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RAF overexpression in RAS -mutant cells. ( A , B ) RAF inhibitors increase the abundance of RAF isoforms in NRAS -mutant cells. ( A ) Growing MEL-JUSO cells were treated with increasing concentrations of Type I RAF inhibitor SB-590885 ( left ) or Type II RAF inhibitor Sorafenib ( right ), as indicated, for 24 h. DMSO served as a control. Cells were lysed and the expression of RAF isoforms and ERK activation were assessed using western blot. One representative replicate is shown. ( B ) Growing MEL-JUSO cells were treated with 4 μM Type II RAF inhibitor TAK-632 for the indicated times and analyzed as in ( A ). n.t.—non treated ( C , D ) Overexpression of RAF isoforms contributes to reactivation of ERK signaling. ( C ) Growing MEL-JUSO cells were treated with 6 μM SB-590885 (blue) or 9 μM Sorafenib (green) for the indicated times. Relative ppERK levels were quantified using the MSD Multi-Spot Assay ELISA System. ( D ) Overexpression of <t>BRAF</t> WT in CaCo-2tet cells (RAS WT ) was induced by addition of doxycycline for 24 h (orange). Uninduced cells (black) were used as control. Subsequently, cells were treated for a further 24 h with increasing amounts of TAK-632. Relative ppERK levels were quantified using Luminex ELISA, as described. The averages of three independent replicates are shown, error bars represent mean ± SEM. ( E ) Dependence of ERK signaling on the RAF abundance for cells with wild-type RAS ([RAS–GTP] = 25 nM). BRAF abundance is set to 50 nM (black) and 200 nM (orange). For both conditions, CRAF and ARAF abundances are set to 50 nM. The model predicts stationary responses of ERK signaling to Type II RAF inhibitor (e.g., TAK-632).

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1) Product Images from "A Combination of Conformation-Specific RAF Inhibitors Overcome Drug Resistance Brought about by RAF Overexpression"

Article Title: A Combination of Conformation-Specific RAF Inhibitors Overcome Drug Resistance Brought about by RAF Overexpression

Journal: Biomolecules

doi: 10.3390/biom13081212

Figure Legend Snippet: RAF overexpression in RAS -mutant cells. ( A , B ) RAF inhibitors increase the abundance of RAF isoforms in NRAS -mutant cells. ( A ) Growing MEL-JUSO cells were treated with increasing concentrations of Type I RAF inhibitor SB-590885 ( left ) or Type II RAF inhibitor Sorafenib ( right ), as indicated, for 24 h. DMSO served as a control. Cells were lysed and the expression of RAF isoforms and ERK activation were assessed using western blot. One representative replicate is shown. ( B ) Growing MEL-JUSO cells were treated with 4 μM Type II RAF inhibitor TAK-632 for the indicated times and analyzed as in ( A ). n.t.—non treated ( C , D ) Overexpression of RAF isoforms contributes to reactivation of ERK signaling. ( C ) Growing MEL-JUSO cells were treated with 6 μM SB-590885 (blue) or 9 μM Sorafenib (green) for the indicated times. Relative ppERK levels were quantified using the MSD Multi-Spot Assay ELISA System. ( D ) Overexpression of BRAF WT in CaCo-2tet cells (RAS WT ) was induced by addition of doxycycline for 24 h (orange). Uninduced cells (black) were used as control. Subsequently, cells were treated for a further 24 h with increasing amounts of TAK-632. Relative ppERK levels were quantified using Luminex ELISA, as described. The averages of three independent replicates are shown, error bars represent mean ± SEM. ( E ) Dependence of ERK signaling on the RAF abundance for cells with wild-type RAS ([RAS–GTP] = 25 nM). BRAF abundance is set to 50 nM (black) and 200 nM (orange). For both conditions, CRAF and ARAF abundances are set to 50 nM. The model predicts stationary responses of ERK signaling to Type II RAF inhibitor (e.g., TAK-632).

Techniques Used: Over Expression, Mutagenesis, Control, Expressing, Activation Assay, Western Blot, Spot Test, Enzyme-linked Immunosorbent Assay, Luminex

Structure-based model predictions: ARAF isoform upregulation results in the increase in the range of paradoxical activation. ( A ) Schematic overview of the processes described in the model. ( B – D ) Dependence of ERK signaling on the ARAF abundance for cells expressing mutant RAS ([RAS–GTP] = 250 nM). ARAF abundance was set to 50 nM (black) and 200 nM (sky blue). For both conditions, BRAF and CRAF abundances were set to 50 nM. Dashed horizontal lines indicate basal ppERK levels for each condition. Vertical dashed lines denote inhibitor concentrations at which dose response curves drop below their basal levels. The model predicts stationary responses of ERK signaling to the Type II RAF inhibitor ( B ), Type I RAF inhibitor ( C ), and Type I½ RAF inhibitor ( D ).

Figure Legend Snippet: Structure-based model predictions: ARAF isoform upregulation results in the increase in the range of paradoxical activation. ( A ) Schematic overview of the processes described in the model. ( B – D ) Dependence of ERK signaling on the ARAF abundance for cells expressing mutant RAS ([RAS–GTP] = 250 nM). ARAF abundance was set to 50 nM (black) and 200 nM (sky blue). For both conditions, BRAF and CRAF abundances were set to 50 nM. Dashed horizontal lines indicate basal ppERK levels for each condition. Vertical dashed lines denote inhibitor concentrations at which dose response curves drop below their basal levels. The model predicts stationary responses of ERK signaling to the Type II RAF inhibitor ( B ), Type I RAF inhibitor ( C ), and Type I½ RAF inhibitor ( D ).

Techniques Used: Activation Assay, Expressing, Mutagenesis

Knockout of one RAF isoform results in overexpression of other RAF isoforms to sustain ERK signaling. ( A ) Model predictions of dose responses and their dependencies on the RAF isoform abundances. Effect of Type I½ and Type II RAF inhibitors on ERK phosphorylation under ARAF knockout and BRAF overexpression. ( left ) [BRAF] = [CRAF] = 50 nM, [ARAF]= 200 nM. ( right ) [BRAF] = 100 nM, [CRAF] = 50 nM, [ARAF] = 0 nM. ( B ) ARAF +/+ or single-cell derived CRISPR/Cas9 knockout ARAF –/– MEL-JUSO cells were lysed, and RAF expression and ERK activation were analyzed by western blot. GAPDH served as a loading control. ( C ) Western blots of BRAF and CRAF expression levels in three replicates were quantified. Bar charts represent relative BRAF or CRAF expression in ARAF –/– cell clones. Error bars represent mean ± SEM. Clone #D (highlighted in red) was used for the dose response curves shown in ( D ). ( D ) Growing ARAF +/+ or ARAF –/– MEL-JUSO cells were treated with increasing concentrations of Vemurafenib or Sorafenib as indicated for 24 h. DMSO served as a control. ERK1/2 activation was assessed using the Magpix ELISA system. Charts represent relative ERK1/2 activation, error bars represent mean ± SEM, n = 3.

Figure Legend Snippet: Knockout of one RAF isoform results in overexpression of other RAF isoforms to sustain ERK signaling. ( A ) Model predictions of dose responses and their dependencies on the RAF isoform abundances. Effect of Type I½ and Type II RAF inhibitors on ERK phosphorylation under ARAF knockout and BRAF overexpression. ( left ) [BRAF] = [CRAF] = 50 nM, [ARAF]= 200 nM. ( right ) [BRAF] = 100 nM, [CRAF] = 50 nM, [ARAF] = 0 nM. ( B ) ARAF +/+ or single-cell derived CRISPR/Cas9 knockout ARAF –/– MEL-JUSO cells were lysed, and RAF expression and ERK activation were analyzed by western blot. GAPDH served as a loading control. ( C ) Western blots of BRAF and CRAF expression levels in three replicates were quantified. Bar charts represent relative BRAF or CRAF expression in ARAF –/– cell clones. Error bars represent mean ± SEM. Clone #D (highlighted in red) was used for the dose response curves shown in ( D ). ( D ) Growing ARAF +/+ or ARAF –/– MEL-JUSO cells were treated with increasing concentrations of Vemurafenib or Sorafenib as indicated for 24 h. DMSO served as a control. ERK1/2 activation was assessed using the Magpix ELISA system. Charts represent relative ERK1/2 activation, error bars represent mean ± SEM, n = 3.

Techniques Used: Knock-Out, Over Expression, Phospho-proteomics, Derivative Assay, CRISPR, Expressing, Activation Assay, Western Blot, Control, Clone Assay, Enzyme-linked Immunosorbent Assay

Our model predicts how combination of two RAF inhibitors overcomes drug resistance brought about by RAF overexpression. ( A ) Predictive simulations demonstrate synergy of inhibiting ERK signaling by combinations of Type I and Type II RAF inhibitors in RAF-overexpressing cells. ( Left panel ) Dependencies of ERK activation on BRAF and ARAF abundances for cells treated with 150·Kd Type I RAF inhibitor (blue), 200·Kd Type II RAF inhibitor (green), or a combination of 75·Kd Type I RAF inhibitor and 100·Kd Type II RAF inhibitor (red). The concentration of CRAF is 50 nM. ( Middle and right panels ) Isolines of steady-state ERK signaling responses to type Type I and Type II RAF inhibitors and their combinations. ( Middle panel ) [BRAF] = [ARAF] = 25 nM. ( Right panel ) [BRAF] = [ARAF] = 100 nM. For both conditions, [CRAF] = 50 nM. ( B ) ppERK signaling responses of growing MEL-JUSO cells to two structurally different RAF inhibitors and combination. ( Left panel ) Time course measured using MSD Multi-Spot Assay ELISA System in cells treated with 6 μM SB-590885 (blue), 9 μM Sorafenib (green), or a combination of 3 μM SB-590885 and 4.5 μM Sorafenib. ( Middle and right panels ) Dose responses to SB-590885, Sorafenib, and combination measured using xMAP assay for 1 h (middle) and 24 h of treatment, respectively.

Figure Legend Snippet: Our model predicts how combination of two RAF inhibitors overcomes drug resistance brought about by RAF overexpression. ( A ) Predictive simulations demonstrate synergy of inhibiting ERK signaling by combinations of Type I and Type II RAF inhibitors in RAF-overexpressing cells. ( Left panel ) Dependencies of ERK activation on BRAF and ARAF abundances for cells treated with 150·Kd Type I RAF inhibitor (blue), 200·Kd Type II RAF inhibitor (green), or a combination of 75·Kd Type I RAF inhibitor and 100·Kd Type II RAF inhibitor (red). The concentration of CRAF is 50 nM. ( Middle and right panels ) Isolines of steady-state ERK signaling responses to type Type I and Type II RAF inhibitors and their combinations. ( Middle panel ) [BRAF] = [ARAF] = 25 nM. ( Right panel ) [BRAF] = [ARAF] = 100 nM. For both conditions, [CRAF] = 50 nM. ( B ) ppERK signaling responses of growing MEL-JUSO cells to two structurally different RAF inhibitors and combination. ( Left panel ) Time course measured using MSD Multi-Spot Assay ELISA System in cells treated with 6 μM SB-590885 (blue), 9 μM Sorafenib (green), or a combination of 3 μM SB-590885 and 4.5 μM Sorafenib. ( Middle and right panels ) Dose responses to SB-590885, Sorafenib, and combination measured using xMAP assay for 1 h (middle) and 24 h of treatment, respectively.

Techniques Used: Over Expression, Activation Assay, Concentration Assay, Spot Test, Enzyme-linked Immunosorbent Assay

Imaging:

Article Title: A Combination of Conformation-Specific RAF Inhibitors Overcome Drug Resistance Brought about by RAF Overexpression
Article Snippet: Lysates were then resolved by SDS PAGE (10% PAA) in the Mini Protean Tetra system (Bio-Rad, Hercules, CA, USA), and proteins were transferred on a polyvinylidene difluoride membrane (PVDF, MilliporeSigma, Burlington, CA, USA). .. Protein visualization was performed by the iBrightTM CL750 Imaging System (InvitrogenTM, Thermo Fisher Scientific, Waltham, MA, USA), using horseradish peroxidase-conjugated secondary antibodies (#7074 resp. #7076, from Cell Signaling Technologies, Danvers, MA, USA), and the enhanced chemiluminescence system (GE Healthcare, Piscataway, NJ, USA) for the following antibodies: ARAF (D2P9P) (#75804); GAPDH (14C10) (#2118), both from Cell Signaling Technologies, Danvers, MA, USA; BRAF (F-7) (# sc-5284); CRAF/RAF1 (C-12) (# sc-133), both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA, 75220; and polyclonal rabbit anti-human mitogen-activated protein (MAP) kinase [extra-cellular signal-regulated kinase (ERK) 1 & 2] antibody (#M5670), monoclonal mouse anti-human MAP kinase, activated (diphosphorylated ERK-1 & 2) antibody (#8159), both from Sigma-Aldrich, Burlington, MA, USA. ..

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(A) Basal ppERK levels in parental PANC1 cells (black) and PANC1 cells overexpressing V600E (yellow) and G466E (blue) BRAF mutants, as predicted by our PANC1-specific model. (B) The model-predicted Loewe isobolograms for a combination of type I½ and type II RAFi in PANC1 cell overexpressing BRAF mutant V600E or G466E. (C) Representative western blots showing the BRAF and ppERK levels in PANC1 transfected with an empty vector (EV) or BRAF V600E and treated with trametinib (Tram), encorafenib (Enco), TAK-632 (TAK), or a combination of Enco and TAK. GAPDH was used as a loading control. (D and E) Quantification of the western blots for four biological replicates from two independent experiments. Data represent the mean ± SEM. (F) Representative western blots showing the BRAF and ppERK levels in PANC1 transfected with an empty vector or BRAF G466E and treated with Tram, Enco (type I½), TAK (type II), or a combination of Enco and TAK. GAPDH was used as a loading control. (G and H) Quantification of the western blots for three biological replicates. Data represent the mean ± SEM. Statistical analysis was performed using Student’s t test, one-way, or two-way ANOVA followed by Tukey’s post-hoc tests (letters describing statistical significance are explained in the legend to ). Log transformation was applied before performing the ANOVAs. For Student’s t test: ** p < 0.01 and *** p < 0.001.

Journal: Cell reports

Article Title: Cell-specific models reveal conformation-specific RAF inhibitor combinations that synergistically inhibit ERK signaling in pancreatic cancer cells

doi: 10.1016/j.celrep.2024.114710

Figure Lengend Snippet: (A) Basal ppERK levels in parental PANC1 cells (black) and PANC1 cells overexpressing V600E (yellow) and G466E (blue) BRAF mutants, as predicted by our PANC1-specific model. (B) The model-predicted Loewe isobolograms for a combination of type I½ and type II RAFi in PANC1 cell overexpressing BRAF mutant V600E or G466E. (C) Representative western blots showing the BRAF and ppERK levels in PANC1 transfected with an empty vector (EV) or BRAF V600E and treated with trametinib (Tram), encorafenib (Enco), TAK-632 (TAK), or a combination of Enco and TAK. GAPDH was used as a loading control. (D and E) Quantification of the western blots for four biological replicates from two independent experiments. Data represent the mean ± SEM. (F) Representative western blots showing the BRAF and ppERK levels in PANC1 transfected with an empty vector or BRAF G466E and treated with Tram, Enco (type I½), TAK (type II), or a combination of Enco and TAK. GAPDH was used as a loading control. (G and H) Quantification of the western blots for three biological replicates. Data represent the mean ± SEM. Statistical analysis was performed using Student’s t test, one-way, or two-way ANOVA followed by Tukey’s post-hoc tests (letters describing statistical significance are explained in the legend to ). Log transformation was applied before performing the ANOVAs. For Student’s t test: ** p < 0.01 and *** p < 0.001.

Article Snippet: BRAF Antibody (F-7) , Santa Cruz , sc-5284; RRID:AB_626760.

Techniques: Mutagenesis, Western Blot, Transfection, Plasmid Preparation, Control, Transformation Assay

Journal: Cell reports

Article Title: Cell-specific models reveal conformation-specific RAF inhibitor combinations that synergistically inhibit ERK signaling in pancreatic cancer cells

doi: 10.1016/j.celrep.2024.114710

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: BRAF Antibody (F-7) , Santa Cruz , sc-5284; RRID:AB_626760.

Techniques: Recombinant, Produced, Mass Spectrometry, Multiplex Assay, Bicinchoninic Acid Protein Assay, Proliferation Assay, Cell Viability Assay, Phospho-proteomics, Comparison, Western Blot, Software

Figure 3. Structure-based model predictions: ARAF isoform upregulation results in the increase in the range of paradoxical activation. (A) Schematic overview of the processes described in the model. (B–D) Dependence of ERK signaling on the ARAF abundance for cells expressing mutant RAS ([RAS–GTP] = 250 nM). ARAF abundance was set to 50 nM (black) and 200 nM (sky blue). For both conditions, BRAF and CRAF abundances were set to 50 nM. Dashed horizontal lines indicate basal ppERK levels for each condition. Vertical dashed lines denote inhibitor concentrations at which dose response curves drop below their basal levels. The model predicts stationary responses of ERK signaling to the Type II RAF inhibitor (B), Type I RAF inhibitor (C), and Type I 1

Journal: Biomolecules

Article Title: A Combination of Conformation-Specific RAF Inhibitors Overcome Drug Resistance Brought about by RAF Overexpression.

doi: 10.3390/biom13081212

Figure Lengend Snippet: Figure 3. Structure-based model predictions: ARAF isoform upregulation results in the increase in the range of paradoxical activation. (A) Schematic overview of the processes described in the model. (B–D) Dependence of ERK signaling on the ARAF abundance for cells expressing mutant RAS ([RAS–GTP] = 250 nM). ARAF abundance was set to 50 nM (black) and 200 nM (sky blue). For both conditions, BRAF and CRAF abundances were set to 50 nM. Dashed horizontal lines indicate basal ppERK levels for each condition. Vertical dashed lines denote inhibitor concentrations at which dose response curves drop below their basal levels. The model predicts stationary responses of ERK signaling to the Type II RAF inhibitor (B), Type I RAF inhibitor (C), and Type I 1

Article Snippet: Protein visualization was performed by the iBrightTM CL750 Imaging System (InvitrogenTM, Thermo Fisher Scientific, Waltham, MA, USA), using horseradish peroxidase-conjugated secondary antibodies (#7074 resp. #7076, from Cell Signaling Technologies, Danvers, MA, USA), and the enhanced chemiluminescence system (GE Healthcare, Piscataway, NJ, USA) for the following antibodies: ARAF (D2P9P) (#75804); GAPDH (14C10) (#2118), both from Cell Signaling Technologies, Danvers, MA, USA; BRAF (F-7) (# sc-5284); CRAF/RAF1 (C-12) (# sc-133), both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA, 75220; and polyclonal rabbit anti-human mitogen-activated protein (MAP) kinase [extra-cellular signalregulated kinase (ERK) 1 & 2] antibody (#M5670), monoclonal mouse anti-human MAP kinase, activated (diphosphorylated ERK-1 & 2) antibody (#8159), both from Sigma-Aldrich, Burlington, MA, USA.

Techniques: Activation Assay, Expressing, Mutagenesis

Journal: Biomolecules

Article Title: A Combination of Conformation-Specific RAF Inhibitors Overcome Drug Resistance Brought about by RAF Overexpression

doi: 10.3390/biom13081212

Figure Lengend Snippet: RAF overexpression in RAS -mutant cells. ( A , B ) RAF inhibitors increase the abundance of RAF isoforms in NRAS -mutant cells. ( A ) Growing MEL-JUSO cells were treated with increasing concentrations of Type I RAF inhibitor SB-590885 ( left ) or Type II RAF inhibitor Sorafenib ( right ), as indicated, for 24 h. DMSO served as a control. Cells were lysed and the expression of RAF isoforms and ERK activation were assessed using western blot. One representative replicate is shown. ( B ) Growing MEL-JUSO cells were treated with 4 μM Type II RAF inhibitor TAK-632 for the indicated times and analyzed as in ( A ). n.t.—non treated ( C , D ) Overexpression of RAF isoforms contributes to reactivation of ERK signaling. ( C ) Growing MEL-JUSO cells were treated with 6 μM SB-590885 (blue) or 9 μM Sorafenib (green) for the indicated times. Relative ppERK levels were quantified using the MSD Multi-Spot Assay ELISA System. ( D ) Overexpression of BRAF WT in CaCo-2tet cells (RAS WT ) was induced by addition of doxycycline for 24 h (orange). Uninduced cells (black) were used as control. Subsequently, cells were treated for a further 24 h with increasing amounts of TAK-632. Relative ppERK levels were quantified using Luminex ELISA, as described. The averages of three independent replicates are shown, error bars represent mean ± SEM. ( E ) Dependence of ERK signaling on the RAF abundance for cells with wild-type RAS ([RAS–GTP] = 25 nM). BRAF abundance is set to 50 nM (black) and 200 nM (orange). For both conditions, CRAF and ARAF abundances are set to 50 nM. The model predicts stationary responses of ERK signaling to Type II RAF inhibitor (e.g., TAK-632).

Article Snippet: Protein visualization was performed by the iBrightTM CL750 Imaging System (InvitrogenTM, Thermo Fisher Scientific, Waltham, MA, USA), using horseradish peroxidase-conjugated secondary antibodies (#7074 resp. #7076, from Cell Signaling Technologies, Danvers, MA, USA), and the enhanced chemiluminescence system (GE Healthcare, Piscataway, NJ, USA) for the following antibodies: ARAF (D2P9P) (#75804); GAPDH (14C10) (#2118), both from Cell Signaling Technologies, Danvers, MA, USA; BRAF (F-7) (# sc-5284); CRAF/RAF1 (C-12) (# sc-133), both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA, 75220; and polyclonal rabbit anti-human mitogen-activated protein (MAP) kinase [extra-cellular signal-regulated kinase (ERK) 1 & 2] antibody (#M5670), monoclonal mouse anti-human MAP kinase, activated (diphosphorylated ERK-1 & 2) antibody (#8159), both from Sigma-Aldrich, Burlington, MA, USA.

Techniques: Over Expression, Mutagenesis, Control, Expressing, Activation Assay, Western Blot, Spot Test, Enzyme-linked Immunosorbent Assay, Luminex

Journal: Biomolecules

Article Title: A Combination of Conformation-Specific RAF Inhibitors Overcome Drug Resistance Brought about by RAF Overexpression

doi: 10.3390/biom13081212

Figure Lengend Snippet: Structure-based model predictions: ARAF isoform upregulation results in the increase in the range of paradoxical activation. ( A ) Schematic overview of the processes described in the model. ( B – D ) Dependence of ERK signaling on the ARAF abundance for cells expressing mutant RAS ([RAS–GTP] = 250 nM). ARAF abundance was set to 50 nM (black) and 200 nM (sky blue). For both conditions, BRAF and CRAF abundances were set to 50 nM. Dashed horizontal lines indicate basal ppERK levels for each condition. Vertical dashed lines denote inhibitor concentrations at which dose response curves drop below their basal levels. The model predicts stationary responses of ERK signaling to the Type II RAF inhibitor ( B ), Type I RAF inhibitor ( C ), and Type I½ RAF inhibitor ( D ).

Article Snippet: Protein visualization was performed by the iBrightTM CL750 Imaging System (InvitrogenTM, Thermo Fisher Scientific, Waltham, MA, USA), using horseradish peroxidase-conjugated secondary antibodies (#7074 resp. #7076, from Cell Signaling Technologies, Danvers, MA, USA), and the enhanced chemiluminescence system (GE Healthcare, Piscataway, NJ, USA) for the following antibodies: ARAF (D2P9P) (#75804); GAPDH (14C10) (#2118), both from Cell Signaling Technologies, Danvers, MA, USA; BRAF (F-7) (# sc-5284); CRAF/RAF1 (C-12) (# sc-133), both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA, 75220; and polyclonal rabbit anti-human mitogen-activated protein (MAP) kinase [extra-cellular signal-regulated kinase (ERK) 1 & 2] antibody (#M5670), monoclonal mouse anti-human MAP kinase, activated (diphosphorylated ERK-1 & 2) antibody (#8159), both from Sigma-Aldrich, Burlington, MA, USA.

Techniques: Activation Assay, Expressing, Mutagenesis

Journal: Biomolecules

Article Title: A Combination of Conformation-Specific RAF Inhibitors Overcome Drug Resistance Brought about by RAF Overexpression

doi: 10.3390/biom13081212

Figure Lengend Snippet: Knockout of one RAF isoform results in overexpression of other RAF isoforms to sustain ERK signaling. ( A ) Model predictions of dose responses and their dependencies on the RAF isoform abundances. Effect of Type I½ and Type II RAF inhibitors on ERK phosphorylation under ARAF knockout and BRAF overexpression. ( left ) [BRAF] = [CRAF] = 50 nM, [ARAF]= 200 nM. ( right ) [BRAF] = 100 nM, [CRAF] = 50 nM, [ARAF] = 0 nM. ( B ) ARAF +/+ or single-cell derived CRISPR/Cas9 knockout ARAF –/– MEL-JUSO cells were lysed, and RAF expression and ERK activation were analyzed by western blot. GAPDH served as a loading control. ( C ) Western blots of BRAF and CRAF expression levels in three replicates were quantified. Bar charts represent relative BRAF or CRAF expression in ARAF –/– cell clones. Error bars represent mean ± SEM. Clone #D (highlighted in red) was used for the dose response curves shown in ( D ). ( D ) Growing ARAF +/+ or ARAF –/– MEL-JUSO cells were treated with increasing concentrations of Vemurafenib or Sorafenib as indicated for 24 h. DMSO served as a control. ERK1/2 activation was assessed using the Magpix ELISA system. Charts represent relative ERK1/2 activation, error bars represent mean ± SEM, n = 3.

Article Snippet: Protein visualization was performed by the iBrightTM CL750 Imaging System (InvitrogenTM, Thermo Fisher Scientific, Waltham, MA, USA), using horseradish peroxidase-conjugated secondary antibodies (#7074 resp. #7076, from Cell Signaling Technologies, Danvers, MA, USA), and the enhanced chemiluminescence system (GE Healthcare, Piscataway, NJ, USA) for the following antibodies: ARAF (D2P9P) (#75804); GAPDH (14C10) (#2118), both from Cell Signaling Technologies, Danvers, MA, USA; BRAF (F-7) (# sc-5284); CRAF/RAF1 (C-12) (# sc-133), both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA, 75220; and polyclonal rabbit anti-human mitogen-activated protein (MAP) kinase [extra-cellular signal-regulated kinase (ERK) 1 & 2] antibody (#M5670), monoclonal mouse anti-human MAP kinase, activated (diphosphorylated ERK-1 & 2) antibody (#8159), both from Sigma-Aldrich, Burlington, MA, USA.

Techniques: Knock-Out, Over Expression, Phospho-proteomics, Derivative Assay, CRISPR, Expressing, Activation Assay, Western Blot, Control, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: A Combination of Conformation-Specific RAF Inhibitors Overcome Drug Resistance Brought about by RAF Overexpression

doi: 10.3390/biom13081212

Figure Lengend Snippet: Our model predicts how combination of two RAF inhibitors overcomes drug resistance brought about by RAF overexpression. ( A ) Predictive simulations demonstrate synergy of inhibiting ERK signaling by combinations of Type I and Type II RAF inhibitors in RAF-overexpressing cells. ( Left panel ) Dependencies of ERK activation on BRAF and ARAF abundances for cells treated with 150·Kd Type I RAF inhibitor (blue), 200·Kd Type II RAF inhibitor (green), or a combination of 75·Kd Type I RAF inhibitor and 100·Kd Type II RAF inhibitor (red). The concentration of CRAF is 50 nM. ( Middle and right panels ) Isolines of steady-state ERK signaling responses to type Type I and Type II RAF inhibitors and their combinations. ( Middle panel ) [BRAF] = [ARAF] = 25 nM. ( Right panel ) [BRAF] = [ARAF] = 100 nM. For both conditions, [CRAF] = 50 nM. ( B ) ppERK signaling responses of growing MEL-JUSO cells to two structurally different RAF inhibitors and combination. ( Left panel ) Time course measured using MSD Multi-Spot Assay ELISA System in cells treated with 6 μM SB-590885 (blue), 9 μM Sorafenib (green), or a combination of 3 μM SB-590885 and 4.5 μM Sorafenib. ( Middle and right panels ) Dose responses to SB-590885, Sorafenib, and combination measured using xMAP assay for 1 h (middle) and 24 h of treatment, respectively.

Article Snippet: Protein visualization was performed by the iBrightTM CL750 Imaging System (InvitrogenTM, Thermo Fisher Scientific, Waltham, MA, USA), using horseradish peroxidase-conjugated secondary antibodies (#7074 resp. #7076, from Cell Signaling Technologies, Danvers, MA, USA), and the enhanced chemiluminescence system (GE Healthcare, Piscataway, NJ, USA) for the following antibodies: ARAF (D2P9P) (#75804); GAPDH (14C10) (#2118), both from Cell Signaling Technologies, Danvers, MA, USA; BRAF (F-7) (# sc-5284); CRAF/RAF1 (C-12) (# sc-133), both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA, 75220; and polyclonal rabbit anti-human mitogen-activated protein (MAP) kinase [extra-cellular signal-regulated kinase (ERK) 1 & 2] antibody (#M5670), monoclonal mouse anti-human MAP kinase, activated (diphosphorylated ERK-1 & 2) antibody (#8159), both from Sigma-Aldrich, Burlington, MA, USA.

Techniques: Over Expression, Activation Assay, Concentration Assay, Spot Test, Enzyme-linked Immunosorbent Assay

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braf f 7 (Cell Signaling Technology Inc)

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