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Proteintech mouse anti raf1 mab

The expression of <t>Raf1</t> in AgRP neurons of DIO mice was significantly elevated. A and B: Western blotting analysis of the protein levels of RAF1 in the hypothalamus of mice fed an NCD or an HFD ( n = 3 mice). C: Representative FISH staining shows co-localization of Raf1 and Agrp mRNA in the hypothalamus of mice fed an NCD or an HFD ( n = 3 mice; scale bars, 100 μm). D: Quantification of relative fluorescence intensity of Raf1 mRNA FISH staining in AgRP neurons of NCD- and HFD-fed mice ( n = 3 mice; NCD, N = 149; HFD, N = 96). E: Representative FISH staining shows co-localization of Raf1 and Pomc mRNA in the hypothalamus of mice fed an NCD or an HFD (scale bars, 100 μm). Arrows in C and E indicate the co-localization of the genes. F: Quantification of relative fluorescence intensity of Raf1 mRNA FISH staining in POMC neurons of NCD- and HFD-fed mice ( n = 3 mice; NCD, N = 166; HFD, N = 176). N represents the cell number, and n represents the mouse number. Data are presented as the mean ± standard error of the mean. * P < 0.05 and *** P < 0.001 by unpaired Student's t -tests and non-parametric tests (B, D, and F). Abbreviations: AgRP, agouti-related peptide; DIO, diet-induced obesity; FISH, fluorescence in situ hybridization; HFD, high-fat diet; NCD, normal chow diet; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; POMC, pro-opiomelanocortin.

Mouse Anti Raf1 Mab, supplied by Proteintech, used in various techniques. Bioz Stars score: 95/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway"

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

Journal: Journal of Biomedical Research

doi: 10.7555/JBR.39.20250114

The expression of Raf1 in AgRP neurons of DIO mice was significantly elevated. A and B: Western blotting analysis of the protein levels of RAF1 in the hypothalamus of mice fed an NCD or an HFD ( n = 3 mice). C: Representative FISH staining shows co-localization of Raf1 and Agrp mRNA in the hypothalamus of mice fed an NCD or an HFD ( n = 3 mice; scale bars, 100 μm). D: Quantification of relative fluorescence intensity of Raf1 mRNA FISH staining in AgRP neurons of NCD- and HFD-fed mice ( n = 3 mice; NCD, N = 149; HFD, N = 96). E: Representative FISH staining shows co-localization of Raf1 and Pomc mRNA in the hypothalamus of mice fed an NCD or an HFD (scale bars, 100 μm). Arrows in C and E indicate the co-localization of the genes. F: Quantification of relative fluorescence intensity of Raf1 mRNA FISH staining in POMC neurons of NCD- and HFD-fed mice ( n = 3 mice; NCD, N = 166; HFD, N = 176). N represents the cell number, and n represents the mouse number. Data are presented as the mean ± standard error of the mean. * P < 0.05 and *** P < 0.001 by unpaired Student's t -tests and non-parametric tests (B, D, and F). Abbreviations: AgRP, agouti-related peptide; DIO, diet-induced obesity; FISH, fluorescence in situ hybridization; HFD, high-fat diet; NCD, normal chow diet; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; POMC, pro-opiomelanocortin.

Figure Legend Snippet: The expression of Raf1 in AgRP neurons of DIO mice was significantly elevated. A and B: Western blotting analysis of the protein levels of RAF1 in the hypothalamus of mice fed an NCD or an HFD ( n = 3 mice). C: Representative FISH staining shows co-localization of Raf1 and Agrp mRNA in the hypothalamus of mice fed an NCD or an HFD ( n = 3 mice; scale bars, 100 μm). D: Quantification of relative fluorescence intensity of Raf1 mRNA FISH staining in AgRP neurons of NCD- and HFD-fed mice ( n = 3 mice; NCD, N = 149; HFD, N = 96). E: Representative FISH staining shows co-localization of Raf1 and Pomc mRNA in the hypothalamus of mice fed an NCD or an HFD (scale bars, 100 μm). Arrows in C and E indicate the co-localization of the genes. F: Quantification of relative fluorescence intensity of Raf1 mRNA FISH staining in POMC neurons of NCD- and HFD-fed mice ( n = 3 mice; NCD, N = 166; HFD, N = 176). N represents the cell number, and n represents the mouse number. Data are presented as the mean ± standard error of the mean. * P < 0.05 and *** P < 0.001 by unpaired Student's t -tests and non-parametric tests (B, D, and F). Abbreviations: AgRP, agouti-related peptide; DIO, diet-induced obesity; FISH, fluorescence in situ hybridization; HFD, high-fat diet; NCD, normal chow diet; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; POMC, pro-opiomelanocortin.

Techniques Used: Expressing, Western Blot, Staining, Fluorescence, In Situ Hybridization

Overexpression of Raf1 in AgRP neurons promoted obesity and related metabolic disorders. A: Schematic diagram of bilateral injections of AAV-DIO- Raf1 -HA and its control AAV-DIO-mCherry into the ARC of Agrp - IRES - Cre ; Npy - hrGFP mice. B: Representative IF staining of mCherry and RAF1-HA in AgRP neurons ( n = 3 mice; scale bars, 100 μm). C: Representative image of control and AgRP- Raf1 -OE mice fed an NCD. D–L: Various metabolic indicators, including body weight gain curves ( n = 8–9 mice; D), food intake ( n = 8–9 mice; E), body mass ( n = 6–7 mice; F), representative H&E staining images of liver, BAT, and WAT ( n = 3 mice; scale bars: 100 μm; G), droplet area of WAT ( n = 3 mice; H), respiratory exchange ratio ( n = 4 mice; I), energy expenditure ( n = 4 mice; J), GTT ( n = 6 mice; K), and serum insulin and leptin levels ( n = 4–6 mice; L) in both control and AgRP- Raf1 -OE mice fed an NCD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired t -tests (E, H, and L), two-way ANOVA with Bonferroni's post hoc test (D and K), and multiple t -tests (F, I, and J). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, double-floxed inverted open reading frame; GTT, glucose tolerance test; H&E: hematoxylin and eosin; IF, immunofluorescence; NCD, normal chow diet; NPY, neuropeptide Y; ns, not significant; rAAV-DIO- Raf1 /mCherry, recombinant adeno-associated virus encoding Raf1 -HA or mCherry; RAF1, v-raf-leukemia viral oncogene 1; OE, overexpression; WAT, white adipose tissue.

Figure Legend Snippet: Overexpression of Raf1 in AgRP neurons promoted obesity and related metabolic disorders. A: Schematic diagram of bilateral injections of AAV-DIO- Raf1 -HA and its control AAV-DIO-mCherry into the ARC of Agrp - IRES - Cre ; Npy - hrGFP mice. B: Representative IF staining of mCherry and RAF1-HA in AgRP neurons ( n = 3 mice; scale bars, 100 μm). C: Representative image of control and AgRP- Raf1 -OE mice fed an NCD. D–L: Various metabolic indicators, including body weight gain curves ( n = 8–9 mice; D), food intake ( n = 8–9 mice; E), body mass ( n = 6–7 mice; F), representative H&E staining images of liver, BAT, and WAT ( n = 3 mice; scale bars: 100 μm; G), droplet area of WAT ( n = 3 mice; H), respiratory exchange ratio ( n = 4 mice; I), energy expenditure ( n = 4 mice; J), GTT ( n = 6 mice; K), and serum insulin and leptin levels ( n = 4–6 mice; L) in both control and AgRP- Raf1 -OE mice fed an NCD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired t -tests (E, H, and L), two-way ANOVA with Bonferroni's post hoc test (D and K), and multiple t -tests (F, I, and J). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, double-floxed inverted open reading frame; GTT, glucose tolerance test; H&E: hematoxylin and eosin; IF, immunofluorescence; NCD, normal chow diet; NPY, neuropeptide Y; ns, not significant; rAAV-DIO- Raf1 /mCherry, recombinant adeno-associated virus encoding Raf1 -HA or mCherry; RAF1, v-raf-leukemia viral oncogene 1; OE, overexpression; WAT, white adipose tissue.

Techniques Used: Over Expression, Control, Staining, Immunofluorescence, Recombinant, Virus

Slight promotion of obesity development was observed in AgRP- Raf1 -OE mice under HFD feeding. A: Representative image of control and AgRP- Raf1 -OE mice fed an HFD. B–J: Various metabolic indicators including body weight gain curves ( n = 10 mice; B), food intake ( n = 10 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 4 mice; G), energy expenditure ( n = 4 mice; H), GTT ( n = 6 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -OE mice fed an HFD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05 and ** P < 0.01 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t -tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; BAT, brown adipose tissue; DIO, double-floxed inverted open reading frame; GTT, glucose tolerance test; H&E, hematoxylin and eosin; HFD, high-fat diet; ns, not significant; OE, overexpression; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Figure Legend Snippet: Slight promotion of obesity development was observed in AgRP- Raf1 -OE mice under HFD feeding. A: Representative image of control and AgRP- Raf1 -OE mice fed an HFD. B–J: Various metabolic indicators including body weight gain curves ( n = 10 mice; B), food intake ( n = 10 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 4 mice; G), energy expenditure ( n = 4 mice; H), GTT ( n = 6 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -OE mice fed an HFD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05 and ** P < 0.01 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t -tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; BAT, brown adipose tissue; DIO, double-floxed inverted open reading frame; GTT, glucose tolerance test; H&E, hematoxylin and eosin; HFD, high-fat diet; ns, not significant; OE, overexpression; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Techniques Used: Control, Staining, Over Expression

Screening and in vivo validation of sgRNA. A: Gel electrophoresis shows the cleavage effect of sgRNA targeting the Raf1 gene. B: The schematic diagrams show sgRNA1 and sgRNA2. C and D: Western blotting analysis of RAF1 protein levels in the hypothalamus after control and knockdown AAV injection. β-Actin served as the loading control ( n = 5 mice). rAAV-saCas9-sgRNA1, rAAV-saCas9-sgRNA2, and rAAV-saCas9-sgControl (recombinant adeno-associated viruses encoding sgRNA1, sgRNA2, and a non-targeting control sgRNA, respectively) were used. E: A schematic diagram of bilateral injection of Cre-dependent AAV-DIO-saCas9-sgRNAs and its control into the ARC of Agrp - IRES - Cre ; Npy - hrGFP mice. F: Representative IF staining of the HA-Tag signifying the expression of saCas9 in AgRP neurons ( n = 3 mice; scale bars, 100 μm). Data are presented as the mean ± standard error of the mean. *** P < 0.001 by unpaired t -tests (D). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; DIO, double-floxed inverted open reading frame; NPY, neuropeptide Y; RAF1, v-raf-leukemia viral oncogene 1; sgRNA, single guide RNA.

Figure Legend Snippet: Screening and in vivo validation of sgRNA. A: Gel electrophoresis shows the cleavage effect of sgRNA targeting the Raf1 gene. B: The schematic diagrams show sgRNA1 and sgRNA2. C and D: Western blotting analysis of RAF1 protein levels in the hypothalamus after control and knockdown AAV injection. β-Actin served as the loading control ( n = 5 mice). rAAV-saCas9-sgRNA1, rAAV-saCas9-sgRNA2, and rAAV-saCas9-sgControl (recombinant adeno-associated viruses encoding sgRNA1, sgRNA2, and a non-targeting control sgRNA, respectively) were used. E: A schematic diagram of bilateral injection of Cre-dependent AAV-DIO-saCas9-sgRNAs and its control into the ARC of Agrp - IRES - Cre ; Npy - hrGFP mice. F: Representative IF staining of the HA-Tag signifying the expression of saCas9 in AgRP neurons ( n = 3 mice; scale bars, 100 μm). Data are presented as the mean ± standard error of the mean. *** P < 0.001 by unpaired t -tests (D). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; DIO, double-floxed inverted open reading frame; NPY, neuropeptide Y; RAF1, v-raf-leukemia viral oncogene 1; sgRNA, single guide RNA.

Techniques Used: In Vivo, Biomarker Discovery, Nucleic Acid Electrophoresis, Western Blot, Control, Knockdown, Injection, Recombinant, Staining, Expressing

Raf1 knockout in AgRP neurons in mice did not alter their metabolic phenotypes under NCD feeding. A: Representative image of control and AgRP- Raf1 -KO mice fed an NCD. B–J: Various metabolic indicators including body weight gain curves ( n = 8 mice; B), food intake ( n = 8 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 3–4 mice; G), energy expenditure ( n = 3–4; H), GTT ( n = 6 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -KO mice fed an NCD were measured. Data are presented as the mean ± standard error of the mean. ns P > 0.05 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t -tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, diet-induced obesity; GTT, glucose tolerance test; H&E, hematoxylin and eosin; KO, knockout; NCD, normal chow diet; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Figure Legend Snippet: Raf1 knockout in AgRP neurons in mice did not alter their metabolic phenotypes under NCD feeding. A: Representative image of control and AgRP- Raf1 -KO mice fed an NCD. B–J: Various metabolic indicators including body weight gain curves ( n = 8 mice; B), food intake ( n = 8 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 3–4 mice; G), energy expenditure ( n = 3–4; H), GTT ( n = 6 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -KO mice fed an NCD were measured. Data are presented as the mean ± standard error of the mean. ns P > 0.05 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t -tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, diet-induced obesity; GTT, glucose tolerance test; H&E, hematoxylin and eosin; KO, knockout; NCD, normal chow diet; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Techniques Used: Knock-Out, Control, Staining

Raf1 knockout in AgRP neurons protected against DIO. A: Representative image of control and AgRP- Raf1 -KO mice fed an HFD. B–J: Various metabolic indicators, including body weight gain curves ( n = 8–10 mice; B), food intake ( n = 10 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 4 mice; G), energy expenditure ( n = 4 mice; H), GTT ( n = 6–7 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -KO mice fed an HFD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.01 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t-tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, diet-induced obesity; GTT, glucose tolerance test; H&E, hematoxylin and eosin; HFD, high-fat diet; KO, knockout; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Figure Legend Snippet: Raf1 knockout in AgRP neurons protected against DIO. A: Representative image of control and AgRP- Raf1 -KO mice fed an HFD. B–J: Various metabolic indicators, including body weight gain curves ( n = 8–10 mice; B), food intake ( n = 10 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 4 mice; G), energy expenditure ( n = 4 mice; H), GTT ( n = 6–7 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -KO mice fed an HFD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.01 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t-tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, diet-induced obesity; GTT, glucose tolerance test; H&E, hematoxylin and eosin; HFD, high-fat diet; KO, knockout; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Techniques Used: Knock-Out, Control, Staining

Raf1 regulated MAPK signaling under insulin stimulation. A: Relative mRNA levels of Raf1 , Agrp , and Npy in the hypothalamus of control and AgRP- Raf1 -OE mice fed an NCD ( n = 5–6 mice). B: Western blotting analysis of protein levels of FLAG, MEK1/2, pMEK1/2, ERK1/2, pERK1/2, CREB, and pCREB in the N42 Raf1 -overexpression cells. β-Actin served as the internal control ( n = 3). C: Phosphorylation levels of MEK1/2, ERK1/2, and CREB proteins in the N42 Raf1 -overexpression cells. D: Western blotting analysis of protein levels of RAF1, MEK1/2, pMEK1/2, ERK1/2, pERK1/2, and pCREB in the N42 Raf1 -knockout cells. β-Actin served as the internal control ( n = 3). E: Phosphorylation levels of MEK1/2, ERK1/2, and CREB proteins in the N42 Raf1 -knockout cells. F: Representative IF staining of pCREB in AgRP neurons of control and AgRP- Raf1 -OE mice following 2 mU insulin (icv) stimulation ( n = 3 mice; scale bars, 100 μm). G: Fluorescence intensity quantification of pCREB co-localized with HA/mCherry ( n = 3 mice; AAV-DIO-mCherry, N = 77; AAV-DIO- Raf1 -HA, N = 105). N represents the cell number, and n represents the mouse number. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired t -tests (A, C, E, and G). Abbreviations: AgRP, agouti-related peptide; CREB, cAMP response element-binding protein; ERK1/2, extracellular signal-regulated kinases 1 and 2; icv, intra-cerebroventricular injection; IF, immunofluorescence; MEK1/2, mitogen-activated protein kinase kinases 1 and 2; NCD, normal chow diet; NPY, neuropeptide Y; OE, overexpression; p-CREB, phospho-CREB; p-ERK1/2, phospho-ERK1/2; p-MEK1/2, phospho-MEK1/2; RAF1, v-raf-leukemia viral oncogene 1.

Figure Legend Snippet: Raf1 regulated MAPK signaling under insulin stimulation. A: Relative mRNA levels of Raf1 , Agrp , and Npy in the hypothalamus of control and AgRP- Raf1 -OE mice fed an NCD ( n = 5–6 mice). B: Western blotting analysis of protein levels of FLAG, MEK1/2, pMEK1/2, ERK1/2, pERK1/2, CREB, and pCREB in the N42 Raf1 -overexpression cells. β-Actin served as the internal control ( n = 3). C: Phosphorylation levels of MEK1/2, ERK1/2, and CREB proteins in the N42 Raf1 -overexpression cells. D: Western blotting analysis of protein levels of RAF1, MEK1/2, pMEK1/2, ERK1/2, pERK1/2, and pCREB in the N42 Raf1 -knockout cells. β-Actin served as the internal control ( n = 3). E: Phosphorylation levels of MEK1/2, ERK1/2, and CREB proteins in the N42 Raf1 -knockout cells. F: Representative IF staining of pCREB in AgRP neurons of control and AgRP- Raf1 -OE mice following 2 mU insulin (icv) stimulation ( n = 3 mice; scale bars, 100 μm). G: Fluorescence intensity quantification of pCREB co-localized with HA/mCherry ( n = 3 mice; AAV-DIO-mCherry, N = 77; AAV-DIO- Raf1 -HA, N = 105). N represents the cell number, and n represents the mouse number. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired t -tests (A, C, E, and G). Abbreviations: AgRP, agouti-related peptide; CREB, cAMP response element-binding protein; ERK1/2, extracellular signal-regulated kinases 1 and 2; icv, intra-cerebroventricular injection; IF, immunofluorescence; MEK1/2, mitogen-activated protein kinase kinases 1 and 2; NCD, normal chow diet; NPY, neuropeptide Y; OE, overexpression; p-CREB, phospho-CREB; p-ERK1/2, phospho-ERK1/2; p-MEK1/2, phospho-MEK1/2; RAF1, v-raf-leukemia viral oncogene 1.

Techniques Used: Control, Western Blot, Over Expression, Phospho-proteomics, Knock-Out, Staining, Fluorescence, Binding Assay, Injection, Immunofluorescence

Role of hypothalamic AgRP neuron Raf1 in energy homeostasis regulation. This graphic abstract illustrates the pivotal role of Raf1 within hypothalamic AgRP neurons in governing energy homeostasis. Specifically, Raf1 exerts its regulatory function via the MAPK-mediated modulation of Agrp and Npy expression. In normal physiological conditions, this mechanism contributes to the maintenance of energy balance. Under HFD challenges, the RAF1-MAPK-AGRP/NPY axis becomes dysregulated, leading to disruptions in energy homeostasis. Abbreviations: DIO, diet-induced obesity; HFD, high-fat diet; KO, knockout; OE, overexpression.

Figure Legend Snippet: Role of hypothalamic AgRP neuron Raf1 in energy homeostasis regulation. This graphic abstract illustrates the pivotal role of Raf1 within hypothalamic AgRP neurons in governing energy homeostasis. Specifically, Raf1 exerts its regulatory function via the MAPK-mediated modulation of Agrp and Npy expression. In normal physiological conditions, this mechanism contributes to the maintenance of energy balance. Under HFD challenges, the RAF1-MAPK-AGRP/NPY axis becomes dysregulated, leading to disruptions in energy homeostasis. Abbreviations: DIO, diet-induced obesity; HFD, high-fat diet; KO, knockout; OE, overexpression.

Techniques Used: Expressing, Knock-Out, Over Expression

Incubation:

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway
Article Snippet: Equal quantities of protein were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). .. The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld). .. After overnight incubation in a shaker at 4 °C, goat anti-mouse IgG (H+L) HRP (1∶10000, Cat. #BS12478, Bioworld) and goat anti-rabbit IgG (H+L) HRP (1∶10000, Cat. #BS13278, Bioworld) were incubated simultaneously.

Image Search Results

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: The expression of Raf1 in AgRP neurons of DIO mice was significantly elevated. A and B: Western blotting analysis of the protein levels of RAF1 in the hypothalamus of mice fed an NCD or an HFD ( n = 3 mice). C: Representative FISH staining shows co-localization of Raf1 and Agrp mRNA in the hypothalamus of mice fed an NCD or an HFD ( n = 3 mice; scale bars, 100 μm). D: Quantification of relative fluorescence intensity of Raf1 mRNA FISH staining in AgRP neurons of NCD- and HFD-fed mice ( n = 3 mice; NCD, N = 149; HFD, N = 96). E: Representative FISH staining shows co-localization of Raf1 and Pomc mRNA in the hypothalamus of mice fed an NCD or an HFD (scale bars, 100 μm). Arrows in C and E indicate the co-localization of the genes. F: Quantification of relative fluorescence intensity of Raf1 mRNA FISH staining in POMC neurons of NCD- and HFD-fed mice ( n = 3 mice; NCD, N = 166; HFD, N = 176). N represents the cell number, and n represents the mouse number. Data are presented as the mean ± standard error of the mean. * P < 0.05 and *** P < 0.001 by unpaired Student's t -tests and non-parametric tests (B, D, and F). Abbreviations: AgRP, agouti-related peptide; DIO, diet-induced obesity; FISH, fluorescence in situ hybridization; HFD, high-fat diet; NCD, normal chow diet; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; POMC, pro-opiomelanocortin.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated overnight at 4 °C with primary antibodies, including mouse anti-RAF1 mAb (1∶1000, Cat. #66592-1-Ig, Proteintech, Wuhan, China), rabbit anti-ERK1/2 polyclonal antibody (1∶500, Cat. #BS2265, Bioworld, China), rabbit anti-ERK1/2 (phospho-T202/Y204) polyclonal antibody (1∶5000, Cat. #AP0484, Bioworld), rabbit anti-MEK1/2 polyclonal antibody (1∶500, Cat. #BS3599, Bioworld), rabbit anti-MEK1/2 (phospho-S2218/222) polyclonal antibody (1∶500, Cat. #BS4733, Bioworld), rabbit anti-CREB1 polyclonal antibody (1∶1000, Cat. #12208-1-AP, Proteintech), anti-CREB (phospho S133) (1∶1000, Cat. #ab32096, Abcam, Cambridge, UK), and mouse anti-β-actin mAb (1∶5000, Cat. #BS6007M, Bioworld).

Techniques: Expressing, Western Blot, Staining, Fluorescence, In Situ Hybridization

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Overexpression of Raf1 in AgRP neurons promoted obesity and related metabolic disorders. A: Schematic diagram of bilateral injections of AAV-DIO- Raf1 -HA and its control AAV-DIO-mCherry into the ARC of Agrp - IRES - Cre ; Npy - hrGFP mice. B: Representative IF staining of mCherry and RAF1-HA in AgRP neurons ( n = 3 mice; scale bars, 100 μm). C: Representative image of control and AgRP- Raf1 -OE mice fed an NCD. D–L: Various metabolic indicators, including body weight gain curves ( n = 8–9 mice; D), food intake ( n = 8–9 mice; E), body mass ( n = 6–7 mice; F), representative H&E staining images of liver, BAT, and WAT ( n = 3 mice; scale bars: 100 μm; G), droplet area of WAT ( n = 3 mice; H), respiratory exchange ratio ( n = 4 mice; I), energy expenditure ( n = 4 mice; J), GTT ( n = 6 mice; K), and serum insulin and leptin levels ( n = 4–6 mice; L) in both control and AgRP- Raf1 -OE mice fed an NCD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired t -tests (E, H, and L), two-way ANOVA with Bonferroni's post hoc test (D and K), and multiple t -tests (F, I, and J). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, double-floxed inverted open reading frame; GTT, glucose tolerance test; H&E: hematoxylin and eosin; IF, immunofluorescence; NCD, normal chow diet; NPY, neuropeptide Y; ns, not significant; rAAV-DIO- Raf1 /mCherry, recombinant adeno-associated virus encoding Raf1 -HA or mCherry; RAF1, v-raf-leukemia viral oncogene 1; OE, overexpression; WAT, white adipose tissue.

Techniques: Over Expression, Control, Staining, Immunofluorescence, Recombinant, Virus

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Slight promotion of obesity development was observed in AgRP- Raf1 -OE mice under HFD feeding. A: Representative image of control and AgRP- Raf1 -OE mice fed an HFD. B–J: Various metabolic indicators including body weight gain curves ( n = 10 mice; B), food intake ( n = 10 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 4 mice; G), energy expenditure ( n = 4 mice; H), GTT ( n = 6 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -OE mice fed an HFD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05 and ** P < 0.01 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t -tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; BAT, brown adipose tissue; DIO, double-floxed inverted open reading frame; GTT, glucose tolerance test; H&E, hematoxylin and eosin; HFD, high-fat diet; ns, not significant; OE, overexpression; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Techniques: Control, Staining, Over Expression

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Screening and in vivo validation of sgRNA. A: Gel electrophoresis shows the cleavage effect of sgRNA targeting the Raf1 gene. B: The schematic diagrams show sgRNA1 and sgRNA2. C and D: Western blotting analysis of RAF1 protein levels in the hypothalamus after control and knockdown AAV injection. β-Actin served as the loading control ( n = 5 mice). rAAV-saCas9-sgRNA1, rAAV-saCas9-sgRNA2, and rAAV-saCas9-sgControl (recombinant adeno-associated viruses encoding sgRNA1, sgRNA2, and a non-targeting control sgRNA, respectively) were used. E: A schematic diagram of bilateral injection of Cre-dependent AAV-DIO-saCas9-sgRNAs and its control into the ARC of Agrp - IRES - Cre ; Npy - hrGFP mice. F: Representative IF staining of the HA-Tag signifying the expression of saCas9 in AgRP neurons ( n = 3 mice; scale bars, 100 μm). Data are presented as the mean ± standard error of the mean. *** P < 0.001 by unpaired t -tests (D). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; DIO, double-floxed inverted open reading frame; NPY, neuropeptide Y; RAF1, v-raf-leukemia viral oncogene 1; sgRNA, single guide RNA.

Techniques: In Vivo, Biomarker Discovery, Nucleic Acid Electrophoresis, Western Blot, Control, Knockdown, Injection, Recombinant, Staining, Expressing

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Raf1 knockout in AgRP neurons in mice did not alter their metabolic phenotypes under NCD feeding. A: Representative image of control and AgRP- Raf1 -KO mice fed an NCD. B–J: Various metabolic indicators including body weight gain curves ( n = 8 mice; B), food intake ( n = 8 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 3–4 mice; G), energy expenditure ( n = 3–4; H), GTT ( n = 6 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -KO mice fed an NCD were measured. Data are presented as the mean ± standard error of the mean. ns P > 0.05 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t -tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, diet-induced obesity; GTT, glucose tolerance test; H&E, hematoxylin and eosin; KO, knockout; NCD, normal chow diet; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Techniques: Knock-Out, Control, Staining

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Raf1 knockout in AgRP neurons protected against DIO. A: Representative image of control and AgRP- Raf1 -KO mice fed an HFD. B–J: Various metabolic indicators, including body weight gain curves ( n = 8–10 mice; B), food intake ( n = 10 mice; C), body mass ( n = 6 mice; D), representative H&E staining images (liver, BAT, and WAT) ( n = 3 mice; scale bars, 100 μm; E), droplet area of WAT ( n = 3 mice; F), respiratory exchange ratio ( n = 4 mice; G), energy expenditure ( n = 4 mice; H), GTT ( n = 6–7 mice; I), and serum insulin and leptin levels ( n = 4 mice; J) in both control and AgRP- Raf1 -KO mice fed an HFD were measured. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.01 by unpaired Student's t -tests and nonparametric tests (C, F, and J), two-way ANOVA with Bonferroni's post hoc test (B and I), and multiple t-tests (D, G, and H). Abbreviations: AgRP, agouti-related peptide; ARC, arcuate nucleus; BAT, brown adipose tissue; DIO, diet-induced obesity; GTT, glucose tolerance test; H&E, hematoxylin and eosin; HFD, high-fat diet; KO, knockout; ns, not significant; RAF1, v-raf-leukemia viral oncogene 1; WAT, white adipose tissue.

Techniques: Knock-Out, Control, Staining

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Raf1 regulated MAPK signaling under insulin stimulation. A: Relative mRNA levels of Raf1 , Agrp , and Npy in the hypothalamus of control and AgRP- Raf1 -OE mice fed an NCD ( n = 5–6 mice). B: Western blotting analysis of protein levels of FLAG, MEK1/2, pMEK1/2, ERK1/2, pERK1/2, CREB, and pCREB in the N42 Raf1 -overexpression cells. β-Actin served as the internal control ( n = 3). C: Phosphorylation levels of MEK1/2, ERK1/2, and CREB proteins in the N42 Raf1 -overexpression cells. D: Western blotting analysis of protein levels of RAF1, MEK1/2, pMEK1/2, ERK1/2, pERK1/2, and pCREB in the N42 Raf1 -knockout cells. β-Actin served as the internal control ( n = 3). E: Phosphorylation levels of MEK1/2, ERK1/2, and CREB proteins in the N42 Raf1 -knockout cells. F: Representative IF staining of pCREB in AgRP neurons of control and AgRP- Raf1 -OE mice following 2 mU insulin (icv) stimulation ( n = 3 mice; scale bars, 100 μm). G: Fluorescence intensity quantification of pCREB co-localized with HA/mCherry ( n = 3 mice; AAV-DIO-mCherry, N = 77; AAV-DIO- Raf1 -HA, N = 105). N represents the cell number, and n represents the mouse number. Data are presented as the mean ± standard error of the mean. * P < 0.05, ** P < 0.01, and *** P < 0.001 by unpaired t -tests (A, C, E, and G). Abbreviations: AgRP, agouti-related peptide; CREB, cAMP response element-binding protein; ERK1/2, extracellular signal-regulated kinases 1 and 2; icv, intra-cerebroventricular injection; IF, immunofluorescence; MEK1/2, mitogen-activated protein kinase kinases 1 and 2; NCD, normal chow diet; NPY, neuropeptide Y; OE, overexpression; p-CREB, phospho-CREB; p-ERK1/2, phospho-ERK1/2; p-MEK1/2, phospho-MEK1/2; RAF1, v-raf-leukemia viral oncogene 1.

Techniques: Control, Western Blot, Over Expression, Phospho-proteomics, Knock-Out, Staining, Fluorescence, Binding Assay, Injection, Immunofluorescence

Journal: Journal of Biomedical Research

Article Title: RAF1 in AgRP neurons involved in the regulation of energy metabolism via the MAPK signaling pathway

doi: 10.7555/JBR.39.20250114

Figure Lengend Snippet: Role of hypothalamic AgRP neuron Raf1 in energy homeostasis regulation. This graphic abstract illustrates the pivotal role of Raf1 within hypothalamic AgRP neurons in governing energy homeostasis. Specifically, Raf1 exerts its regulatory function via the MAPK-mediated modulation of Agrp and Npy expression. In normal physiological conditions, this mechanism contributes to the maintenance of energy balance. Under HFD challenges, the RAF1-MAPK-AGRP/NPY axis becomes dysregulated, leading to disruptions in energy homeostasis. Abbreviations: DIO, diet-induced obesity; HFD, high-fat diet; KO, knockout; OE, overexpression.

Techniques: Expressing, Knock-Out, Over Expression

(A) 293T cells transfected with Flag-RAF1 and CFP-KSR1 in the presence or absence of Myc-Erbin co-expression were lysed and immunoprecipitated with Flag-affinity beads. The presence of KSR1 and Erbin in the immunoprecipitates were detected by Western blot. The arrow indicates the endogenous KSR1 co-immunoprecipitated by Flag-RAF1. (B) The relative amount of KSR1 co-immunoprecipitated with RAF1 in the presence or absence of Myc-Erbin co-expression was quantified and expressed graphically (n=3, * p<0.05). (C) Erbin interacts with endogenous KSR1. Endogenous Erbin was immunoprecipitated from LIM2405 cell lysates and the presence of KSR1 in the immunoprecipitates was detected by Western blot. (D) Endogenous RAF1 was immunoprecipitated from sh-Control and sh-Erbin Caco2 and LIM2405 cells. The levels of KSR1 co-immunoprecipitated with RAF1 were detected by Western blot. (E) The expression of KSR1 and Erbin were silenced individually or in combination in SW480 cells using corresponding shRNA lentivirus. Cell lysates were analyzed for the phosphorylation status of ERK. The relative levels of ERK phosphorylation in different cells were quantified by normalizing ECL signals generated by p-ERK to that of total ERK.

Journal: Cancer research

Article Title: Erbin suppresses KSR1-mediated RAS/RAF signaling and tumorigenesis in colorectal cancer

doi: 10.1158/0008-5472.CAN-17-3629

Figure Lengend Snippet: (A) 293T cells transfected with Flag-RAF1 and CFP-KSR1 in the presence or absence of Myc-Erbin co-expression were lysed and immunoprecipitated with Flag-affinity beads. The presence of KSR1 and Erbin in the immunoprecipitates were detected by Western blot. The arrow indicates the endogenous KSR1 co-immunoprecipitated by Flag-RAF1. (B) The relative amount of KSR1 co-immunoprecipitated with RAF1 in the presence or absence of Myc-Erbin co-expression was quantified and expressed graphically (n=3, * p<0.05). (C) Erbin interacts with endogenous KSR1. Endogenous Erbin was immunoprecipitated from LIM2405 cell lysates and the presence of KSR1 in the immunoprecipitates was detected by Western blot. (D) Endogenous RAF1 was immunoprecipitated from sh-Control and sh-Erbin Caco2 and LIM2405 cells. The levels of KSR1 co-immunoprecipitated with RAF1 were detected by Western blot. (E) The expression of KSR1 and Erbin were silenced individually or in combination in SW480 cells using corresponding shRNA lentivirus. Cell lysates were analyzed for the phosphorylation status of ERK. The relative levels of ERK phosphorylation in different cells were quantified by normalizing ECL signals generated by p-ERK to that of total ERK.

Article Snippet: The phospho-AKT (p-AKT for Ser473), pan-Akt, phospho-RAF1 (p-RAF1 for Ser338), total RAF1, phospho-MEK1/2 (p-MEK for Ser217/221), total MEK1/2, phospho-ERK1/2 (p-ERK for Thr202/Tyr204), total ERK1/2, and E-cadherin antibodies were from Cell Signaling.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, shRNA, Generated

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