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Article Title:

doi:

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:

Article Title:

doi:

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

Journal: Cell reports

Article Title: Hypothalamic steroid receptor coactivator-2 regulates adaptations to fasting and overnutrition

doi: 10.1016/j.celrep.2021.110075

Figure Lengend Snippet: (A) Dual immunofluorescence for GFP (green) and SRC-2 (red) in the ARH of POMC-EGFP mice (controls, upper panels) and pomcSRC-2-KO/POMC-EGFP mice (lower panels). Arrowheads point to double-labeled neurons. Scale bar represents 100 μm. 3V, 3 rd ventricle; ARH, arcuate nucleus of the hypothalamus; ME, median eminence; VMH, ventromedial hypothalamic nucleus. (B and C) Changes in body weight (B), fat mass, or lean mass (C) of chow-fed male control and pomcSRC-2-KO littermates (3 to 4 months old) after a 24-h fasting. Data are presented as mean ± SEM (% baseline before fasting). n = 6 or 10 mice per group. **p < 0.01 in t tests. (D) Daily energy expenditure measured by CLAMS in male control and pomcSRC-2-KO littermates (3 months old) during ad libitum fed, fasted, and refed periods. Data are presented as mean ± SEM. n = 8 or 9 mice per group. *p < 0.05 and ***p < 0.001 in two-way ANOVA analyses repeated measurement followed by Sidak tests. (E) Representative microscopic images showing a recorded POMC neuron (GFP-labeled in POMC-EGFP mice). Scale bar represents 10 μm. (F) Representative action potential traces in POMC neurons from fasted control versus pomcSRC-2-KO mice. (G and H) Quantifications of firing rate (G) and resting membrane potential (H) in POMC neurons from fasted control versus pomcSRC-2-KO mice. Data are presented as mean ± SEM. n = 17 or 21 neurons from 3 mice per group. *p < 0.05 in t tests. (I) Representative traces for mIPSC recorded in POMC neurons from fasted control versus pomcSRC-2-KO mice. (J and K) Quantifications of frequency (J) and amplitude (K) of mIPSC. Data are presented as mean ± SEM. n = 15 or 17 neurons from 3 mice per group. *p < 0.05 in t tests. (L) Relative mRNA levels of indicated genes measured in POMC neurons isolated from control versus pomcSRC-2-KO mice. Data are presented as mean ± SEM. n = 8 samples per group. *p < 0.05 in t tests.

Article Snippet: The blot was probed with anti-Flag-HRP (1:3,000–10,000, A8592, Sigma), rabbit polyclonal anti-SRC-2 antibody (1:1,000, ab10491, Abcam) or mouse monoclonal anti-β-Actin-HRP (1:5,000, #12262, Cell Signaling), and the secondary antibody was goat anti-rabbit IgG (Jackson ImmunoResearch), followed by development with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Techniques: Immunofluorescence, Labeling, Isolation

Journal: Cell reports

Article Title: Hypothalamic steroid receptor coactivator-2 regulates adaptations to fasting and overnutrition

doi: 10.1016/j.celrep.2021.110075

Figure Lengend Snippet: (A) Effects of SRC-2 on POMC-luciferase activity in “fed” and fasted cells. Data are presented as mean ± SEM. n = 4 or 5 repeated experiments with 6 biological replicates per group in each experiment. *p < 0.05 and ***p < 0.001 in two-way ANOVA analyses followed by Sidak tests. (B) Effects of SRC-2 on POMC-luciferase activity in fasted cells with or without FoxO1 knockdown. Data are presented as mean ± SEM. n = 4–7 repeated experiments with 6 biological replicates per group in each experiment. ***p < 0.001 in two-way ANOVA analyses followed by Sidak tests. (C) Hypothalamic mRNAs in fasted control and pomcSRC-2-KO mice. Data are presented as mean ± SEM. n = 14 or 15 mice per group. *p < 0.05 in t tests. (D) Interaction of endogenous SRC-2 and FoxO1 in the hypothalamus from chow- or HFD-fed male mice. (E) Body weight curves in male control and pomcSRC-2-KO littermates fed HFD ad libitum since 7 weeks of age. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05 in two-way ANOVA analyses repeated measurement followed by Sidak tests. (F) Fat or lean mass in 14-week-old male control and pomcSRC-2-KO littermates after 7 weeks HFD feeding. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05 in t tests. (G) Cumulative HFD intake in male control and pomcSRC-2-KO littermates fed HFD ad libitum since 7 weeks of age. Data are presented as mean ± SEM. n = 8 mice per group. *p < 0.05 in two-way ANOVA analyses repeated measurement followed by Sidak tests. (H) Changes in body weight during a 3-week HFD feeding in mice receiving stereotaxic injections of AAV-FLEX-FoxO1 AAA or AAV-GFP into the ARH. Data are presented as mean ± SEM. n = 6–10 mice per group. * or #, p < 0.05 between POMC-Cre+ AAV-FLEX-FoxO1 AAA versus WT+ AAV-FLEX-FoxO1 AAA or versus pomcSRC-2-KO+AAV-FLEX-FoxO1 AAA in two-way ANOVA analyses repeated measurement followed by Sidak tests. (I and J) Changes in body weight (I) and fat mass (J) at the end of the 3-week HFD feeding in mice described in (H). Data are presented as mean ± SEM. n = 6–10 mice per group. *p < 0.05 in two-way ANOVA analyses followed by Sidak tests; p = 0.09 in t tests. (K) Cumulative HFD intake in mice described in (H). Data are presented as mean ± SEM. n = 6–10 mice per group. * or #, p < 0.05 between POMC-Cre+ AAV-FLEX-FoxO1 AAA versus WT+ AAV-FLEX-FoxO1 AAA or versus pomcSRC-2-KO+AAV-FLEX-FoxO1 AAA in two-way ANOVA analyses repeated measurement followed by Sidak tests.

Article Snippet: The blot was probed with anti-Flag-HRP (1:3,000–10,000, A8592, Sigma), rabbit polyclonal anti-SRC-2 antibody (1:1,000, ab10491, Abcam) or mouse monoclonal anti-β-Actin-HRP (1:5,000, #12262, Cell Signaling), and the secondary antibody was goat anti-rabbit IgG (Jackson ImmunoResearch), followed by development with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Techniques: Luciferase, Activity Assay

Journal: Cell reports

Article Title: Hypothalamic steroid receptor coactivator-2 regulates adaptations to fasting and overnutrition

doi: 10.1016/j.celrep.2021.110075

Figure Lengend Snippet:

Article Snippet: The blot was probed with anti-Flag-HRP (1:3,000–10,000, A8592, Sigma), rabbit polyclonal anti-SRC-2 antibody (1:1,000, ab10491, Abcam) or mouse monoclonal anti-β-Actin-HRP (1:5,000, #12262, Cell Signaling), and the secondary antibody was goat anti-rabbit IgG (Jackson ImmunoResearch), followed by development with the SuperSignal West Pico Chemiluminescent Substrate (Pierce).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Luciferase, Transgenic Assay

Journal:

Article Title: GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

doi: 10.1242/jcs.009878

Figure Lengend Snippet: Agarose-bound GST fusion proteins of ERRγ (1ug) were used to pull down whole cell lysates containing a fixed amount of SRC1 (A) or SRC2 (B), mixed with increasing amounts of the wild-type GNL3L (A1, B1) or the dI mutant lacking the ERRγ-interacting domain (A2, B2). Whole cell proteins in each sample were adjusted to the same amount. In the agarose-retained portions (R), the interaction between GNL3L and ERRγ can reduce the amount of SRC1 and SRC2 bound by ERRγ in a dose-dependent manner, but the dI mutant fails to do so. Conversely, when GST-ERRγ fusion proteins were used to pull down the same amount of GNL3L in the presence of increasing amounts of SRC1 (C) or SRC2 (D), SRC1 and SRC2 were able to reduce the amount of GNL3L bound by ERRγ in a dose-dependent way as well. Proteins in the agarose-bound fraction and in the supernatant are indicated by (R) and (S), respectively.

Article Snippet: Western analyses were performed using the mouse anti-Myc, rabbit anti-HA, rabbit anti-SRC1 (abcam, ab2859, 500X), and mouse anti-SRC2 (BD Transduction Laboratories, clone 29, 250X) antibodies.

Techniques: Mutagenesis

Journal:

Article Title: GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

doi: 10.1242/jcs.009878

Figure Lengend Snippet: (A) The GNL3L effect on the DNA binding of ERRγ was examined by electrophoretic mobility shift assays (EMSA) using ERE-containing probes and whole cell lysates expressing the indicated recombinant proteins. Compared to the probe alone (lane 1) and the vector-transfected control sample (lane 2), the ERRγ-specific DNA-protein complex can be identified in lane 3 (arrow b), competed by excess non-labeled probes (lane 4), and supershifted by anti-Myc antibody (lane 5, arrow a). Coexpression of GNL3L produces fast-moving complexes (lane 6, arrows d and e), which can be supershifted by anti-Myc antibody (lane 7, arrow c) but not by anti-HA antibody (lane 8). GNL3L itself cannot bind the ERE probe (lane 9). The intensity of the fast-moving complex d is reduced by a deletion of the ERRγ-binding I-domain of GNL3L (lanes 10–12). (B) The fast-moving complex d and the slow-moving complex b were retrieved from the EMSA gel, fractionated in SDS-denaturing PAGE, and analyzed for their ERRγ (α-Myc), GNL3L (α-HA), SRC1, and SRC2 protein components by western blottings. Our results indicate that the increase in the electrophoretic mobility of the ERRγ-DNA complex by GNL3L coexpression can be explained by a loss of SRC1 binding (arrow) and diminished SRC2 binding, rather than by protein cleavage of ERRγ.

Article Snippet: Western analyses were performed using the mouse anti-Myc, rabbit anti-HA, rabbit anti-SRC1 (abcam, ab2859, 500X), and mouse anti-SRC2 (BD Transduction Laboratories, clone 29, 250X) antibodies.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Expressing, Recombinant, Plasmid Preparation, Transfection, Labeling, Western Blot

Journal:

Article Title: GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

doi: 10.1242/jcs.009878

Figure Lengend Snippet: (A) Using the same cell-based reporter system as described in Fig. 4, we show that the ERE-specific transcriptional activity in cells coexpressing ERRγ and SRC1 (8.0±0.3) is 1.7 times higher than that of the ERRγ-expressing sample (4.8±0.3). When coexpressed with the wild-type GNL3L (WT), this ERRγ and SRC1-mediated ERE-specific transcriptional activity is reduced by 55 and 70 percent compared to the sample expressing both ERRγ and SRC1 in a dose-dependent manner. This inhibitory effect of GNL3L on the SRC1-mediated coactivation of ERRγ requires the I-domain of GNL3L, as a deletion of this domain (dI) fails to suppress the transcriptional activity of ERRγ and SRC1 (P value = 0.17). (B) Using the same approach, we show that GNL3L can also suppress the coactivator function of SRC2 on the ERRγ-dependent transcriptional activity in a dose-dependent (54% reduction for 100ng of GNL3L and 71% reduction for 200ng of GNL3L) and I-domain-dependent (P value = 0.58) manner. Error bars represent stand error of mean (s.e.m.). ***, P value < 0.0001.

Article Snippet: Western analyses were performed using the mouse anti-Myc, rabbit anti-HA, rabbit anti-SRC1 (abcam, ab2859, 500X), and mouse anti-SRC2 (BD Transduction Laboratories, clone 29, 250X) antibodies.

Techniques: Activity Assay, Expressing

Journal:

Article Title: GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

doi: 10.1242/jcs.009878

Figure Lengend Snippet: Our data reveal a novel mechanism that regulates the activity of ERR family genes by a nucleolar GTP-binding protein GNL3L. GNL3L decreases the transcriptional activity of ERR proteins. This event takes place in the nucleoplasm and does not require the nucleolar localization of GNL3L. The interaction between GNL3L and ERRγ displaces coactivators such as SRC1 and SRC2 from the ERRγ complex. The SRC-depleted ERRγ protein binds DNA without GNL3L, resulting in transcriptional inhibition. In this model, the nucleolar accumulation of GNL3L does not appear to affect its ability to suppress the transcriptional function of ERR proteins (grey arrows). Abbreviations for protein domains of GNL3L and ERR are explained in Fig. 2A and 2D.

Article Snippet: Western analyses were performed using the mouse anti-Myc, rabbit anti-HA, rabbit anti-SRC1 (abcam, ab2859, 500X), and mouse anti-SRC2 (BD Transduction Laboratories, clone 29, 250X) antibodies.

Techniques: Activity Assay, Binding Assay, Inhibition