raf1 antibody  (Cell Signaling Technology Inc)

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

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) Schematic of Raf1 BioID and localization workflow. ( B ) SAINT plots demonstrating all hits with a positive SAINT score for proteins enriched in the proximal proteome of Raf1 in dependent MM485 or AsPC1 cells compared to Raf1-independent CHL1 and BxPC3 and eGFP controls. Red are select known Raf1 interactors and cyan indicates proteins of interest involved in metabolic processes. Dashed line drawn at SAINT score of 0.8 ( C ) Combined Cell Component, Biological Process, and Molecular Function Gene Ontology Enrichment in SAINT ≥ 0.8 proteins. Benjamini-Hochberg adjusted pValues are used. ( D ) Network of Raf1 proximal proteins with SAINT ≥ 0.9 in both pancreatic and melanoma cell lines of interest. Edges between non-Raf1 proteins represent known interactions. Proteins with known mitochondrial localization labeled with blue. ( E ) Mito-tag Mitochondrial isolation of MM485 melanoma cells. WCL denotes whole cell lysate, Mito indicates mitochondrial fraction, and PK denotes proteinase K treatment to digest outer mitochondrial membrane (OMM) proteins. Lamin A/C is a nuclear (nuc) protein, p70S6 kinase is a cytoplasmic (cyto) protein, and TOMM20 spans the mitochondrial outer mitochondrial membrane (OMM) ( F ) STED microscopy demonstrating inner-mitochondrial Raf1 (yellow arrows). Image stack side-view (XZ and YZ) are cropped laterally and then mean-projected along X or Y. Inset displays a Y-projection of a particular mitochondria of interest in XZ.

Article Snippet: Samples treated with primary mouse Raf1 (12552, Cell Signaling Technology) or Rabbit TOMM20 (ab78547, Abcam) antibodies overnight at 1:250 concentration.

Techniques: Labeling, Isolation, Membrane, Microscopy

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) Expression of Raf1-BasuHA and eGFP-BasuHA in CHL1 cells. Input is cells post labeling, which occurs with addition of exogenous biotin. Unbound lanes are beads after elution of biotinylated peptides. ( B ) BioID western of eluted proteins blotted for MEK and Ras. ( C ) Concentration of previously concentrated Raf1-BasuHA lentivirus applied to CHL1 cells. Blotted for Raf1 and HA. ( D ) Expression of Raf1-BasuHA in MM485, AsPC1, and BxPC3 cells with varying lentivirus concentration. ( E ) Blots produced from AsPC1, BxPC3, CHL1, and MM485cells expressing Raf1-BasuHA or eGFP-BasuHA with biotin labeling. 5% of streptavidin beads ran on gel for 5% bound. Unbound is running of lysate after elution. ( F ) Buffer-based isolation of mitochondria from MM485 and CHL1 cells and treatment of isolated mitochondria with proteinase K. ( G ) Mito-tag isolation of AsPC1 mitochondria ad blotted for endogenous Raf1.

Article Snippet: Samples treated with primary mouse Raf1 (12552, Cell Signaling Technology) or Rabbit TOMM20 (ab78547, Abcam) antibodies overnight at 1:250 concentration.

Techniques: Expressing, Labeling, Western Blot, Concentration Assay, Produced, Isolation

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) Schematic indicating 13 C glutamine tracing for both reductive and oxidative TCA. ( B ) 13C-Glutamine tracing of M+5 Glutamine (Gln), Glutamate (Glu), and αKetoglutarate (αKG) with siRNA knockdown of GLS or Raf1; n = 3, **** P < 0.0001, ** P < 0.01. ( C ) Mass isotopologues of citrate pool in MM485 cells; n = 3,**** P < 0.0001. ( D ) Glutamine to glutamate conversion as measured by luciferase-based Glutamine Glo assay with a non-targeting siRNA as a negative control. All Raf1 knockdowns rescued with differentially localized proteins. siCntrl (n =6) is nontargeting siRNA, siGLS (n=6), siRaf1 (n=6), and rescue with PM Raf1 (n=3), Mito Raf1 (n=6), or Mito Raf1 K375A kinase dead construct (n=3); **** P < 0.0001. ( E ) Western blot of MM485 cells blotting for ERK activation, effective Raf1 knockdown, Raf1 rescue, and successful GLS knockdown.( F ) Quantitation of 2 western blots with phosphoERK normalized to total ERK.

Article Snippet: Samples treated with primary mouse Raf1 (12552, Cell Signaling Technology) or Rabbit TOMM20 (ab78547, Abcam) antibodies overnight at 1:250 concentration.

Techniques: Knockdown, Luciferase, Glo Assay, Negative Control, Construct, Western Blot, Activation Assay, Quantitation Assay

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) siRNA knockdown of Raf1 and GLS in MM485 and CHL1 cells. ( B ) Incorporation of M+5 L-malic acid and L-citric acid at 0 minute, 40 minute, 1 hour, 6 hour, and 24 hour timepoints. Timecourse to determine optimal labeling time. ( C ) Percent labeled 2-HG in CHL1 and MM485 cells; * P < 0.05, *** P < 0.001, **** P < 0.0001. ( D ) Percent labeled isotopologues of reduced glutathione.

Article Snippet: Samples treated with primary mouse Raf1 (12552, Cell Signaling Technology) or Rabbit TOMM20 (ab78547, Abcam) antibodies overnight at 1:250 concentration.

Techniques: Knockdown, Labeling

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) PLA between Raf1 and GLS with shRNA knockdown of Raf1 with control. Scale bar represents 20 µm. Includes quantitation in bar graphs quantify particles per nucleus; *** P < 0.001 (B) Co-immunoprecipitation of Flag-6XHis-HA tagged MAPK component proteins with a flag antibody with appropriate inputs. ( C ) co-IP of plasma membrane and mitochondrial Raf1 with immunoblot for GLS with appropriate inputs. ( D ) Microscale thermophoresis of labeled 6X His-Raf1 against GLS, MEK1, or PCK2 to produce affinity constants of 183 nM, 196 nM, and no binding respectively. ( E ) Crosslinking mass spectrometry circos plot between crosslinked GLS and Raf1. Pink indicates the interface between Raf1 and GLS according to docking experiments. (A) ( F ) Molecular docking between Raf1 in blue and GLS in green. Interfacing amino acids indicated in salmon.

Article Snippet: Samples treated with primary mouse Raf1 (12552, Cell Signaling Technology) or Rabbit TOMM20 (ab78547, Abcam) antibodies overnight at 1:250 concentration.

Techniques: shRNA, Knockdown, Control, Quantitation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Clinical Proteomics, Membrane, Western Blot, Microscale Thermophoresis, Labeling, Binding Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) shRNA knockdown of Raf1, GLS, or an empty vector (EV) assessed via western blot with GAPDH as a loading control. ( B ) Immunofluorescence of shRNA knockdown of Raf-1, GLS, or an empty vector (EV). Primary antibodies indicated above images. ( C ) Quantitation of immunofluorescence in previous panel via the corrected total cell fluorescence sum across fields of view of several replicates. Presented for both Raf1 and GLS compared to non-targeting short hairpin RNAs ( D ) Immunoprecipitation of GLS-FHH with antiHA antibody followed by immunoblot for Raf1. ( E ) Purification of proteins with a BSA standard curve for in vitro microscale thermophoresis stained with InstantBlue coomassie stain. ( F ) BS3 crosslinking of Raf1 and GLS at three different concentrations of crosslinker. ( G ) Bar graph indicating crosslinks between lysines on GLS and Raf1 by intensity from 200 kD and 400 kD isolated bands.

Article Snippet: Samples treated with primary mouse Raf1 (12552, Cell Signaling Technology) or Rabbit TOMM20 (ab78547, Abcam) antibodies overnight at 1:250 concentration.

Techniques: shRNA, Knockdown, Plasmid Preparation, Western Blot, Control, Immunofluorescence, Quantitation Assay, Fluorescence, Immunoprecipitation, Purification, In Vitro, Microscale Thermophoresis, Staining, Isolation

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) Expression of Raf1 WT Raf1, PM Raf1, Mito Raf1, and Mito Raf1 K375A in MM485 cells to be injected subcutaneously in mice. All constructs are FHH tagged and GAPDH used as a loading control. ( B ) Cell-titer blue growth assay measuring cell growth in 2D via fluorescence of localized Raf1 constructs in MM485 cells. Y axis is foldchange in signal over day 0 average for each construct.

Article Snippet: Samples treated with primary mouse Raf1 (12552, Cell Signaling Technology) or Rabbit TOMM20 (ab78547, Abcam) antibodies overnight at 1:250 concentration.

Techniques: Expressing, Injection, Construct, Control, Growth Assay, Fluorescence

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) Subcutaneous tumor growth with overexpression of 4 Raf1 constructs: WT Raf1 (n = 14), PM Raf1 (n = 8), Mito Raf1 (n = 8), and Mito Raf1 Kinase Dead (K375A) (n = 8); * P < 0.05, **** P < 0.0001. ( B ) Western blot of phosphorylated ERK and total ERK protein in cells expressing empty vector or mitochondrial Raf1 K375A. ( C ) With quantitation; P = ns. ( D ) PLA of spontaneous tumor microarrays for 3 normal and 3 squamous cell carcinoma (SCC) samples, including one matched. Scale bar represents 50 µm. ( E ) Density plot of puncta count per cell for PLA across several patient samples: 6 normal skin samples, and 26 SCC samples. Mean puncta count per cell is 6.928 for normal skin and 14.266 for SCC; **** P < 0.0001.

Article Snippet: Samples treated with primary mouse Raf1 (12552, Cell Signaling Technology) or Rabbit TOMM20 (ab78547, Abcam) antibodies overnight at 1:250 concentration.

Techniques: Over Expression, Construct, Western Blot, Expressing, Plasmid Preparation, Quantitation Assay

Journal: bioRxiv

Article Title: Mitochondrial Raf1 Regulates Glutamine Catabolism

doi: 10.1101/2024.03.08.581297

Figure Lengend Snippet: (A) Proximity ligation assays for normal patient tissues not depicted previously. PLA signal depicted in red and nuclei stained with DAPI depicted in blue. Numbers correspond to Image Number present in D. ( B ) Proximity ligation assays for 3 squamous cell carcinoma patient samples. Numbers correspond to Image Number present in D. ( C ) Per-image quantitation of PLA punctae per cell in the skin tissue microarray as violin plots. Normal tissues depicted in grey and SCC in red. ( D ) Per-image quantitation of PLA punctae per cell in multicancer tissue microarray. ( E ) Aggregate PLA counts per cell across tissues organized via tumor type presented in a density plot. ( F ) A model of the ways in which Raf1 activation of GLS contributes to tumorigenesis. The black lines have been directly demonstrated by our data, whereas the grey portions are the expected result of Raf1-induced glutaminolysis based on the corpus of literature around glutamine metabolism in cancer.

Article Snippet: Samples treated with primary mouse Raf1 (12552, Cell Signaling Technology) or Rabbit TOMM20 (ab78547, Abcam) antibodies overnight at 1:250 concentration.

Techniques: Ligation, Staining, Quantitation Assay, Microarray, Activation Assay