braf f7  (Cell Signaling Technology Inc)

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: Cell reports

Article Title: Acetylation-dependent regulation of BRAF oncogenic function

doi: 10.1016/j.celrep.2021.110250

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mice monoclonal Anti-BRAF (F7) , Santa Cruz , Cat#sc-5284; RRID: AB_2721130.

Techniques: Virus, Recombinant, Cell Fractionation, Mutagenesis, Western Blot, Functional Assay, Plasmid Preparation, Software

Journal: Oncogene

Article Title: BRAF V600E drives dedifferentiation in small intestinal and colonic organoids and cooperates with mutant p53 and Apc loss in transformation

doi: 10.1038/s41388-020-01414-9

Figure Lengend Snippet: a Heatmap of the log2 fold changes (induced vs. non-induced, color coded) of the genes listed in the HALLMARK_P53_PATHWAY MSigDB gene set. b Heatmap of selected differentially regulated transcripts. Color code represents the log2 fold change (induced vs. non-induced). Asterisks marked genes have already been found to be among the top 50 of differentially regulated genes in human CRC cell line spheroids upon BRAF V600E inhibition (; blue = downregulated; red = upregulated). c Western blots (WB) of SI and COL organoids using the indicated antibodies. GAPDH and HSP90 serve as loading controls. Each subpanel identified by its pERK detection reflects a distinct biological experiment. d MUC2 IF staining of formalin-fixed paraffin-embedded (FFPE) SI and COL organoid sections shows enhanced mucin production within mutant organoids, with highest levels in double-mutant ones. Scale bars: 50 µm.

Article Snippet: Standard western blot analysis was performed using the following primary antibodies against: AMACR (2A10) (1:1000, #3207, Cell Signaling), BRAF F7 (1:1000, sc-5284, Santa Cruz), BRAF V600E (VE1) (1:100, kindly provided by Prof. A. v. Deimling and 1:750, ab228461, Abcam), CAV1 (D46G3) (1:1000, #3267, Cell Signaling), cleaved Caspase-3 (1:1000, #9661, Cell Signaling), CTSE (1:1000, ab36996, Abcam), DUSP6 (1:1000, LS-B5975, LifeSpan BioSciences), ECAD (1:1000, 610181, BD Biosciences), EPHA2 (1:1000, #6997, Cell Signaling), c-Fos (K-25) (1:1000, sc-253, Santa Cruz), GAPDH (1:2000, ab9489, Abcam), HSP90 (1:1000, #4874, Cell Signaling), MEK1/2 (1:1000, #9122, Cell Signaling), MYOF (D-11) (1:1000, sc-376879, Santa Cruz), NR2E3 (1:1000, sc-374513, Santa Cruz), OLFM4 (D6Y5A) (1:1000, #39141, Cell Signaling), Phospho-c-Fos (Ser32) (D82C12) (1:1000, #5348, Cell Signaling), Phospho-FRA1 (Ser265) (D22B1) (1:1000, #5841, Cell Signaling), Phospho-MEK1/2 (pS217/221) (1:1000, #9121, Cell Signaling), Phospho-P44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:2000, #9101, Cell Signaling), P44/42 MAPK (Erk1/2) (1:2000, #9102, Cell Signaling), 14–3–3 (1:1000, sc-1657, Santa Cruz), Vinculin (1:1000, #4650, Cell Signaling).

Techniques: Inhibition, Western Blot, Staining, Formalin-fixed Paraffin-Embedded, Mutagenesis

Journal: Oncogene

Article Title: BRAF V600E drives dedifferentiation in small intestinal and colonic organoids and cooperates with mutant p53 and Apc loss in transformation

doi: 10.1038/s41388-020-01414-9

Figure Lengend Snippet: a Gene set enrichment analysis (GSEA) of our transcriptomic data with genes that are up- (left) and downregulated (right), respectively, in Cdx1/Cdx2 double-knockout (DKO) mice (GSE24633). b GSEA of our transcriptomic data against genes that are up- (left) and downregulated (right), respectively, in mouse fetal intestinal spheroids. c GSEAs showing the comparison of the LGR5-independent fetal signature to human data sets. The left panel shows the enrichment of the fetal spheroids signature in TCGA BRAF V600E mutant vs. healthy colon. The right panel shows the enrichment of the Popovici signature, i.e., 314 differentially expressed probe sets between WT and BRAFm samples, in fetal spheroids. Note, a positive fold change of the “Popovici genes” indicates higher expression in WT vs BRAFm, which results in an inverse correlation to the fetal signature. NES normalized enrichment score, PV P -value.

Article Snippet: Standard western blot analysis was performed using the following primary antibodies against: AMACR (2A10) (1:1000, #3207, Cell Signaling), BRAF F7 (1:1000, sc-5284, Santa Cruz), BRAF V600E (VE1) (1:100, kindly provided by Prof. A. v. Deimling and 1:750, ab228461, Abcam), CAV1 (D46G3) (1:1000, #3267, Cell Signaling), cleaved Caspase-3 (1:1000, #9661, Cell Signaling), CTSE (1:1000, ab36996, Abcam), DUSP6 (1:1000, LS-B5975, LifeSpan BioSciences), ECAD (1:1000, 610181, BD Biosciences), EPHA2 (1:1000, #6997, Cell Signaling), c-Fos (K-25) (1:1000, sc-253, Santa Cruz), GAPDH (1:2000, ab9489, Abcam), HSP90 (1:1000, #4874, Cell Signaling), MEK1/2 (1:1000, #9122, Cell Signaling), MYOF (D-11) (1:1000, sc-376879, Santa Cruz), NR2E3 (1:1000, sc-374513, Santa Cruz), OLFM4 (D6Y5A) (1:1000, #39141, Cell Signaling), Phospho-c-Fos (Ser32) (D82C12) (1:1000, #5348, Cell Signaling), Phospho-FRA1 (Ser265) (D22B1) (1:1000, #5841, Cell Signaling), Phospho-MEK1/2 (pS217/221) (1:1000, #9121, Cell Signaling), Phospho-P44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:2000, #9101, Cell Signaling), P44/42 MAPK (Erk1/2) (1:2000, #9102, Cell Signaling), 14–3–3 (1:1000, sc-1657, Santa Cruz), Vinculin (1:1000, #4650, Cell Signaling).

Techniques: Double Knockout, Comparison, Mutagenesis, Expressing

Journal: Oncogene

Article Title: BRAF V600E drives dedifferentiation in small intestinal and colonic organoids and cooperates with mutant p53 and Apc loss in transformation

doi: 10.1038/s41388-020-01414-9

Figure Lengend Snippet: a , b GSEA of the delta log2 fold changes of double-mutant vs. BRAF V600E -only organoids was performed. Shown are enrichment heatmaps for p53-related gene sets ( a ) and for the top ten significantly ( P < 0.05) regulated chemical and genetic perturbations (CGP) ( b ). On both heatmaps, color code and circle size represent NES. c Representative bright-field (BF) images of three independent experiments show colony growth capacity of COL organoids. Quantification is shown in ( d ), d Quantification of colony growth capacity, normalized to the corresponding non-induced control. The longest straight lines of the crypts were measured. e Control or 4-HT-induced COL crypts were disaggregated, and grown on PolyHEMA-coated culture dishes for 6 days before BF images were taken and the diameters of the formed cell clusters were measured. Representative BF pictures of ≥3 independent experiments that are quantified in ( f ), are shown. f Quantification of anchorage-independent growth. The longest straight lines of the cell clusters were measured. Note that neither non-induced controls nor p53 R172H -mutant organoids were able to form cell clusters on PolyHEMA. g Control or 4-HT-induced COL organoids were grown in diluted (50%) Matrigel. BF images were taken at day 7, and organoids attached to the plastic surface were counted (highlighted by dashed lines). Representative pictures of ≥3 independent experiments are shown, which are quantified in ( h ). Higher-magnification BF images of “invaded” organoids are shown in Supplementary Fig. S . h Quantification of “invaded” organoids. In ( c , e , g ), scale bars: 50 µm. In ( d , f , h ), symbol colors refer to donor mice, symbol shapes refer to independent experiments. Data are presented as mean ± SD, and statistical significance was determined by one-way ANOVA (corrected for multiple comparison by Bonferroni). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Article Snippet: Standard western blot analysis was performed using the following primary antibodies against: AMACR (2A10) (1:1000, #3207, Cell Signaling), BRAF F7 (1:1000, sc-5284, Santa Cruz), BRAF V600E (VE1) (1:100, kindly provided by Prof. A. v. Deimling and 1:750, ab228461, Abcam), CAV1 (D46G3) (1:1000, #3267, Cell Signaling), cleaved Caspase-3 (1:1000, #9661, Cell Signaling), CTSE (1:1000, ab36996, Abcam), DUSP6 (1:1000, LS-B5975, LifeSpan BioSciences), ECAD (1:1000, 610181, BD Biosciences), EPHA2 (1:1000, #6997, Cell Signaling), c-Fos (K-25) (1:1000, sc-253, Santa Cruz), GAPDH (1:2000, ab9489, Abcam), HSP90 (1:1000, #4874, Cell Signaling), MEK1/2 (1:1000, #9122, Cell Signaling), MYOF (D-11) (1:1000, sc-376879, Santa Cruz), NR2E3 (1:1000, sc-374513, Santa Cruz), OLFM4 (D6Y5A) (1:1000, #39141, Cell Signaling), Phospho-c-Fos (Ser32) (D82C12) (1:1000, #5348, Cell Signaling), Phospho-FRA1 (Ser265) (D22B1) (1:1000, #5841, Cell Signaling), Phospho-MEK1/2 (pS217/221) (1:1000, #9121, Cell Signaling), Phospho-P44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:2000, #9101, Cell Signaling), P44/42 MAPK (Erk1/2) (1:2000, #9102, Cell Signaling), 14–3–3 (1:1000, sc-1657, Santa Cruz), Vinculin (1:1000, #4650, Cell Signaling).

Techniques: Mutagenesis, Control, Comparison

Journal: Oncogene

Article Title: BRAF V600E drives dedifferentiation in small intestinal and colonic organoids and cooperates with mutant p53 and Apc loss in transformation

doi: 10.1038/s41388-020-01414-9

Figure Lengend Snippet: a COL organoids with the indicted genotypes were treated with 3 µM 4-HT for 24 h. Representative microscopy pictures at days 2 and 6 (see also Supplementary Video ) and MTT staining at day 7 are shown. b COL organoids with the indicated genotypes were induced with 3 µM 4-HT and cultured without the growth factors (GFs) EGF, R-Spondin, Noggin, and Wnt3a. BF images and MTT staining at day 9 are shown. c COL organoids with the indicated genotypes were treated with DMSO or indicated trametinib concentrations 1 day after induction with 3 µM 4-HT. Representative MTT staining at day 9 after 4-HT induction is shown. Note that the dark-blue colonies indicate metabolic activity. d Quantification of trametinib treatment shown in ( c ) of two (for Apc Δ/Δ ) and three (for Braf V600E/+ ,Apc Δ/Δ and Braf V600E/+ ,Apc Δ/Δ ,Trp53 R172H/+ ) independent experiments. Colony count was normalized to corresponding DMSO control, and statistical significance was determined by two-way ANOVA (corrected for multiple comparison by Bonferroni). * P ≤ 0.05; ** P ≤ 0.01. In ( a , b ), scale bars: 50 µm.

Article Snippet: Standard western blot analysis was performed using the following primary antibodies against: AMACR (2A10) (1:1000, #3207, Cell Signaling), BRAF F7 (1:1000, sc-5284, Santa Cruz), BRAF V600E (VE1) (1:100, kindly provided by Prof. A. v. Deimling and 1:750, ab228461, Abcam), CAV1 (D46G3) (1:1000, #3267, Cell Signaling), cleaved Caspase-3 (1:1000, #9661, Cell Signaling), CTSE (1:1000, ab36996, Abcam), DUSP6 (1:1000, LS-B5975, LifeSpan BioSciences), ECAD (1:1000, 610181, BD Biosciences), EPHA2 (1:1000, #6997, Cell Signaling), c-Fos (K-25) (1:1000, sc-253, Santa Cruz), GAPDH (1:2000, ab9489, Abcam), HSP90 (1:1000, #4874, Cell Signaling), MEK1/2 (1:1000, #9122, Cell Signaling), MYOF (D-11) (1:1000, sc-376879, Santa Cruz), NR2E3 (1:1000, sc-374513, Santa Cruz), OLFM4 (D6Y5A) (1:1000, #39141, Cell Signaling), Phospho-c-Fos (Ser32) (D82C12) (1:1000, #5348, Cell Signaling), Phospho-FRA1 (Ser265) (D22B1) (1:1000, #5841, Cell Signaling), Phospho-MEK1/2 (pS217/221) (1:1000, #9121, Cell Signaling), Phospho-P44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:2000, #9101, Cell Signaling), P44/42 MAPK (Erk1/2) (1:2000, #9102, Cell Signaling), 14–3–3 (1:1000, sc-1657, Santa Cruz), Vinculin (1:1000, #4650, Cell Signaling).

Techniques: Microscopy, Staining, Cell Culture, Activity Assay, Control, Comparison

Journal: Oncogene

Article Title: BRAF V600E drives dedifferentiation in small intestinal and colonic organoids and cooperates with mutant p53 and Apc loss in transformation

doi: 10.1038/s41388-020-01414-9

Figure Lengend Snippet: From left to right: Expression of oncogenic BRAF V600E induces a fetal gene signature in adult wildtype colonic organoids, but also leads to rapid disintegration and subsequent cell death. Co-expression of p53 R172H extends organoid survival and conveys proliferative and invasive properties. Additional loss of APC prevents the collapse of the intestinal stem cell (ISC) niche, thereby promoting the survival of the mutant organoids. Importantly, additional APC loss confers growth factor independence and modulates the sensitivity to MEK inhibitor (MEKi) treatment. AJ adherens junctions, TJ tight junctions.

Article Snippet: Standard western blot analysis was performed using the following primary antibodies against: AMACR (2A10) (1:1000, #3207, Cell Signaling), BRAF F7 (1:1000, sc-5284, Santa Cruz), BRAF V600E (VE1) (1:100, kindly provided by Prof. A. v. Deimling and 1:750, ab228461, Abcam), CAV1 (D46G3) (1:1000, #3267, Cell Signaling), cleaved Caspase-3 (1:1000, #9661, Cell Signaling), CTSE (1:1000, ab36996, Abcam), DUSP6 (1:1000, LS-B5975, LifeSpan BioSciences), ECAD (1:1000, 610181, BD Biosciences), EPHA2 (1:1000, #6997, Cell Signaling), c-Fos (K-25) (1:1000, sc-253, Santa Cruz), GAPDH (1:2000, ab9489, Abcam), HSP90 (1:1000, #4874, Cell Signaling), MEK1/2 (1:1000, #9122, Cell Signaling), MYOF (D-11) (1:1000, sc-376879, Santa Cruz), NR2E3 (1:1000, sc-374513, Santa Cruz), OLFM4 (D6Y5A) (1:1000, #39141, Cell Signaling), Phospho-c-Fos (Ser32) (D82C12) (1:1000, #5348, Cell Signaling), Phospho-FRA1 (Ser265) (D22B1) (1:1000, #5841, Cell Signaling), Phospho-MEK1/2 (pS217/221) (1:1000, #9121, Cell Signaling), Phospho-P44/42 MAPK (Erk1/2) (Thr202/Tyr204) (1:2000, #9101, Cell Signaling), P44/42 MAPK (Erk1/2) (1:2000, #9102, Cell Signaling), 14–3–3 (1:1000, sc-1657, Santa Cruz), Vinculin (1:1000, #4650, Cell Signaling).

Techniques: Expressing, Mutagenesis