anti mek 1⁄2  (Cell Signaling Technology Inc)

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibiting BRAF Oncogene-Mediated Radioresistance Effectively Radiosensitizes BRAF V600E Mutant Thyroid Cancer Cells by Constraining DNA Double-strand Break Repair

doi: 10.1158/1078-0432.CCR-18-3625

Figure Lengend Snippet: (A) BRAFV600E mutant 8505C and BCPAP cells were cultured in media containing 100 nM vemurafenib (BRAFi) 3 hrs prior to 4Gy dose of irradiation. At the indicated time points in hours following IR, cell lysates were made for immunoblotting of γ-H2AX and GAPDH. The 0 time point indicates no IR, but treated with 100 nM vemurafenib for 3 hrs. (B) BRAFV600E mutant 8505C and BCPAP cells were cultured in media containing 100 nM vemurafenib 3 hrs prior to 1Gy dose of irradiation. At the indicated time points following IR, the cells were prepared for immunofluorescence analysis of γ-H2AX nuclear foci. Mean percentage of cells with greater than 10 γ-H2AX foci and s.e.m. at each time point after IR from two experiments of no less than 100 cells per condition are shown. Representative γ-H2AX foci of the control and vemurafenib treated at 24 h post-IR are shown. (C) DNA damage was quantified as the mean tail-moment taken from no less than 75 cells at different time points relative to 10 Gy in 8505C cells treated with/without 100 nM vemurafenib (Vem). Representative set of photomicrographs of control and vemurafenib treated comets at 24 h post-IR are shown. **p<0.01.

Article Snippet: Total ERK-1⁄2, phospho-ERK-1⁄2 (Thr202/Tyr204), total MEK-1⁄2, phospho-MEK-1⁄2 (Ser217/221), total BRAF, phospho-BRAF (Ser445), phospho-H2AX, XLF/NHEJ-1, and GAPDH primary antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Mutagenesis, Cell Culture, Irradiation, Western Blot, Immunofluorescence, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Inhibiting BRAF Oncogene-Mediated Radioresistance Effectively Radiosensitizes BRAF V600E Mutant Thyroid Cancer Cells by Constraining DNA Double-strand Break Repair

doi: 10.1158/1078-0432.CCR-18-3625

Figure Lengend Snippet: (A) Correlation of XLF expression with BRAFV600E mutation in TCGA thyroid cancer (THCA) data. Box plot showing the relationship between XLF expression and BRAFV600E mutation status. Expression was measured from primary tumor and is reported as log2 of the normalized count plus 1. A linear model shows a significant association (p=2.4e−14) of XLF expression with BRAFV600E mutation status in thyroid cancer. (B) A panel of thyroid cancer cell lines with BRAF wild type (U-Hth-74, TPC1) or V600E mutation (8505C, BCPAP, SW-1736) were analyzed for XLF protein by immunoblotting (left) and XLF mRNA levels by quantitative RT-PCR (right). (C) Exponentially growing BCPAP cells were treated with 100 nM vemurafenib or vehicle control (Ctrl) and analyzed for XLF mRNA levels by quantitative RT-PCR (left) and XLF protein by immunoblotting (right). **p<0.01 Vem vs. Ctrl. (D) TPC-1 cells expressing BRAF V600E (TPC-1 V600E) were cultured in media containing 100 nM vemurafenib (Vem) 3 hrs prior to irradiation with 0 (no IR), 2, 4, 6 and 8 Gy doses, followed by radiation clonogenic survival assays. Inset shows the upregulation of XLF and BRAFV600E in TPC-1 V600E cells by immunoblotting. **p<0.01 Vem vs. DMSO. BRAFV600E specific antibody confirms V600E expression. (E) Stable TPC-1 cells expressing BRAFV600E (TPC-1 V600E) or empty vector control (TPC-1 EMP) were transfected with siRNA of control (sictr) or XLF (siXLF) for 24 hrs. Then the cells were irradiated with 0 (no IR), 2, 4, 6 and 8 Gy doses, followed by radiation clonogenic survival assays. Inset shows the knockdown of XLF in TPC-1 V600E cells by immunoblotting with GAPDH as loading control. **p<0.01, ***p<0.001 V600E sictr vs. V600V siXLF. (F) BRAFV600E mutant 8505C cells were transfected with siRNA of control (sictr) or XLF (siXLF) for 24 hrs. Then the cells were irradiated with 0 (no IR), 2, 4, 6 and 8 Gy doses, followed by radiation clonogenic survival assays. Inset shows the knockdown of XLF in 8505C cells by immunoblotting with GAPDH as loading control. Each dose was prepared in triplicate per experiment, and no less than 2 experiments were performed. *p<0.05, ***p<0.001, ****p<0.0001

Article Snippet: Total ERK-1⁄2, phospho-ERK-1⁄2 (Thr202/Tyr204), total MEK-1⁄2, phospho-MEK-1⁄2 (Ser217/221), total BRAF, phospho-BRAF (Ser445), phospho-H2AX, XLF/NHEJ-1, and GAPDH primary antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Mutagenesis, Western Blot, Quantitative RT-PCR, Control, Cell Culture, Irradiation, Plasmid Preparation, Transfection, Knockdown

Journal: Cell host & microbe

Article Title: Listeria Adhesion Protein Induces Intestinal Epithelial Barrier Dysfunction for Bacterial Translocation

doi: 10.1016/j.chom.2018.03.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-MEK 1⁄2 , Cell Signaling , Cat # 8727, RRID:AB_10829473.

Techniques: Virus, Isolation, Recombinant, Modification, Ab Array, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, BIA-KA, Extraction, Membrane, Protein Extraction, Luciferase, Endotoxin Assay, LDH Cytotoxicity Assay, shRNA, Control, Plasmid Preparation, Transgenic Assay, Knock-Out, Software

Journal: Nature Communications

Article Title: SHOC2 phosphatase-dependent RAF dimerization mediates resistance to MEK inhibition in RAS-mutant cancers

doi: 10.1038/s41467-019-10367-x

Figure Lengend Snippet: SHOC2 is required for feedback relief ERK activation induced by MEKi’s. a SHOC2 deletion impairs ERK-reactivation after treatment with Selumetinib. Indicated cells were treated with 1 µM Selumetinib and lysates collected at indicated time points. b Quantification of P-BRAF/BRAF over time for cell lines shown in ( a ) relative to NT control. c Quantification of P-ERK/ERK over time for cell lines shown in ( a ) relative to NT control. d – f SHOC2 deletion impairs MEK, but not PanRAF induced ERK-reactivation. A549 and A427 cells were pre-treated for 12 h with either 1 µM Selumetinib ( d ) / 100 nM Trametinib ( e ) / or 2.5 µM LY3009120 ( f ). Cells were either lysed at this point (NT - Non Treated, NW - Non washed) or the inhibitor was washed-out for the indicated time points before lysate collection. Lysates were used to perform RAS-RBD pull downs and the additional cell lysate probed with indicated antibodies. g H520 cells or h H522 cells, which have no known driver mutations in the ERK pathway show a reduced dependency on SHOC2 for MEKi-induced ERK-reactivation. Parental or SHOC2 KO H520/H522 cells were treated as ( e )

Article Snippet: MEK 1⁄2 , Cell Signaling Technology , 4694 , Rabbit , 1:1000.

Techniques: Activation Assay, Control

Journal: Nature Communications

Article Title: SHOC2 phosphatase-dependent RAF dimerization mediates resistance to MEK inhibition in RAS-mutant cancers

doi: 10.1038/s41467-019-10367-x

Figure Lengend Snippet: SHOC2 is required for RAF dimerization induced by MEKi’s. a SHOC2 depletion abrogates MEKi-induced RAF dimerization and impairs ERK pathway reactivation after MEKi withdrawal. shSCR of shSHOC2 H358 cells were pre-treated with 1 µM Selumetinib for 12 h, before the inhibitor was washed-out at indicated time points and lysates used to perform endogenous RAF IPs. (NT - Non Treated, NW - Non washed). Con = IgG control IP. b As ( a ) using A549 and HCC4006 cells with a single wash-out time point of 30 min. c SHOC2 is required for MEK but not PanRAFi-induced RAF dimerization. Parental and SHOC2 KO H358 cells were pre-treated with 1 µM Selumetinib, 100 nM Trametinib 2.5 μM LY3009120 and subject to endogenous RAF IPs as ( a ). d SHOC2 is required for ERK inhibitor induced RAF dimerization. As ( c ), H358 cells were treated with 1 μM Selumetinib and 2 μM LY3214996. e B & C but not ARAF knockdown partially diminish MEKi induced signalling rebound and ERK reactivation. H358 cells transfected with indicated siRNAs were treated 3 days later with 1 µM Selumetinib for 12 h before the inhibitor was washed-out for 30 min. (NT - Non Treated, NW - Non washed). f Quantification of P-ERK and P-T380 RSK in ( e ). g B & C, but not ARAF knockdown partially sensitise H358 cells to Selumetinib. Viability curves for Selumetinib of H358 cells transfected with siRNAs as in ( e ). h Schematic to illustrate the requirement of the SHOC2 phosphatase complex for feedback relief ERK-activation on MEKi treatment. ERK activity in RAS-mutant cells is maintained at steady state by negative feedbacks at multiple levels including RTK and RAF pathway nodes. MEKi treatment leads to feedback relief ERK-pathway activation that is both dependent upon RAS-GTP and SHOC2 phosphatase-dependent ‘S259’ dephosphorylation for RAF dimerization. Following inhibitor withdrawal, release of this ‘primed’ P-MEK (phosphorylated but unable to activate ERK when inhibitor-bound) generates a wave of ERK phosphorylation that is dampened by negative feedbacks. Even in the presence of mutant RAS in SHOC2 KO cells MEKi induced feedback relief RAF dimerization is prevented, leading to reduced P-MEK rebound and more potent and durable ERK inhibition

Article Snippet: MEK 1⁄2 , Cell Signaling Technology , 4694 , Rabbit , 1:1000.

Techniques: Control, Knockdown, Transfection, Activation Assay, Activity Assay, Mutagenesis, De-Phosphorylation Assay, Phospho-proteomics, Inhibition