Review

rabbit anti phospho braf antibody (Bioss)

Name: rabbit anti phospho braf antibody
Brand: Bioss
Rating: 94 (1 reviews)

Bioss is a verified supplier

Bioss manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Bioss rabbit anti phospho braf antibody
Rabbit Anti Phospho Braf Antibody, supplied by Bioss, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit anti phospho braf antibody/product/Bioss
Average 94 stars, based on 1 article reviews

rabbit anti phospho braf antibody - by Bioz Stars, 2026-02

94/100 stars

Images

Image Search Results

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).

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

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 ).

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 ).

Techniques: 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.

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.

Techniques: 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.

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.

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

Figure 2. Detection of MKRN1-BRAF fusion by next-generation sequencing. (A) Representative part of NGS sequencing of the MKRN1–BRAF fusion in THJ-16T cells. Green part, BRAF gene; blue part, MKRN1 gene; red part, the MKRN1-BRAF fusion. (B) Schematics of wild-type BRAF (green), wild-type MKRN1 (blue), BRAFV600E (yellow), and the fused MKRN1-BRAF proteins. The zinc ﬁnger domains of MKRN1 and the serine-threonine (S/T) kinase domain of BRAF remain intact in the fused protein. WT, wild-type; ex, exon; RBD, Ras-binding domain; CRD, cysteine-rich domain.

Journal: International journal of molecular sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Figure 2. Detection of MKRN1-BRAF fusion by next-generation sequencing. (A) Representative part of NGS sequencing of the MKRN1–BRAF fusion in THJ-16T cells. Green part, BRAF gene; blue part, MKRN1 gene; red part, the MKRN1-BRAF fusion. (B) Schematics of wild-type BRAF (green), wild-type MKRN1 (blue), BRAFV600E (yellow), and the fused MKRN1-BRAF proteins. The zinc ﬁnger domains of MKRN1 and the serine-threonine (S/T) kinase domain of BRAF remain intact in the fused protein. WT, wild-type; ex, exon; RBD, Ras-binding domain; CRD, cysteine-rich domain.

Article Snippet: The membrane was blocked with 5% skimmed milk in tris-buffered saline (TBS-T) and incubated overnight at 4 ◦C with total rabbit polyclonal anti-ERK1/2 (1:500, WL01864, Wanleibio, Shenyang, China), rabbit polyclonal anti-phosphorylated ERK1/2 (1:200, WLP1512, Wanleibio, Shenyang, China), total mouse monoclonal anti-MEK1/2 (1:1000, sc-81504, Santa Cruz Biotech, CA, Canada), mouse monoclonal anti-phosphorylated MEK1/2 (1:1000, sc-81503, Santa Cruz Biotech, CA, Canada), rabbit monoclonal anti-N-term BRAF (1:1000, #14814, Cell Signaling, Topsfield, MA, USA), rabbit polyclonal anti-C-term BRAF (1:1000, OM160689, Omnimabs, Alhambra, CA, USA), rabbit polyclonal anti-phosphorylated BRAF (1:1000, #2696, Cell Signaling, MA, USA), rabbit polyclonal anti-phosphorylated STAT3 (1:500, WLP2412, Wanleibio, Shenyang, China), total rabbit polyclonal anti-STAT3 (1:300, WL03208, Wanleibio, Shenyang, China), rabbit polyclonal anti-IL-6 (1:1000, WL02841, Wanleibio, Shenyang, China), and rabbit polyclonal anti-GAPDH (1;5000, 10494-1-AP, Proteintech, Wuhan, China), followed by incubation with HRP-conjugated goat anti-rabbit IgG (SE134, Solarbio Life Sciences, Beijing, China) or HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, Wuhan, China).

Techniques: Next-Generation Sequencing, Sequencing, Binding Assay

Figure 3. Identiﬁcation of mutant BRAF expression by reverse transcription–polymerase chain reaction and Sanger sequencing. Reverse transcription–polymerase chain reaction (RT-PCR) detected BRAF exon 15 and MKRN1-BRAF transcripts in ATC cells. Sanger sequencing chromatograph of the BRAF exon 15 and MKRN1–BRAF fusion in THJ-11T (A), THJ-16T (B), THJ-21T (C), and Nthyori 3-1 cells (A,C). The arrow shows the breakpoint of the fusion between MKRN1 (NM_001145125, end of exon 3) and BRAF (NM_004333, start of exon 10).

Journal: International journal of molecular sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Figure 3. Identiﬁcation of mutant BRAF expression by reverse transcription–polymerase chain reaction and Sanger sequencing. Reverse transcription–polymerase chain reaction (RT-PCR) detected BRAF exon 15 and MKRN1-BRAF transcripts in ATC cells. Sanger sequencing chromatograph of the BRAF exon 15 and MKRN1–BRAF fusion in THJ-11T (A), THJ-16T (B), THJ-21T (C), and Nthyori 3-1 cells (A,C). The arrow shows the breakpoint of the fusion between MKRN1 (NM_001145125, end of exon 3) and BRAF (NM_004333, start of exon 10).

Techniques: Mutagenesis, Expressing, Reverse Transcription, Polymerase Chain Reaction, Sequencing, Reverse Transcription Polymerase Chain Reaction

Figure 4. Differential expression of pBRAF, BRAF, pMEK, MEK, pERK, ERK in Nthyori 3-1, THJ-11T, THJ-16T, and THJ-21T cells. (A) Western blotting analyses of pBRAF, BRAF, pMEK, MEK, pERK, and ERK levels in Nthyori 3-1, THJ-16T, and THJ-21T cells. GAPDH served as a loading control. (B) Immunocytochemical staining (scale bar, 5 µm) of pERK performed on Nthyori 3-1, THJ-11T, THJ-16T, and THJ-21T cells.

Journal: International journal of molecular sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Figure 4. Differential expression of pBRAF, BRAF, pMEK, MEK, pERK, ERK in Nthyori 3-1, THJ-11T, THJ-16T, and THJ-21T cells. (A) Western blotting analyses of pBRAF, BRAF, pMEK, MEK, pERK, and ERK levels in Nthyori 3-1, THJ-16T, and THJ-21T cells. GAPDH served as a loading control. (B) Immunocytochemical staining (scale bar, 5 µm) of pERK performed on Nthyori 3-1, THJ-11T, THJ-16T, and THJ-21T cells.

Techniques: Quantitative Proteomics, Western Blot, Control, Staining

Figure 5. Differential pBRAF, BRAF, pMEK, MEK, pERK, ERK expression in THJ-11T, THJ-16T, and THJ-21T cells without and with resveratrol (R), trametinib (T), dabrafenib and trametinib (D+T), dabrafenib (D) and/or trametinib (T) in combination with resveratrol (R) treatment. (A) Western blotting was performed on the sample proteins of THJ-11T, THJ-16T, and THJ-21T cells before and after 48h drug treatment, GAPDH served as a loading control. (B) pERK immunocytochemical staining (scale bar, 5 µm) performed on THJ-11T, THJ-16T, and THJ-21T cells after 48h drugs treatment.

Journal: International journal of molecular sciences

Article Title: Dual Inhibition of BRAF-MAPK and STAT3 Signaling Pathways in Resveratrol-Suppressed Anaplastic Thyroid Cancer Cells with BRAF Mutations.

doi: 10.3390/ijms232214385

Figure Lengend Snippet: Figure 5. Differential pBRAF, BRAF, pMEK, MEK, pERK, ERK expression in THJ-11T, THJ-16T, and THJ-21T cells without and with resveratrol (R), trametinib (T), dabrafenib and trametinib (D+T), dabrafenib (D) and/or trametinib (T) in combination with resveratrol (R) treatment. (A) Western blotting was performed on the sample proteins of THJ-11T, THJ-16T, and THJ-21T cells before and after 48h drug treatment, GAPDH served as a loading control. (B) pERK immunocytochemical staining (scale bar, 5 µm) performed on THJ-11T, THJ-16T, and THJ-21T cells after 48h drugs treatment.

Techniques: Expressing, Western Blot, Control, Staining

a Structure of the SMP complex is shown as a surface. SHOC2 and MRAS are colored pink and blue, respectively. The surface of PP1CA is shown as an electrostatic surface as calculated by APBS. The three active site channels – acidic, hydrophobic and C-terminal are shown as yellow, cyan and green lines, respectively. Mn 2+ ion is shown as a gray sphere. b The CABS-dock server was used to generate a 15-mer peptide of the CR2-pS region of BRAF and dock into the PP1CA structure of the SMP complex. All 202 peptides from the top cluster of solutions are presented as ribbons. The vast majority being placed in the active site, with all peptides placed with their N- and C-termini in the acidic and hydrophobic active site channels. c Fluorescent western blot of CRAF either untreated or treated with SMP or lambda phosphatase (λP). Right-hand panels show the total CRAF present (red), while left-hand panels reveal CRAF by specific phosphoserine antibodies (green) targeting pS259 (top), pS43 (middle), and pS621 (bottom). Lambda phosphatase removes all phosphates, while the SMP complex only removes pS259. d Sequence alignments of the CRAF pS43, CR2-pS of ARAF, BRAF and CRAF, and CR3-pS of ARAF, BRAF and CRAF. The phosphoserine in each case is boxed in black at position 0. e The top docked CR2-pS peptide of BRAF is displayed as a ribbon in the active site with the PP1CA surface shown in electrostatic surface representation. S365 of BRAF present in the active site is colored magenta. The docked model suggests that A366 of BRAF would be placed inside the narrow negatively charged active site channel. This residue is an aspartic acid in the pS43 of CRAF and a glutamic acid in the CR3-pS peptides, offering a possible reason for the selectivity of the SMP complex for CR2-pS phosphopeptides. f Fluorescent Western blot of CRAF either untreated or treated with λP, PP1CA, SMP or SKP. Phosphoserine-specific antibodies for pS259 and pS621 are shown in red. Total CRAF is shown in green. SMP and SKP complexes specifically dephosphorylate pS259 of CRAF.P, PP1CA, SMP or SKP. Phosphoserine-specific antibodies for pS259 and pS621 are shown in red. Total CRAF is shown in green. SMP and SKP complexes specifically dephosphorylate pS259 of CRAF. g Comparison of dephosphorylation activity ( EC50) of PP1CA and SMP complex on BRAF and RAF substrates derived from Li-COR quantification of bands from Supplementary Fig. 6c. h Model showing the role of the SMP complex in the RAF activation process.

Journal: bioRxiv

Article Title: Structure of the SHOC2–MRAS–PP1C complex provides insights into RAF activation and Noonan syndrome

doi: 10.1101/2022.05.10.491335

Figure Lengend Snippet: a Structure of the SMP complex is shown as a surface. SHOC2 and MRAS are colored pink and blue, respectively. The surface of PP1CA is shown as an electrostatic surface as calculated by APBS. The three active site channels – acidic, hydrophobic and C-terminal are shown as yellow, cyan and green lines, respectively. Mn 2+ ion is shown as a gray sphere. b The CABS-dock server was used to generate a 15-mer peptide of the CR2-pS region of BRAF and dock into the PP1CA structure of the SMP complex. All 202 peptides from the top cluster of solutions are presented as ribbons. The vast majority being placed in the active site, with all peptides placed with their N- and C-termini in the acidic and hydrophobic active site channels. c Fluorescent western blot of CRAF either untreated or treated with SMP or lambda phosphatase (λP). Right-hand panels show the total CRAF present (red), while left-hand panels reveal CRAF by specific phosphoserine antibodies (green) targeting pS259 (top), pS43 (middle), and pS621 (bottom). Lambda phosphatase removes all phosphates, while the SMP complex only removes pS259. d Sequence alignments of the CRAF pS43, CR2-pS of ARAF, BRAF and CRAF, and CR3-pS of ARAF, BRAF and CRAF. The phosphoserine in each case is boxed in black at position 0. e The top docked CR2-pS peptide of BRAF is displayed as a ribbon in the active site with the PP1CA surface shown in electrostatic surface representation. S365 of BRAF present in the active site is colored magenta. The docked model suggests that A366 of BRAF would be placed inside the narrow negatively charged active site channel. This residue is an aspartic acid in the pS43 of CRAF and a glutamic acid in the CR3-pS peptides, offering a possible reason for the selectivity of the SMP complex for CR2-pS phosphopeptides. f Fluorescent Western blot of CRAF either untreated or treated with λP, PP1CA, SMP or SKP. Phosphoserine-specific antibodies for pS259 and pS621 are shown in red. Total CRAF is shown in green. SMP and SKP complexes specifically dephosphorylate pS259 of CRAF.P, PP1CA, SMP or SKP. Phosphoserine-specific antibodies for pS259 and pS621 are shown in red. Total CRAF is shown in green. SMP and SKP complexes specifically dephosphorylate pS259 of CRAF. g Comparison of dephosphorylation activity ( EC50) of PP1CA and SMP complex on BRAF and RAF substrates derived from Li-COR quantification of bands from Supplementary Fig. 6c. h Model showing the role of the SMP complex in the RAF activation process.

Article Snippet: Western blots were prepared as described above and probed using antibodies against pS365 BRAF (in-house antibody), pS259 CRAF (CST #9421), BRAF F7 (SC #5284), CRAF (BD #610152), SHOC2 (in-house antibody) and PP1CA E-9 (SC #7482).

Techniques: Western Blot, Sequencing, Residue, Comparison, De-Phosphorylation Assay, Activity Assay, Derivative Assay, Activation Assay

Journal: Cancer cell

Article Title: MAST1 drives cisplatin resistance in human cancers by rewiring cRaf independent MEK activation

doi: 10.1016/j.ccell.2018.06.012

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-phospho BRaf (S445) antibody , Cell Signaling Technology , Cat#2696; RRID: AB_390721.

Techniques: Recombinant, Negative Control, Viability Assay, Kinase Assay, Extraction, Ab Array, Activation Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription, shRNA, Sequencing, Plasmid Preparation, Software

Review

Structured Review

Images

Similar Products

Image Search Results