r d systems cat af8125  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Activating transcription factor 3 regulates hepatic apolipoprotein A4 upon metabolic stress

doi: 10.1016/j.jbc.2025.108468

Figure Lengend Snippet: Serum protein screening shows ApoA4 upregulation in several models of dysregulated hepatic metabolism. Coomassie-stained SDS-PAGE gels of serum proteins, followed by Western blots for ApoA4 with ponceau stained albumin bands for loading control. A , serum was derived from male mice, fasted 24 h, with WT, Cpt2 L−/− , PCx L−/− , PDH L−/− genotypes. n = 4. B , comparison of WT, TSC1 L−/− , Pparα −/− , and InsR L−/− genotypes; n = 4. C , serum derived from female mice, fasted 24 h, with WT, PCx L−/− , Cpt2 L−/− , PDH L−/− , and TSC1 L−/− genotypes; n = 3. D , serum derived from WT male mice in various dietary states: Chow-fed 4 h fast, chow-fed 24 h fast, high-fat diet–fed 4 h fast, and ketogenic diet–fed 4 h fast; n = 4. Relative values for Western blot band intensities are illustrated. Statistical significance was determined by ordinary one-way ANOVA with the Brown-Forsythe test, Bartlett's test, and Bonferroni multiple comparisons test, conducted with GraphPad prism; n = 4. Data expressed as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. ApoA4, apolipoprotein A4; Cpt2, carnitine palmitoyltransferase 2; PCx, pyruvate carboxylase; InsR, insulin receptor; PDH, pyruvate dehydrogenase; TSC1, tuberous sclerosis complex 1.

Article Snippet: After another 3 × 5 m TBST washes, blots were incubated with primary antibodies in 3% bovine serum albumin overnight at 4 °C; ApoA4 (#AF8125, R&D systems), Atf3 (#33593, Cell Signaling), and HSC70(sc-7298, Santa Cruz).

Techniques: Staining, SDS Page, Western Blot, Control, Derivative Assay, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Activating transcription factor 3 regulates hepatic apolipoprotein A4 upon metabolic stress

doi: 10.1016/j.jbc.2025.108468

Figure Lengend Snippet: Changes in serum ApoA4 levels vary across PCx L−/− and Cpt2 L−/− mice fed chow, high-fat, and ketogenic diets. Coomassie-stained SDS-PAGE gel of serum proteins, followed by Western blots for ApoA4 with ponceau-stained albumin bands for loading control. Serum was collected from male mice, fasted 4 h, in the following groups (n = 4): ( A ) PCx L f/f (phenotypically WT) on chow or HFD, PCx L−/− (liver knockout) on chow or HFD; ( B ) PCx L f/f on chow or KD, PCx L−/− on chow or KD; ( C ) Cpt2 L f/f on chow or HFD, Cpt2 L−/− on chow or HFD; ( D ) Cpt2 L f/f on chow or KD, and Cpt2 L−/− on chow or KD. Relative values for Western blot band intensities are illustrated. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test, conducted with GraphPad prism; n = 4. Data expressed as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. ApoA4, apolipoprotein A4; Cpt2, carnitine palmitoyltransferase 2; HFD, high-fat diet; KD, ketogenic diet; PCx, pyruvate carboxylase.

Article Snippet: After another 3 × 5 m TBST washes, blots were incubated with primary antibodies in 3% bovine serum albumin overnight at 4 °C; ApoA4 (#AF8125, R&D systems), Atf3 (#33593, Cell Signaling), and HSC70(sc-7298, Santa Cruz).

Techniques: Staining, SDS Page, Western Blot, Control, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Activating transcription factor 3 regulates hepatic apolipoprotein A4 upon metabolic stress

doi: 10.1016/j.jbc.2025.108468

Figure Lengend Snippet: Relative liver ApoA4 mRNA levels increase for PCx L−/− and Cpt2 L−/− mice in different dietary states, as determined by reverse transcriptase-quantitative polymerase chain reaction . Dietary states described as: chow diet, 4 h fast; chow diet, 24 h fast; high-fat diet, 4 h fast; and ketogenic diet, 4 h fast. A – F , liver mRNA (ApoA1, ApoA4, ApoA5, ApoB, ApoC3, and ApoE, respectively) of PCx L f/f and PCx L−/− mice with described dietary states, relative to chow diet 4 h fast PCx L f/f . G – L , liver mRNA (ApoA1, ApoA4, ApoA5, ApoB, ApoC3, and ApoE, respectively) of Cpt2 L f/f and Cpt2 L−/− mice with described dietary states, relative to chow diet 4 h fast Cpt2 L f/f . Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test, conducted using GraphPad prism. Data are expressed as mean ± SD. Single-letter difference denotes p < 0.05 between groups. ApoA4, apolipoprotein A4; Cpt2, carnitine palmitoyltransferase 2; PCx, pyruvate carboxylase.

Article Snippet: After another 3 × 5 m TBST washes, blots were incubated with primary antibodies in 3% bovine serum albumin overnight at 4 °C; ApoA4 (#AF8125, R&D systems), Atf3 (#33593, Cell Signaling), and HSC70(sc-7298, Santa Cruz).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Activating transcription factor 3 regulates hepatic apolipoprotein A4 upon metabolic stress

doi: 10.1016/j.jbc.2025.108468

Figure Lengend Snippet: Hepatic ApoA4 upregulation is most prominent with a 24 h fast in the absence of fatty acid oxidation. A , Western blots comparing serum ApoA4 levels in 24 h-fasted WT, PCx L−/− , Cpt2 L−/− , and PCx/Cpt2 L−/−;−/− (double liver KO) mice. Ponceau-stained serum albumin bands were used as loading controls. B , reverse transcriptase-quantitative polymerase chain reaction comparison of ApoA4 mRNA from liver homogenate across 24 h-fasted WT (n = 5), PCx L−/− (n = 5), Cpt2 L−/− (n = 6), and PCx/Cpt2 L−/−;−/− mice (n = 6). In groups with n = 5, one outlier was excluded prior to statistical analysis due to aberrant Cq values for control genes (CycloA and 18s). Data are expressed as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. ApoA4, apolipoprotein A4; Cpt2, carnitine palmitoyltransferase 2; PCx, pyruvate carboxylase.

Article Snippet: After another 3 × 5 m TBST washes, blots were incubated with primary antibodies in 3% bovine serum albumin overnight at 4 °C; ApoA4 (#AF8125, R&D systems), Atf3 (#33593, Cell Signaling), and HSC70(sc-7298, Santa Cruz).

Techniques: Western Blot, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison, Control

Journal: The Journal of Biological Chemistry

Article Title: Activating transcription factor 3 regulates hepatic apolipoprotein A4 upon metabolic stress

doi: 10.1016/j.jbc.2025.108468

Figure Lengend Snippet: Atf3 overexpression in WT mice significantly upregulates hepatic ApoA4 in females, but not males with a 24 h fast. A , coomassie-stained SDS-PAGE gels of serum proteins, followed by Western blots for ApoA4. Both male and female groups are shown for hepatic Atf3 or eGFP-overexpressing mice. Serum proteins are compared under a 4 h fast, followed by a 24 h fast replicate using the same mice ( B ) reverse transcriptase-quantitative polymerase chain reaction was used to assess hepatic mRNA abundance of apolipoproteins in 24 h-fasted female (n = 3) ( C ) or male (n = 4) ( D ) Atf3-overexpressing mice compared to GFP controls. Relevant genes in the CREB/Atf family were also assessed by reverse transcriptase-quantitative polymerase chain reaction in female ( E ) and male ( F ) groups. Data are expressed as mean ± SD and analyzed using Welch’s t test with no correction for multiple comparisons. All statistical tests were conducted using GraphPad prism. ApoA4, apolipoprotein A4; AFT, activating transcription factor; Cpt2, carnitine palmitoyltransferase 2; PCx, pyruvate carboxylase.

Article Snippet: After another 3 × 5 m TBST washes, blots were incubated with primary antibodies in 3% bovine serum albumin overnight at 4 °C; ApoA4 (#AF8125, R&D systems), Atf3 (#33593, Cell Signaling), and HSC70(sc-7298, Santa Cruz).

Techniques: Over Expression, Staining, SDS Page, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Activating transcription factor 3 regulates hepatic apolipoprotein A4 upon metabolic stress

doi: 10.1016/j.jbc.2025.108468

Figure Lengend Snippet: Hepatic Atf3 L−/− /Cpt2 L−/− double KOs demonstrate a reduced induction of hepatic ApoA4 in females, with a transcriptional profile distinct from Cpt2 L−/− single knockouts. A , validation of Atf3 L−/− /Cpt2 L−/− double KO model using reverse transcriptase-quantitative polymerase chain reaction to show significantly reduced levels of hepatic Atf3 and Cpt2 mRNA. B , reverse transcriptase-quantitative polymerase chain reaction of liver mRNA for ApoA4 and its documented regulators, CREB3L3, CREB3, and ERRα. C , Western blots comparing serum ApoA4 levels in Atf3 L−/− /Cpt2 L−/− males and females compared to Cpt2 L−/− mice (n = 4). Ponceau-stained serum albumin bands were used as loading controls. D , serum triglyceride assay comparing floxed, Cpt2 L−/− , and Atf3 L−/− /Cpt2 L−/− levels. Data analyzed using ordinary one-way ANOVA with the Brown-Forsythe test, Bartlett's test, and Bonferroni multiple comparisons test, conducted with GraphPad PRISM; n = 6. AFT, activating transcription factor; ApoA4, apolipoprotein A4; Cpt2, carnitine palmitoyltransferase 2; ERRα, estrogen-related receptor alpha; PCx, pyruvate carboxylase.

Article Snippet: After another 3 × 5 m TBST washes, blots were incubated with primary antibodies in 3% bovine serum albumin overnight at 4 °C; ApoA4 (#AF8125, R&D systems), Atf3 (#33593, Cell Signaling), and HSC70(sc-7298, Santa Cruz).

Techniques: Biomarker Discovery, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Staining