primary antibodies to mouse foxp1 abe68  (Millipore)

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Foxp1 expression in adipocytes is induced by adrenergic stimuli. a H&E staining and immunofluorescence (IF) analysis for the Foxp1 expression in BAT and sWAT from wild type mice at age of 4 weeks. DAPI, blue staining for nucleus; green color for Foxp1 expression. Bar, 50 μm. b Western blotting showed the four isoforms (A, B, D, C) of Foxp1 protein in BAT and sWAT from wild type mice at age of 2 months. c IHC analysis of FOXP1 expression in biopsies from PHEO patients and normal controls. Bar, 10 μm. d qPCR analysis of expression of Foxp1 and brown adipocyte markers ( C/ebpα , Pparγ , and Ucp1 ) during the time course of brown adipocyte differentiation from SVFs. e qPCR analysis of Foxp1 and β3-AR expression in BAT in mice with overnight 4 °C cold exposure. n = 3 biologically independent samples. f Western blotting of Foxp1 in BAT from mice above ( e ). g , h qPCR analysis of Foxp1 and β3-AR expression in brown adipocytes differentiated from murine ( g ) and human SVF ( h ) during an 8-hour CL-316,243 (0.1 μM) treatment as indicated. n = 3 biologically independent experiments. i Foxp1 expression profile in adipocytes derived from 3T3-L1 cells during an 8-h time course, stimulated by CL-316,243 (0.5 μM) with or without SB202190 (p38 kinase inhibitor, 10 μM), FR180204 (Erk1/2 inhibitors, 1 μM) and SCH772984 (Erk1/2 inhibitors, 10 μM), respectively. n = 3 biologically independent experiments. j Western blotting for Foxp1 in brown adipocytes derived from SVF, which were stimulated by CL-316,243 (0.1 μM) with or without SCH772984 (10 μM) for 8 h. * P < 0.05; ** P < 0.01; *** P < 0.001; error bar, mean ± SEM

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 h at room temperature, then incubated overnight at 4 °C with primary antibodies to mouse Foxp1 (Millipore, ABE68, 1:100), Ucp1 (Abcam, ab10893, 1:50), Prdm16 (Abcam, ab106410, 1:50).

Techniques: Expressing, Staining, Immunofluorescence, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Foxp1 deficiency promotes brown and beige adipocyte activation. a Representative dorsal view of BAT depot in Foxp1 fl/fl and Foxp1 Myf5 ∆/∆ mice at postnatal day 7. Bar, 1 cm. b HE (upper panel) and IHC staining (lower panel) with UCP1 antibody for BAT in ( a ). Bar, 10 μm. c , d qPCR analysis of Foxp1 and BAT-selective markers for BAT depot. n = 5 biologically independent samples. e Representative view of 3-month-old Foxp1 fl/fl and Foxp1 Ad ∆/∆ mice. Bar, 2 cm. f Representative view of BAT (upper panel) and sWAT depot (lower panel) in ( e ). g HE staining for BAT and sWAT in mutant mice. Bar, 50 μm. h IHC analysis with UCP1 antibody for BAT and sWAT upon 6-h cold exposure. Bar, 50 μm. i – k qPCR for expression of Foxp1 and BAT-selective genes in BAT and sWAT from Foxp1 fl/fl and Foxp1 Ad ∆/∆ mutant mice. n = 4 biologically independent mice/each group. * P < 0.05; ** P < 0.01; *** P < 0.001; error bar, mean ± SEM

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 h at room temperature, then incubated overnight at 4 °C with primary antibodies to mouse Foxp1 (Millipore, ABE68, 1:100), Ucp1 (Abcam, ab10893, 1:50), Prdm16 (Abcam, ab106410, 1:50).

Techniques: Activation Assay, Immunohistochemistry, Staining, Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Foxp1 deletion elevates energy expenditure and resists to HFD-induced obesity. a , b VO 2 and VCO 2 of Foxp1 fl/fl and Foxp1 Ad ∆/∆ mice in metabolic cages at age of 3 months old. n = 7 biologically independent mice/each group. c , d Quantification of O 2 and CO 2 consumption in light and dark. e Dorsal view of Foxp1 fl/fl and Foxp1 Ad ∆/∆ mice after 8-week feeding with HFD at age of 6 months old. Bar, 2 cm. f Representative depot of BAT and sWAT in HFD-fed mice ( e ). Bar, 1 cm. g Body weight of HFD-fed mice. h Relative adiposity of HFD-fed mice. i Statistics of rectal temperature of HFD-fed mice. j , k GTT and ITT of HFD-fed mice. * P < 0.05; ** P < 0.01; *** P < 0.001; n = 7 biologically independent mice/each group; error bar, mean ± SEM

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 h at room temperature, then incubated overnight at 4 °C with primary antibodies to mouse Foxp1 (Millipore, ABE68, 1:100), Ucp1 (Abcam, ab10893, 1:50), Prdm16 (Abcam, ab106410, 1:50).

Techniques:

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Overexpression of Foxp1 represses adaptive thermogenesis and is sensitive to HFD-induce obesity. a Representative bioluminescence imaging of two stains of aP2-Foxp1;Ucp1-Luc transgenic mice after 6-hour cold exposure. n = 3 biologically independent mice/each group. Bar, 2 cm. b Representative picture of BAT depot in aP2-Foxp1 transgenic mice. Bar, 0.5 cm. c H&E staining for adipose sections of BAT and sWAT from transgenic mice in ( b ). Bar, 10 μm. d IHC with UCP1 antibody for adipose sections from transgenic mice after 6-h cold exposure. Bar, 10 μm. e Intracellular structure of adipocytes in BAT and sWAT from aP2-Foxp1 mice, as showed by transmission electronic microscope (TEM). Bar, 2 μm. f Assessment of mitochondria DNA abundance by qPCR for the BAT and sWAT from transgenic mice. n = 4 biologically independent mice/each group. g qPCR analysis for Ucp1 expression in adipose tissues of transgenic mice. n = 4 biologically independent samples. h Western blotting for UCP1, HSL, and phosphorylated HSL in BAT and sWAT. i Representative dorsal view of aP2-Foxp1 transgenic mice subjected to 8-week HFD feeding since at age of 3 months. Bar, 2 cm. j Relative adiposity in transgenic mice ( i ). n = 4 biologically independent mice/each group. k The heat map of relative expression of adipogenic genes in three strains of aP2-Foxp1 transgenic mice at age of 3 weeks, as evaluated by RNA-seq. l qPCR analysis validated the BAT-selective genes expression in BAT from ( k ). n = 3 biologically independent mice/each group; * P < 0.05; ** P < 0.01; *** P < 0.001; error bar, mean ± SEM

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 h at room temperature, then incubated overnight at 4 °C with primary antibodies to mouse Foxp1 (Millipore, ABE68, 1:100), Ucp1 (Abcam, ab10893, 1:50), Prdm16 (Abcam, ab106410, 1:50).

Techniques: Over Expression, Imaging, Transgenic Assay, Staining, Transmission Assay, Microscopy, Expressing, Western Blot, RNA Sequencing Assay

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Foxp1 represses β3-AR transcription in adipocytes. a, b β3-AR expression in BAT and sWAT were assessed by qPCR and western blotting in aP2-Foxp1 transgenic mice at 2 month old. n = 3 biologically independent samples/each group. c, d β3-AR expression in BAT and sWAT were assessed by qPCR and western blotting in Foxp1 aP2 ∆/∆ knockout mice at 2 months old. n = 3 biologically independent samples/each group. e ChIP-seq profile showed the Foxp1 binding sites within Adrb3 gene promoter region. Red arrows indicated the potential Foxp1 binding sites. Adrb3 gene was reversely transcribed. ChIP was based on SVF cells derived from BAT. f Upper panel showed the location of Foxp1 binding site at −2251 bp upstream of β3-AR gene promoter with a schematic drawing. ChIP-PCR confirmed the relative enrichment of Foxp1 binding sites (lower panel). n = 3 biologically independent samples/each group. g Basic OCR of SVF-derived brown adipocytes from Foxp1 aP2 ∆/∆ mice, which were transfected β3-AR -shRNA lentivirus or administrated with β3-AR inhibitor L748337 (10 μM) for 1 h. n = 3 biologically independent experiments. h Representative infrared imaging of aP2-Foxp1 transgenic mice with or without 6-hour CL-316,243 (10 μM) exposure. Bar, 1 cm. i Quantification of average skin temperature in boxed regions in ( h ). n = 4 biologically independent mice/each group. Samples were isolated from mice at age of 2 months. j CL-stimulated β3-AR desensitization in SVF-derived brown adipocytes from aP2-Foxp1 transgenic mice and Foxp1 aP2 ∆/∆ knockout mice. β3-AR expression was assessed by qPCR during the 6-h time course of CL-316243 (0.1 μM) treatment. n = 3 biologically independent samples/each group. * P < 0.05; ** P < 0.01; *** P < 0.001; error bar, mean ± SEM

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 h at room temperature, then incubated overnight at 4 °C with primary antibodies to mouse Foxp1 (Millipore, ABE68, 1:100), Ucp1 (Abcam, ab10893, 1:50), Prdm16 (Abcam, ab106410, 1:50).

Techniques: Expressing, Western Blot, Transgenic Assay, Knock-Out, ChIP-sequencing, Binding Assay, Derivative Assay, Transfection, shRNA, Imaging, Isolation

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Foxp1 physically interacts with Prdm16-C/ebpβ complex in brown adipocytes. a , b Co-immunoprecipitation validated the physical interaction between Foxp1, C/ebpβ and Prdm16 proteins in cell lysates from 293T cell lines transfected with indicated plasmids ( a ), or brown adipocytes induced from stromal vascular fractions ( b ). c Immunofluorescence analysis showed the co-localization of Foxp1 and Prdm16 in the nucleus of BAT from wild type mice at age of 1 month old. Bar, 10 μm. d Luciferase reporter assay showed the transactivation of PPARγ -Luc by Foxp1 and Prdm16 protein in 3T3-L1 cell lines. n = 3 biologically independent samples/each group. e Luciferase reporter assay showed the transactivation of β3-AR -Luc by Foxp1, C/ebpβ and Prdm16 protein in 293T cell lines. n = 3 biologically independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001; error bar, mean ± SEM. f Diagram depicting the mechanisms that Foxp1 represses brown/beige adipocyte differentiation and adaptive thermogenesis. Expression of Foxp1 is induced by β3-AR/cAMP/Erk1/2 cascades. Conversely, Foxp1 suppresses β3-AR transcription by counteracting the activity of Prdm16-C/ebpβ complex

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 h at room temperature, then incubated overnight at 4 °C with primary antibodies to mouse Foxp1 (Millipore, ABE68, 1:100), Ucp1 (Abcam, ab10893, 1:50), Prdm16 (Abcam, ab106410, 1:50).

Techniques: Immunoprecipitation, Transfection, Immunofluorescence, Luciferase, Reporter Assay, Expressing, Activity Assay