anti raf1  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Medicine

Article Title: STOML2 interacts with PHB to activate the MEK/ERK signaling pathway and mediates autophagy-related proteins in the progression of hepatocellular carcinoma

doi: 10.3892/ijmm.2025.5709

Figure Lengend Snippet: Sorafenib (an RAF1 inhibitor) significantly attenuated STOML2-induced cell cycle progression, migration and autophagy, while promoting apoptosis in HCC cells. (A) Cell cycle distribution analysis of Huh7-STOML2 and control cells following treatment with sorafenib (20 μ M, 24 h) or DMSO. Representative images from three independent experiments are shown. (B) Wound healing assay showing the migratory capacity of Huh7-STOML2 cells treated with sorafenib or DMSO. Representative images from three independent experiments are shown. (C) Flow cytometric analysis of apoptosis in STOML2-overexpressing Huh7 and control cells after treatment with sorafenib or DMSO (unpaired Student's t-test, **** P<0.0001). Representative flow cytometry plots and quantitative data (mean ± SD) from three independent experiments are shown. (D) Transwell assay showing the invasive capacity of Huh7-STOML2 and control cells following sorafenib treatment or DMSO. Data are presented as mean ± SD (unpaired Student's t-test, *** P<0.001). Western blot analysis of (E) MAPK signaling pathway proteins, and (F) p62 and Beclin1 in Huh7-STOML2 cells after treatment with sorafenib or DMSO. Representative images from three independent experiments are shown. p-, phosphorylated; STOML2, stomatin-like protein 2.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with primary antibodies (1:500) specific to the target proteins STOML2 (cat. no. 10348-1-AP; Proteintech Group, Inc.), PHB (cat. no. GB113098-100; Wuhan Servicebio Technology Co., Ltd.), p62 (cat. no. 18420-1-AP; Proteintech Group, Inc.), Beclin1 (cat. no. 11306-1-AP; Proteintech Group, Inc.), RAF1 (cat. no. 26863-1-AP; Proteintech Group, Inc.), phosphorylated (p)-RAF1 (cat. no. 9427; Cell Signaling Technology, Inc.), MEK1/2 (cat. no. 11049-1-AP; Proteintech Group, Inc.), p-MEK1/2 (cat. no. 9154; Cell Signaling Technology, Inc.), ERK1/2 (cat. no. 11257-1-AP; Proteintech Group, Inc.), p-ERK1/2 (cat. no. 80031-1-RR; Proteintech Group, Inc.) and GAPDH (cat. no. 60004-1-Ig; Proteintech Group, Inc.).

Techniques: Migration, Control, Wound Healing Assay, Flow Cytometry, Transwell Assay, Western Blot

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Human cytomegalovirus (HCMV) infection induces the AMP-activated protein kinase-specific phosphorylation of RAF1. ( A ) Schematic depicting the RAF1 signaling pathway. ( B ) Western blot analysis of RAF1 protein over the course of infection (120 h) in the absence and presence of 5 µM compound C (CC) in MRC5 fibroblasts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and UL26 is a control for successful viral infection. ( C–E ) Quantification of Western blots in ( B ), where 0 h post-infection (hpi) is representative of mock infection (avg ± standard error of the mean, n = 2), comparing phospho-RAF1 to total RAF1 for ( C ) P-RAF1-S338 and ( D ) P-RAF1-S621, and ( E ) total RAF1 levels relative to GAPDH. ( F ) Two-dimensional gel electrophoresis of RAF1 protein during mock infection and HCMV infection with or without CC treatment, MOI = 3.0 at 48 hpi in MRC5 fibroblasts ( n = 1).

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: Infection, Phospho-proteomics, Western Blot, Control, Two-Dimensional Gel Electrophoresis, Electrophoresis

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Phosphorylation of S621 enhances RAF1 binding to 14-3-3 during HCMV infection. ( A ) Overexpression of Flag-RAF1-WT (wild type) or Flag-RAF1-S621A via lentiviral transduction were compared to control MRC5 fibroblasts and assessed for protein expression by western blot using antibodies specific to Flag, RAF1, and S621- or S338-phosphorylated RAF1 and total or phospho-MEK1/2 and -ERK1/2 (arrows indicate doublet). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control ( n = 1). ( B ) Empty vector (EV), Flag-RAF1-WT, and -S621A transduced cells were mock ( M )- or HCMV ( H )-infected with AD169-WT at an MOI of 3.0 for 72 h in the presence or absence of 5 µM Compound C (CC) prior to Flag pulldown and co-precipitation of endogenous 14-3-3. Numbers indicate the ratio of 14-3-3 to Flag protein pulled down in the representative blot. ( C ) Ratios of 14-3-3 to Flag pulled down during HCMV infection lanes in ( B ) were quantified along with a second blot (not shown) (avg, n = 2). ( D ) Cells were infected at an MOI of 3.0 for 120 h or ( E ) at an MOI of 0.01 for 9 days (avg ±SEM, n = 4), and viral titers were assessed by TCID50. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparisons test.

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: Phospho-proteomics, Binding Assay, Infection, Over Expression, Transduction, Control, Expressing, Western Blot, Plasmid Preparation

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Reported IC50s of regorafenib and sorafenib to recombinant RAF pathway proteins ( 50 , 51 )

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: Recombinant

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Pharmacological inhibition of the RAF1 pathway inhibits HCMV infection. ( A, B ) Regorafenib (Reg) or ( C, D ) sorafenib (Sor) treated ( A, C ) MRC5 or ( B, D ) HFF fibroblasts were treated with the indicated concentration of drugs or DMSO control at the time of infection. ( A–D ) Cells were infected at an MOI of 0.1 with AD169-GFP for 5 days, then fixed. GFP-positive cells were counted per area of the well and plotted against drug concentration [avg ± standard error of the mean (SEM), n = 3]. IC50 values were calculated. The dotted line indicates the average maximal GFP in untreated cells. ( E–H ) Fibroblasts were infected at an MOI of 3.0 in the presence of DMSO or 2.17 µM regorafenib or sorafenib added at the time of viral adsorption. UN is an untreated control. Viral titers from ( E ) MRC5 or ( F ) HFF cells were assessed by TCID50 (avg ± SEM, n = 3) 120 hpi. The dotted line indicates the limit of detection. In ( G ) MRC5 or ( H ) HFF cells, at 24 h post-infection (hpi), phosphorylation of MEK1/2 and ERK1/2 (arrows indicate doublet) was assessed by western blot ( n = 1).

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: Inhibition, Infection, Concentration Assay, Control, Adsorption, Phospho-proteomics, Western Blot

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Pharmacological inhibition of the RAF1 pathway does not impact fibroblast cell counts. ( A, B ) MRC5 or ( C, D ) HFF fibroblasts were treated with the indicated concentration of ( A, C ) regorafenib or ( B, D ) sorafenib at the time of infection, AD169-GFP, MOI = 0.1, fixed on Day 5. ( A–D ) Hoechst-stained nuclei were counted and plotted against the concentration (avg ± standard error of the mean, n = 3). The solid gray line indicates the average number of nuclei in untreated HCMV-infected cells, while the dotted gray line indicates the average number of nuclei in untreated mock-infected cells.

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: Inhibition, Concentration Assay, Infection, Staining

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Inhibition of the RAF1 pathway negatively impacts viral DNA and protein accumulation. ( A–C ) MRC5 fibroblasts were treated with 2.17 µM of regorafenib or sorafenib or DMSO control at the time of infection with AD169 at an MOI of 3.0 for the indicated amount of time. ( A ) Viral DNA was quantified by RT-qPCR of the viral gene, IE1 [avg ± standard deviation (SD), n = 12]. ( B ) Samples in ( A ) were compared by area under the curve (AUC) to show the significance between treatments over the course of infection (avg ± SD, n = 12). Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test. ( C ) Viral protein expression levels at 24, 72, and 96 h post-infection (hpi) were assessed by western blot ( n = 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( D–G ) Quantification of western blots in ( C ) (avg ± standard error of the mean, n = 2) normalized to GAPDH and DMSO, 24 hpi sample for viral proteins ( D ) IE1, ( E ) IE2, ( F ) UL44, and ( G ) pp28.

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: Inhibition, Control, Infection, Quantitative RT-PCR, Standard Deviation, Comparison, Expressing, Western Blot

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: RAF1 shRNA-mediated knockdown inhibits HCMV infection. ( A–H ) Indicated shRNA guides were delivered via lentiviral delivery in HFF fibroblast cells. ( A–D ) RAF1 shRNA (shRAF1) were assessed for ( A ) knockdown efficiency in uninfected fibroblasts by RT-qPCR [avg ± standard error of the mean (SEM), n = 3 or 4], ( B ) protein expression was assessed via western blot ( n = 1), and ( C–D ) viral titer was measured by TCID50 (avg ± SEM, n = 4) at ( C ) an MOI of 3.0 at 120 h post-infection, or ( D ) an MOI of 0.01 at 10 days post-infection. ( E–F ) ARAF shRNA (shARAF) were assessed for ( E ) RNA levels via RT-qPCR and ( F ) viral titers via the GFP expression of cells infected at an MOI of 3.0 at 120 h post-infection. ( G–H ) BRAF shRNA (shBRAF) were assessed for ( G ) RNA levels via RT-qPCR and ( H ) viral titers via GFP expression of cells infected at an MOI of 3.0 at 120 h post-infection. ( A, E, G ) RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and shRNA empty vector (shEV) control in all RT-qPCR experiments. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test.

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: shRNA, Knockdown, Infection, Quantitative RT-PCR, Expressing, Western Blot, Plasmid Preparation, Control, Comparison

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: CRISPR-mediated knockout of RAF1 attenuates cell-to-cell spread of HCMV. ( A ) Knockout (KO) scores for CRISPR RAF1 KO cell lines generated. ( B–D ) MRC5 parental (par) and RAF1 polyclonal CRISPR knockout (poly KO) cells were infected at an MOI of 3.0 for ( B ) 96 h, and protein expression was assessed by western blot during mock ( M ) or HCMV ( H ) infection ( n = 1). UL26 is a control for successful viral infection, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein loading control. ( C ) At 72, 96, and 120 h post infection (hpi), viral titers were assessed by TCID50 [avg ± standard error of the mean (SEM), n = 2]. Significance is based on Student’s t -test at 120 hpi. ( D ) MRC5 (avg ± SEM, n = 72) and ( E ) HFF (avg ± SEM, n = 48), parental, and RAF1 polyclonal CRISPR knockout cell lines were infected with AD169-GFP at an MOI of 0.05 for 10 days. Infected cells per area of the well were counted via GFP expression, and significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test.

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: CRISPR, Knock-Out, Generated, Infection, Expressing, Western Blot, Control, Comparison

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Pharmacological inhibition of RAF1 attenuates HCMV TB40/E cell-to-cell spread in fibroblasts and epithelial cells. ( A, B ) MRC5 fibroblasts or ( C ) ARPE19 epithelial cells were treated with 2.17 µM of regorafenib or sorafenib or DMSO control at the time of infection with ( A ) AD169-GFP or ( B, C ) TB40/E-mCherry at an MOI of 0.01. Viral spread was calculated by cells positive for ( A ) GFP or ( B ) mCherry on Day 12 in MRC5 cells or ( C ) mCherry on Days 15 and 30 in ARPE19 cells [avg ± standard error of the mean (SEM), n = 48]. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test. ( D, E ) Mock-infected ARPE19 cells were treated with indicated concentrations of ( D ) regorafenib or ( E ) sorafenib at t = 0. Cells were fixed on Day 1, 5, or 11. Hoechst-stained nuclei were counted and plotted against time (avg ± SEM, n = 3).

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: Inhibition, Control, Infection, Comparison, Staining

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: CRISPR guide sequences

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: CRISPR, Sequencing

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Primers used for genomic PCRs of CRISPR polyclonal knockout cell lines

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: CRISPR, Knock-Out

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: List of antibodies used ^a

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques:

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: List of qPCR primer sequences

Article Snippet: Phospho-Raf1 (c-Raf) (Ser338) , Cell Signaling Technology , 9427.

Techniques: Sequencing

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Human cytomegalovirus (HCMV) infection induces the AMP-activated protein kinase-specific phosphorylation of RAF1. ( A ) Schematic depicting the RAF1 signaling pathway. ( B ) Western blot analysis of RAF1 protein over the course of infection (120 h) in the absence and presence of 5 µM compound C (CC) in MRC5 fibroblasts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and UL26 is a control for successful viral infection. ( C–E ) Quantification of Western blots in ( B ), where 0 h post-infection (hpi) is representative of mock infection (avg ± standard error of the mean, n = 2), comparing phospho-RAF1 to total RAF1 for ( C ) P-RAF1-S338 and ( D ) P-RAF1-S621, and ( E ) total RAF1 levels relative to GAPDH. ( F ) Two-dimensional gel electrophoresis of RAF1 protein during mock infection and HCMV infection with or without CC treatment, MOI = 3.0 at 48 hpi in MRC5 fibroblasts ( n = 1).

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: Infection, Phospho-proteomics, Western Blot, Control, Two-Dimensional Gel Electrophoresis, Electrophoresis

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Phosphorylation of S621 enhances RAF1 binding to 14-3-3 during HCMV infection. ( A ) Overexpression of Flag-RAF1-WT (wild type) or Flag-RAF1-S621A via lentiviral transduction were compared to control MRC5 fibroblasts and assessed for protein expression by western blot using antibodies specific to Flag, RAF1, and S621- or S338-phosphorylated RAF1 and total or phospho-MEK1/2 and -ERK1/2 (arrows indicate doublet). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control ( n = 1). ( B ) Empty vector (EV), Flag-RAF1-WT, and -S621A transduced cells were mock ( M )- or HCMV ( H )-infected with AD169-WT at an MOI of 3.0 for 72 h in the presence or absence of 5 µM Compound C (CC) prior to Flag pulldown and co-precipitation of endogenous 14-3-3. Numbers indicate the ratio of 14-3-3 to Flag protein pulled down in the representative blot. ( C ) Ratios of 14-3-3 to Flag pulled down during HCMV infection lanes in ( B ) were quantified along with a second blot (not shown) (avg, n = 2). ( D ) Cells were infected at an MOI of 3.0 for 120 h or ( E ) at an MOI of 0.01 for 9 days (avg ±SEM, n = 4), and viral titers were assessed by TCID50. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparisons test.

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: Phospho-proteomics, Binding Assay, Infection, Over Expression, Transduction, Control, Expressing, Western Blot, Plasmid Preparation

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Reported IC50s of regorafenib and sorafenib to recombinant RAF pathway proteins ( 50 , 51 )

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: Recombinant

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Pharmacological inhibition of the RAF1 pathway inhibits HCMV infection. ( A, B ) Regorafenib (Reg) or ( C, D ) sorafenib (Sor) treated ( A, C ) MRC5 or ( B, D ) HFF fibroblasts were treated with the indicated concentration of drugs or DMSO control at the time of infection. ( A–D ) Cells were infected at an MOI of 0.1 with AD169-GFP for 5 days, then fixed. GFP-positive cells were counted per area of the well and plotted against drug concentration [avg ± standard error of the mean (SEM), n = 3]. IC50 values were calculated. The dotted line indicates the average maximal GFP in untreated cells. ( E–H ) Fibroblasts were infected at an MOI of 3.0 in the presence of DMSO or 2.17 µM regorafenib or sorafenib added at the time of viral adsorption. UN is an untreated control. Viral titers from ( E ) MRC5 or ( F ) HFF cells were assessed by TCID50 (avg ± SEM, n = 3) 120 hpi. The dotted line indicates the limit of detection. In ( G ) MRC5 or ( H ) HFF cells, at 24 h post-infection (hpi), phosphorylation of MEK1/2 and ERK1/2 (arrows indicate doublet) was assessed by western blot ( n = 1).

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: Inhibition, Infection, Concentration Assay, Control, Adsorption, Phospho-proteomics, Western Blot

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Pharmacological inhibition of the RAF1 pathway does not impact fibroblast cell counts. ( A, B ) MRC5 or ( C, D ) HFF fibroblasts were treated with the indicated concentration of ( A, C ) regorafenib or ( B, D ) sorafenib at the time of infection, AD169-GFP, MOI = 0.1, fixed on Day 5. ( A–D ) Hoechst-stained nuclei were counted and plotted against the concentration (avg ± standard error of the mean, n = 3). The solid gray line indicates the average number of nuclei in untreated HCMV-infected cells, while the dotted gray line indicates the average number of nuclei in untreated mock-infected cells.

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: Inhibition, Concentration Assay, Infection, Staining

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Inhibition of the RAF1 pathway negatively impacts viral DNA and protein accumulation. ( A–C ) MRC5 fibroblasts were treated with 2.17 µM of regorafenib or sorafenib or DMSO control at the time of infection with AD169 at an MOI of 3.0 for the indicated amount of time. ( A ) Viral DNA was quantified by RT-qPCR of the viral gene, IE1 [avg ± standard deviation (SD), n = 12]. ( B ) Samples in ( A ) were compared by area under the curve (AUC) to show the significance between treatments over the course of infection (avg ± SD, n = 12). Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test. ( C ) Viral protein expression levels at 24, 72, and 96 h post-infection (hpi) were assessed by western blot ( n = 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( D–G ) Quantification of western blots in ( C ) (avg ± standard error of the mean, n = 2) normalized to GAPDH and DMSO, 24 hpi sample for viral proteins ( D ) IE1, ( E ) IE2, ( F ) UL44, and ( G ) pp28.

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: Inhibition, Control, Infection, Quantitative RT-PCR, Standard Deviation, Comparison, Expressing, Western Blot

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: RAF1 shRNA-mediated knockdown inhibits HCMV infection. ( A–H ) Indicated shRNA guides were delivered via lentiviral delivery in HFF fibroblast cells. ( A–D ) RAF1 shRNA (shRAF1) were assessed for ( A ) knockdown efficiency in uninfected fibroblasts by RT-qPCR [avg ± standard error of the mean (SEM), n = 3 or 4], ( B ) protein expression was assessed via western blot ( n = 1), and ( C–D ) viral titer was measured by TCID50 (avg ± SEM, n = 4) at ( C ) an MOI of 3.0 at 120 h post-infection, or ( D ) an MOI of 0.01 at 10 days post-infection. ( E–F ) ARAF shRNA (shARAF) were assessed for ( E ) RNA levels via RT-qPCR and ( F ) viral titers via the GFP expression of cells infected at an MOI of 3.0 at 120 h post-infection. ( G–H ) BRAF shRNA (shBRAF) were assessed for ( G ) RNA levels via RT-qPCR and ( H ) viral titers via GFP expression of cells infected at an MOI of 3.0 at 120 h post-infection. ( A, E, G ) RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and shRNA empty vector (shEV) control in all RT-qPCR experiments. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test.

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: shRNA, Knockdown, Infection, Quantitative RT-PCR, Expressing, Western Blot, Plasmid Preparation, Control, Comparison

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: CRISPR-mediated knockout of RAF1 attenuates cell-to-cell spread of HCMV. ( A ) Knockout (KO) scores for CRISPR RAF1 KO cell lines generated. ( B–D ) MRC5 parental (par) and RAF1 polyclonal CRISPR knockout (poly KO) cells were infected at an MOI of 3.0 for ( B ) 96 h, and protein expression was assessed by western blot during mock ( M ) or HCMV ( H ) infection ( n = 1). UL26 is a control for successful viral infection, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein loading control. ( C ) At 72, 96, and 120 h post infection (hpi), viral titers were assessed by TCID50 [avg ± standard error of the mean (SEM), n = 2]. Significance is based on Student’s t -test at 120 hpi. ( D ) MRC5 (avg ± SEM, n = 72) and ( E ) HFF (avg ± SEM, n = 48), parental, and RAF1 polyclonal CRISPR knockout cell lines were infected with AD169-GFP at an MOI of 0.05 for 10 days. Infected cells per area of the well were counted via GFP expression, and significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test.

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: CRISPR, Knock-Out, Generated, Infection, Expressing, Western Blot, Control, Comparison

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Pharmacological inhibition of RAF1 attenuates HCMV TB40/E cell-to-cell spread in fibroblasts and epithelial cells. ( A, B ) MRC5 fibroblasts or ( C ) ARPE19 epithelial cells were treated with 2.17 µM of regorafenib or sorafenib or DMSO control at the time of infection with ( A ) AD169-GFP or ( B, C ) TB40/E-mCherry at an MOI of 0.01. Viral spread was calculated by cells positive for ( A ) GFP or ( B ) mCherry on Day 12 in MRC5 cells or ( C ) mCherry on Days 15 and 30 in ARPE19 cells [avg ± standard error of the mean (SEM), n = 48]. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test. ( D, E ) Mock-infected ARPE19 cells were treated with indicated concentrations of ( D ) regorafenib or ( E ) sorafenib at t = 0. Cells were fixed on Day 1, 5, or 11. Hoechst-stained nuclei were counted and plotted against time (avg ± SEM, n = 3).

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: Inhibition, Control, Infection, Comparison, Staining

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: CRISPR guide sequences

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: CRISPR, Sequencing

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Primers used for genomic PCRs of CRISPR polyclonal knockout cell lines

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: CRISPR, Knock-Out

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: List of antibodies used ^a

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques:

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: List of qPCR primer sequences

Article Snippet: Raf1 (c-Raf) (D4B3J) , Cell Signaling Technology , 53745.

Techniques: Sequencing

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Human cytomegalovirus (HCMV) infection induces the AMP-activated protein kinase-specific phosphorylation of RAF1. ( A ) Schematic depicting the RAF1 signaling pathway. ( B ) Western blot analysis of RAF1 protein over the course of infection (120 h) in the absence and presence of 5 µM compound C (CC) in MRC5 fibroblasts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and UL26 is a control for successful viral infection. ( C–E ) Quantification of Western blots in ( B ), where 0 h post-infection (hpi) is representative of mock infection (avg ± standard error of the mean, n = 2), comparing phospho-RAF1 to total RAF1 for ( C ) P-RAF1-S338 and ( D ) P-RAF1-S621, and ( E ) total RAF1 levels relative to GAPDH. ( F ) Two-dimensional gel electrophoresis of RAF1 protein during mock infection and HCMV infection with or without CC treatment, MOI = 3.0 at 48 hpi in MRC5 fibroblasts ( n = 1).

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: Infection, Phospho-proteomics, Western Blot, Control, Two-Dimensional Gel Electrophoresis, Electrophoresis

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Phosphorylation of S621 enhances RAF1 binding to 14-3-3 during HCMV infection. ( A ) Overexpression of Flag-RAF1-WT (wild type) or Flag-RAF1-S621A via lentiviral transduction were compared to control MRC5 fibroblasts and assessed for protein expression by western blot using antibodies specific to Flag, RAF1, and S621- or S338-phosphorylated RAF1 and total or phospho-MEK1/2 and -ERK1/2 (arrows indicate doublet). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control ( n = 1). ( B ) Empty vector (EV), Flag-RAF1-WT, and -S621A transduced cells were mock ( M )- or HCMV ( H )-infected with AD169-WT at an MOI of 3.0 for 72 h in the presence or absence of 5 µM Compound C (CC) prior to Flag pulldown and co-precipitation of endogenous 14-3-3. Numbers indicate the ratio of 14-3-3 to Flag protein pulled down in the representative blot. ( C ) Ratios of 14-3-3 to Flag pulled down during HCMV infection lanes in ( B ) were quantified along with a second blot (not shown) (avg, n = 2). ( D ) Cells were infected at an MOI of 3.0 for 120 h or ( E ) at an MOI of 0.01 for 9 days (avg ±SEM, n = 4), and viral titers were assessed by TCID50. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparisons test.

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: Phospho-proteomics, Binding Assay, Infection, Over Expression, Transduction, Control, Expressing, Western Blot, Plasmid Preparation

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Reported IC50s of regorafenib and sorafenib to recombinant RAF pathway proteins ( 50 , 51 )

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: Recombinant

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Pharmacological inhibition of the RAF1 pathway inhibits HCMV infection. ( A, B ) Regorafenib (Reg) or ( C, D ) sorafenib (Sor) treated ( A, C ) MRC5 or ( B, D ) HFF fibroblasts were treated with the indicated concentration of drugs or DMSO control at the time of infection. ( A–D ) Cells were infected at an MOI of 0.1 with AD169-GFP for 5 days, then fixed. GFP-positive cells were counted per area of the well and plotted against drug concentration [avg ± standard error of the mean (SEM), n = 3]. IC50 values were calculated. The dotted line indicates the average maximal GFP in untreated cells. ( E–H ) Fibroblasts were infected at an MOI of 3.0 in the presence of DMSO or 2.17 µM regorafenib or sorafenib added at the time of viral adsorption. UN is an untreated control. Viral titers from ( E ) MRC5 or ( F ) HFF cells were assessed by TCID50 (avg ± SEM, n = 3) 120 hpi. The dotted line indicates the limit of detection. In ( G ) MRC5 or ( H ) HFF cells, at 24 h post-infection (hpi), phosphorylation of MEK1/2 and ERK1/2 (arrows indicate doublet) was assessed by western blot ( n = 1).

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: Inhibition, Infection, Concentration Assay, Control, Adsorption, Phospho-proteomics, Western Blot

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Pharmacological inhibition of the RAF1 pathway does not impact fibroblast cell counts. ( A, B ) MRC5 or ( C, D ) HFF fibroblasts were treated with the indicated concentration of ( A, C ) regorafenib or ( B, D ) sorafenib at the time of infection, AD169-GFP, MOI = 0.1, fixed on Day 5. ( A–D ) Hoechst-stained nuclei were counted and plotted against the concentration (avg ± standard error of the mean, n = 3). The solid gray line indicates the average number of nuclei in untreated HCMV-infected cells, while the dotted gray line indicates the average number of nuclei in untreated mock-infected cells.

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: Inhibition, Concentration Assay, Infection, Staining

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Inhibition of the RAF1 pathway negatively impacts viral DNA and protein accumulation. ( A–C ) MRC5 fibroblasts were treated with 2.17 µM of regorafenib or sorafenib or DMSO control at the time of infection with AD169 at an MOI of 3.0 for the indicated amount of time. ( A ) Viral DNA was quantified by RT-qPCR of the viral gene, IE1 [avg ± standard deviation (SD), n = 12]. ( B ) Samples in ( A ) were compared by area under the curve (AUC) to show the significance between treatments over the course of infection (avg ± SD, n = 12). Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test. ( C ) Viral protein expression levels at 24, 72, and 96 h post-infection (hpi) were assessed by western blot ( n = 2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( D–G ) Quantification of western blots in ( C ) (avg ± standard error of the mean, n = 2) normalized to GAPDH and DMSO, 24 hpi sample for viral proteins ( D ) IE1, ( E ) IE2, ( F ) UL44, and ( G ) pp28.

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: Inhibition, Control, Infection, Quantitative RT-PCR, Standard Deviation, Comparison, Expressing, Western Blot

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: RAF1 shRNA-mediated knockdown inhibits HCMV infection. ( A–H ) Indicated shRNA guides were delivered via lentiviral delivery in HFF fibroblast cells. ( A–D ) RAF1 shRNA (shRAF1) were assessed for ( A ) knockdown efficiency in uninfected fibroblasts by RT-qPCR [avg ± standard error of the mean (SEM), n = 3 or 4], ( B ) protein expression was assessed via western blot ( n = 1), and ( C–D ) viral titer was measured by TCID50 (avg ± SEM, n = 4) at ( C ) an MOI of 3.0 at 120 h post-infection, or ( D ) an MOI of 0.01 at 10 days post-infection. ( E–F ) ARAF shRNA (shARAF) were assessed for ( E ) RNA levels via RT-qPCR and ( F ) viral titers via the GFP expression of cells infected at an MOI of 3.0 at 120 h post-infection. ( G–H ) BRAF shRNA (shBRAF) were assessed for ( G ) RNA levels via RT-qPCR and ( H ) viral titers via GFP expression of cells infected at an MOI of 3.0 at 120 h post-infection. ( A, E, G ) RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and shRNA empty vector (shEV) control in all RT-qPCR experiments. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test.

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: shRNA, Knockdown, Infection, Quantitative RT-PCR, Expressing, Western Blot, Plasmid Preparation, Control, Comparison

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: CRISPR-mediated knockout of RAF1 attenuates cell-to-cell spread of HCMV. ( A ) Knockout (KO) scores for CRISPR RAF1 KO cell lines generated. ( B–D ) MRC5 parental (par) and RAF1 polyclonal CRISPR knockout (poly KO) cells were infected at an MOI of 3.0 for ( B ) 96 h, and protein expression was assessed by western blot during mock ( M ) or HCMV ( H ) infection ( n = 1). UL26 is a control for successful viral infection, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein loading control. ( C ) At 72, 96, and 120 h post infection (hpi), viral titers were assessed by TCID50 [avg ± standard error of the mean (SEM), n = 2]. Significance is based on Student’s t -test at 120 hpi. ( D ) MRC5 (avg ± SEM, n = 72) and ( E ) HFF (avg ± SEM, n = 48), parental, and RAF1 polyclonal CRISPR knockout cell lines were infected with AD169-GFP at an MOI of 0.05 for 10 days. Infected cells per area of the well were counted via GFP expression, and significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test.

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: CRISPR, Knock-Out, Generated, Infection, Expressing, Western Blot, Control, Comparison

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Pharmacological inhibition of RAF1 attenuates HCMV TB40/E cell-to-cell spread in fibroblasts and epithelial cells. ( A, B ) MRC5 fibroblasts or ( C ) ARPE19 epithelial cells were treated with 2.17 µM of regorafenib or sorafenib or DMSO control at the time of infection with ( A ) AD169-GFP or ( B, C ) TB40/E-mCherry at an MOI of 0.01. Viral spread was calculated by cells positive for ( A ) GFP or ( B ) mCherry on Day 12 in MRC5 cells or ( C ) mCherry on Days 15 and 30 in ARPE19 cells [avg ± standard error of the mean (SEM), n = 48]. Significance was established by a one-way analysis of variance test, followed by Tukey’s multiple-comparison test. ( D, E ) Mock-infected ARPE19 cells were treated with indicated concentrations of ( D ) regorafenib or ( E ) sorafenib at t = 0. Cells were fixed on Day 1, 5, or 11. Hoechst-stained nuclei were counted and plotted against time (avg ± SEM, n = 3).

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: Inhibition, Control, Infection, Comparison, Staining

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: CRISPR guide sequences

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: CRISPR, Sequencing

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: Primers used for genomic PCRs of CRISPR polyclonal knockout cell lines

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: CRISPR, Knock-Out

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: List of antibodies used ^a

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques:

Journal: Journal of Virology

Article Title: RAF1 promotes successful human cytomegalovirus replication and is regulated by AMPK-mediated phosphorylation during infection

doi: 10.1128/jvi.01866-24

Figure Lengend Snippet: List of qPCR primer sequences

Article Snippet: Phospho-Raf1 (c-Raf) (Ser621) , Thermo Fisher Scientific , 44–504G.

Techniques: Sequencing