorders via excess autophagy

doi: 10.1038/s41467-020-17985-w

Figure Lengend Snippet: a Alignment of LIR/GIM of SRC-1 and TIF2 homologues in various species. b , c Immunoprecipitation assay. HEK293T cells were transfected with the indicated plasmids for 48 h, and then the cells were lysed and immunoprecipitated with anti-FLAG antibody. Precipitates were subjected to immunoblotting with the indicated antibodies. n = 3 independent experiments with similar results. d , e Immunoblotting to detect FLAG in the 3T3-L1 cells overexpressing the indicated plasmids. Twenty-four hours after transfection, the cells were treated with or without 125 nM Baf A1 treatment for 8 h. n = 3 independent experiments. f , g Immunoblotting to detect FLAG in the 3T3-L1 cells. Knockdown was performed for 48 h starting on day 8 following induction of differentiation. The indicated plasmids were transfected for 24 h starting on day 9. Cells were harvested at day 10. n = 3 independent experiments. h Relative mRNA expression of adipogenic genes in 3T3-L1 cells. Knockdown was performed for 48 h starting on day 8 following induction of differentiation. FLAG-W288A-SRC-1 and/or FLAG-W296A TIF2 plasmids were transfected for 24 h starting on day 9. FLAG-only plasmid was used as empty vector. Cells were harvested at day 10. n = 6 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 ( h ), one-way ANOVA followed by Tukey’s test ( d–g ). P value from left to right: <0.0001, <0.0001 ( d ), 0.0013, 0.0019 ( e ), 0.0106, 0.8148 ( f ), 0.0170, 0.7972 ( g ), 0.0268, 0.0082, 0.5758, 0.6119, 0.0040, 0.0810, 0.0462 ( h ). * P < 0.05; ** P < 0.01; *** P < 0.001. N.S. not significant.

Article Snippet: The following antibodies were used for western blotting at the indicated dilutions: rabbit monoclonal anti-Rubicon (CST, #8465, 1:1000), mouse monoclonal anti-ULK1 (Santa Cruz Biotechnology, sc-33182, 1:2000), mouse monoclonal anti-ATG13 (Sigma-Aldrich, SAB4200100, 1:2000), rabbit polyclonal anti-ATG12 (CST, #2011, 1:2000), rabbit monoclonal anti-ATG16L1 (CST, #8089, 1:2000), mouse monoclonal anti-Beclin 1 (BD, 612112, 1:2000), rabbit polyclonal anti-LC3 (MBL, PM036, 1:2000), mouse monoclonal anti-p62 (MBL, M162-3, 1:2000 for IP samples), rabbit polyclonal anti-p62 (MBL, PM045, 1:5000 for other samples), rat monoclonal anti-Adiponectin (R&D Systems, MAB1119, 1:2000), mouse monoclonal anti-FABP4 (Santa Cruz Biotechnology, sc-271529, 1:2000), mouse monoclonal anti-PPARγ (Santa Cruz Biotechnology, sc-7273, 1:2000), rabbit monoclonal anti-SRC-1 (CST, #2191, 1:2000), rabbit polyclonal anti-TIF2 (Bethyl Laboratories, A300-346A, 1:2000), rabbit monoclonal anti-Perilipin1 (CST, #9349, 1:2000), rabbit polyclonal anti-Perilipin2 (Abcam, ab52356, 1:2000), mouse monoclonal anti-Complex III subunit Core 1 (Invitrogen, #459140, 1:2000), rabbit polyclonal anti-Tomm20 (Santa Cruz, sc-11415, 1:2000), rabbit polyclonal anti-Parkin (CST, #2132, 1:2000), rabbit polyclonal anti-PINK1 (Abcam, ab23707, 1:2000), rabbit polyclonal anti-Cleaved Caspase-3 (CST, #9661, 1:2000), rabbit polyclonal anti-FLAG (CST, #2368, 1:2000 for IP samples), mouse monoclonal anti-FLAG (Sigma-Aldrich, F1804, 1:2000 for other samples), mouse monoclonal anti-α-tubulin (Sigma-Aldrich, T5168, 1:25,000), mouse monoclonal anti-β-actin (MBL, M177-3, 1:25,000), HRP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, 111-035-003, 1:2000), HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch, 112-035-003, 1:2000) and HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, 115-035-003, 1:2000).

Techniques: Immunoprecipitation, Transfection, Western Blot, Expressing, Plasmid Preparation, Two Tailed Test