chip buffer  (Proteintech)

Journal: Molecular Metabolism

Article Title: The transcription factor CUX1 exerts opposing roles in human and mouse adipocyte differentiation

doi: 10.1016/j.molmet.2025.102290

Figure Lengend Snippet: ChIP-SICAP identifies the transcription factor CUX1 as a positive regulator of human adipocyte differentiation. (A) Schematic of C/EBPβ ChIP-SICAP experiments in hAPCs. (B) C/EBPβ ChIP-SICAP was performed at day 0 (undifferentiated hAPCs) and one day (day 1) after inducing adipocyte differentiation. Proteins enriched at day 1 vs. day 0 (FC > 3, P value < 0.0005) were annotated. (C) Expression heatmap shows mRNA levels of adipocyte markers in hAPCs in which individual genes (from B) were knocked-out (deleted genes on x-axis) relative to control cells at day 3 of differentiation. (D) SNPs near CUX1 that are associated with altered susceptibility to type 2 diabetes (data from T2D knowledge portal). (E) CUX1 mRNA levels in subcutaneous adipose tissue from non-diabetic (n = 31) and T2D (n = 39) subjects. GADPH was used for normalization. Values represent mean ± SEM. (F) Western blot analysis of CUX1 and GAPDH (loading control) protein levels in control (ctrl) and CUX1 knock-out (KO) hAPCs. CUX1 forms are labelled (see details in ): Full length (FL) and Cut-associated splicing product (CASP). (G) mRNA levels of CUX1 and indicated adipocyte marker genes in ctrl and KO cells on day 14 of differentiation. Values represent mean ± SEM, n = 3 biol. repl. per condition. (H, I) Bodipy staining (H) and TG levels (I) in ctrl and KO cells. Values represent mean ± SEM, n = 3 biol. repl. per condition. E,I, unpaired two-tailed Student's t tests; G, one-way ANOVA followed by Dunnett multiple comparisons. ∗P < 0.05.

Article Snippet: For immunoprecipitation samples, chromatin was incubated overnight at 4 °C with CUX1 antibody (11733-1-AP, Proteintech) in ChIP buffer (1 % Triton X-100, 0.1 % SDS, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0) supplemented with protease inhibitors.

Techniques: Expressing, Control, Western Blot, Knock-Out, Marker, Staining, Two Tailed Test

Journal: Molecular Metabolism

Article Title: The transcription factor CUX1 exerts opposing roles in human and mouse adipocyte differentiation

doi: 10.1016/j.molmet.2025.102290

Figure Lengend Snippet: CUX1 activates PPARG and adipogenic genes in hAPCs. (A) Top enriched pathways (Biological Processes (BP) database) identified from genes that are: downregulated by CUX1 knockout (KO) and upregulated by CUX1-overexpression in hAPCs at day 3 of differentiation. (B) Expression heatmap of adipocyte genes and precursor marker genes ( DPP4 , ICAM1 ) in CUX1 KO and OE hAPCs, relative to control cells (Ctrl) at day 3 of differentiation. (C) ChIP-seq tracks for CUX1 at COPS8 , PPARG, and EBF2 in Ctrll and CUX1 KO hAPCs one day after treatment with differentiation cocktail (day 1). (D) Motif analysis of CUX1 binding regions identified in Ctrl hAPCs. (E) ChIP-qPCR analysis of CUX1 binding at the +66 kb region (labeled in (C)) of PPARG in control and KO hAPCs stimulated with differentiation cocktail for 1 day. Values represent mean ± SEM, n = 3 biol. repl., unpaired two-tailed Student's t test. (F) Transcription assay showing activity of the +66 kb region of PPARG in immortalized hAPCs transfected with control or CUX1-expressing vector. Values represent mean ± SEM, n = 3 biol. repl., one-way ANOVA followed by Dunnett multiple comparisons test. (G) mRNA levels of adipocyte genes in control and KO cells treated with vehicle (control) or 1 μM rosiglitazone at day 14 of differentiation Values represent mean ± SEM, n = 3 biol. repl., two-way ANOVA followed by Sidak's test. (H) Plot showing correlation between CUX1 and PPARG mRNA levels in subcutaneous adipose tissue from healthy, nondiabetic female individuals (n = 39). ∗P < 0.05, ns, not significant.

Article Snippet: For immunoprecipitation samples, chromatin was incubated overnight at 4 °C with CUX1 antibody (11733-1-AP, Proteintech) in ChIP buffer (1 % Triton X-100, 0.1 % SDS, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0) supplemented with protease inhibitors.

Techniques: Knock-Out, Over Expression, Expressing, Marker, Control, ChIP-sequencing, Binding Assay, ChIP-qPCR, Labeling, Two Tailed Test, Transcription Assay, Activity Assay, Transfection, Plasmid Preparation

Journal: Molecular Metabolism

Article Title: The transcription factor CUX1 exerts opposing roles in human and mouse adipocyte differentiation

doi: 10.1016/j.molmet.2025.102290

Figure Lengend Snippet: Cux1 represses mouse adipocyte differentiation. (A – D) APCs were isolated from inguinal white adipose tissue (iWAT) or epididymal WAT (eWAT) of control and APC-selective Cux1 KO mice (male, age 6–8 weeks) 5 days after tamoxifen injection and then induced to differentiate for 9 days. (A, C) Western blot analysis of CUX1 and GADPH (loading control) in cells from iWAT (A) and eWAT (C). (B, D) mRNA levels of adipocyte genes in cells from iWAT (B) and eWAT (D). (E) Western blot analysis of CUX1 and GADPH (loading control) in control and CUX1-overexpressing (OE) hAPCs that were differentiated for 9 days. (F) mRNA levels of adipocyte genes in cells from (E). (G) ChIP-seq tracks for CUX1 in APCs isolated from inguinal WAT of control and KO mice. (H) Motif analysis of CUX1 binding regions in mAPCs stimulated with differentiation cocktail for 1 day. B, D, F, values represent mean ± SEM, n = 3 biol. repl. per condition, one-way ANOVA followed by Dunnett multiple comparisons test. ∗P < 0.05.

Article Snippet: For immunoprecipitation samples, chromatin was incubated overnight at 4 °C with CUX1 antibody (11733-1-AP, Proteintech) in ChIP buffer (1 % Triton X-100, 0.1 % SDS, 150 mM NaCl, 2 mM EDTA, 20 mM Tris–HCl, pH 8.0) supplemented with protease inhibitors.

Techniques: Isolation, Control, Injection, Western Blot, ChIP-sequencing, Binding Assay