craf  (Proteintech)

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.

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: 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.

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: 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.

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: 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.

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: 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.

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: 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.

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: 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.

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, Knock-Out, Over Expression