raf-1 sirna Search Results


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  • 96
    Millipore raf 1 activity
    TNF α induces ERK1/2 activation in a Ras-independent manner and induces <t>Raf-1</t> kinase activity in a FLIP-L-dependent manner. ( a ) Serum-deprived PC12 cells were treated with 100 ng/ml of TNF α or NGF for 5 min, and activated Ras was pulled down using Raf-RBD conjugated agarose beads. GTP-bound Ras was detected by western blot using an anti-pan-Ras antibody. ( b ) Endogenous Raf-1 was immunoprecipitated from PC12 cells treated with TNF α or NGF and immunoprecipitates were incubated with recombinant MEK and ATP in vitro in order to detect Raf-1 kinase activity. Western blot analysis was performed for Raf-1 and P-MEK1. Inputs were blotted using anti-Raf-1, anti-P-ERK1/2, and anti-ERK1/2 antibody as a loading control. ( c ) Raf-1 kinase activity was assessed as in b , in PC12 cells transfected with Neo or SR-I κ B α , ( d ) or after 3 days of PC12 transduction with scrambled sequence (shRNA Scr) or shRNA against FLIP-L (shRNA FLIP-L) lentiviruses. Black lines indicate that intervening lanes have been spliced. ( e ) Serum-deprived PC12 cells were treated with TNF α for the indicated times prior harvesting and subcellular fractionation. Lysates corresponding to cytosolic (C) and membrane fractions (M) were resolved by SDS-PAGE and Raf-1 subcellular localization was assessed by western blot using an anti-Raf-1 antibody. ERK1/2 phosphorylation was also detected to control MAPK/ERK activation following TNF α stimulation. ( f ) PC12 cells were transfected with siRNA targeting Raf-1 or a scrambled sequence. Three days after transfection, cells were treated with TNF α for the indicated time points and western blot was performed to detect P-ERK1/2, Raf-1 and total ERK1/2 as loading control. ( g ) PC12 cells were transfected with pcDNA3-HA-FLIP-L, pcDNA3-Raf-1 or both plasmids. Cells were harvested 24 h later and FLIP-L was immunoprecipitated using a specific anti-FLIP antibody prior western blot using anti-Raf-1 antibody. Transfection efficiency of both plasmids was checked in the inputs. The asterisk indicates nonspecific bands
    Raf 1 Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology raf 1
    RRD-251 inhibits <t>Rb-Raf-1</t> interaction in AoSMCs. (A and B) Treatment with TNFα or PDGF in the presence of RRD-251 inhibits Raf-1 levels in AoSMCs (A) and A10s (B). (C) TNFα and PDGF treatment induced Rb-Raf-1 binding in AoSMCs. (D) RRD-251 inhibits TNFα-induced Rb-Raf-1 interaction.
    Raf 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 831 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher raf 1 small inhibitory rna sirna
    <t>Raf-1</t> is essential for ERK activation by EGF and for PAK165-dependent rescue of ERK activation in cells treated with forskolin and IBMX. (A) Effects of Raf-1 <t>siRNA.</t> HEK293 cells were transfected with GFPERK, vector (−), or PAK165 (+) and
    Raf 1 Small Inhibitory Rna Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery raf1 sirna
    cells were placed in suspension, or adherent cultures were serum-starved for two hours seventy-two hours post transfection with control or <t>Raf1</t> <t>siRNA.</t> Adherent cultures were then stimulated with EGF (1 ng/mL for 5 min) or plated on fibronectin (FN)-coated dishes for the indicated times. Cell lysates were analyzed by western blot with the indicated antisera.
    Raf1 Sirna, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery rat raf 1 selective sirna mixture
    Intrathecal <t>Raf-1-selective</t> <t>siRNA</t> pretreatment attenuates sustained morphine-mediated tactile allodynia. For description of the animal groups see . Paw withdrawal thresholds in response to a series of von Frey filaments applied to the plantar surface
    Rat Raf 1 Selective Sirna Mixture, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery lipid encapsulated raf 1 selective sirna mixture
    A. Experimental design. (Ith: Intrathecal; <t>siRNA:</t> <t>Raf-1</t> siRNA or mismatch siRNA delivery via intrathecal catheter; BL: Baseline; D: Day; IR: Infrared heat). B. Raf-1 siRNA attenuates sustained morphine-mediated thermal hyperalgesia in rats: Male Sprague Dawley rats received intrathecal injections of i-Fect encapsulated Raf-1selective siRNA (Raf-1 siRNA groups); or non-targeting dsRNA (mismatch siRNA groups) or the transfection agent (i-Fect) alone (control) once daily, for 3 days. After pre-treatment, the rats received continous subcutaneous (osmotic minipump) saline (control group, Raf-1 siRNA group and mismatch siRNA group) or morphine (45μg/μl/h) (morphine group, Raf-1 siRNA+morphine group and mismatch siRNA+morphine group) infusions for 6 days. Six animals were included in each treatment group. Thermal hyperalgesia was measured as a decrease in paw withdrawal latencies in a radiant heat paw-withdrawal test.
    Lipid Encapsulated Raf 1 Selective Sirna Mixture, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery smartpool sirna against raf1 siraf
    A. Experimental design. (Ith: Intrathecal; <t>siRNA:</t> <t>Raf-1</t> siRNA or mismatch siRNA delivery via intrathecal catheter; BL: Baseline; D: Day; IR: Infrared heat). B. Raf-1 siRNA attenuates sustained morphine-mediated thermal hyperalgesia in rats: Male Sprague Dawley rats received intrathecal injections of i-Fect encapsulated Raf-1selective siRNA (Raf-1 siRNA groups); or non-targeting dsRNA (mismatch siRNA groups) or the transfection agent (i-Fect) alone (control) once daily, for 3 days. After pre-treatment, the rats received continous subcutaneous (osmotic minipump) saline (control group, Raf-1 siRNA group and mismatch siRNA group) or morphine (45μg/μl/h) (morphine group, Raf-1 siRNA+morphine group and mismatch siRNA+morphine group) infusions for 6 days. Six animals were included in each treatment group. Thermal hyperalgesia was measured as a decrease in paw withdrawal latencies in a radiant heat paw-withdrawal test.
    Smartpool Sirna Against Raf1 Siraf, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti phospho raf 1
    Knockdown (KD) of Plk1 attenuates the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, but not <t>Raf-1</t> phosphorylation. a Representative immunoblots illustrating the effects of Plk1shRNA on Plk1 expression. Blots of HASM cells infected with lentiviruses encoding control shRNA or Plk1 shRNA were probed with antibodies against Plk1 and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells producing Plk1 shRNA were normalized to ratios obtained from cells producing control shRNA. Values are mean ± SE ( n = 4). *Significantly lower Plk1/GAPDH ratios in cells producing Plk1 shRNA compared with cells producing control shRNA ( P
    Anti Phospho Raf 1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho raf 1
    Effects of galectin-1 on the Ras-transmitted downstream signals. Scrambled RNA or galectin-1 shRNA were transfected in HeLa cells. GFP or galectin-1 cDNA were transfected in C33A cells. The cells were irradiated with 6 Gy and harvested 5 or 10 min later. Expressions of activated H-Ras, <t>p-Raf-1,</t> and p-ERK using western blots in HeLa and C33A cells were compared with or without galectin-1 modulation
    Phospho Raf 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti raf 1
    RASAL2 knockdown activates the Ras-ERK pathway (A) The effect of RASAL2 knockdown on Ras activation in SK-OV-3, OVCAR3 and A2780 cells was evaluated by a Ras Activation ELISA Assay Kit. All the data were normalized to the results of cells transfected with scramble-shRNA. The data are shown as the means ± SD (n = 3). (B) Phosphorylation of <t>Raf-1,</t> MEK1/2 and ERK1/2 in SK-OV-3, OVCAR3 and A2780 cells infected with shRASAL2-1, shRASAL2-2 or scramble-shRNA was analyzed by Western blot. Xenograft tumor sections (SK-OV-3) were also examined for levels of p-ERK by IHC (C) and for p-Raf-1 and p-MEK by Western blot (D).
    Anti Raf 1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology raf 1 antibody
    <t>Raf-1</t> signaling is inhibited by RKIP in B-Raf deficient cells (A) Immortalized wild-type (RKIP+/+) and RKIP −/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1) or control siRNA (siCon). After 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP and Raf-1 were detected by western blot. (B) RKIP was stably depleted in WT and Raf-1−/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, and Raf-1 were detected by western blot. (C) Immortalized wild-type and RKIP−/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1), B-Raf siRNA (siB-Raf), double B-Raf and Raf-1 siRNA (siRaf-1+siB-Raf) or control siRNA (siCon); after 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP, Raf-1 and B-Raf were detected by western blot. *, p
    Raf 1 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rabbit anti raf 1 antibodies
    <t>Raf-1</t> signaling is inhibited by RKIP in B-Raf deficient cells (A) Immortalized wild-type (RKIP+/+) and RKIP −/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1) or control siRNA (siCon). After 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP and Raf-1 were detected by western blot. (B) RKIP was stably depleted in WT and Raf-1−/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, and Raf-1 were detected by western blot. (C) Immortalized wild-type and RKIP−/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1), B-Raf siRNA (siB-Raf), double B-Raf and Raf-1 siRNA (siRaf-1+siB-Raf) or control siRNA (siCon); after 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP, Raf-1 and B-Raf were detected by western blot. *, p
    Rabbit Anti Raf 1 Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Horizon Discovery gene specific sirna pools
    BRAF and <t>BCORL1</t> mutations in resistant cells. (A) Whole-exome sequencing copy number variation analysis revealed focal imbalances of the distal region of chromosome 7 in A375-R1 cells compared to parental cells. BRAF locus (zoomed area) lies within the amplified region but shows loss of exons 2 to 8. (B) Sanger sequencing of the deleted BRAF allele showing in-frame junction of exon 1 with exon 9 in A375-R1 cells. (C) PCR amplification of the region across the break point reveals the presence of two BRAF transcripts in A375-R1 cells: full-length wild-type (1216-bp band) and truncated (213-bp band). (D) Anti-BRAF Western blotting shows the presence of a smaller band of approximately 47kDa in A375-R1 cells. Actin is shown as a loading control. (E) <t>siRNA-mediated</t> silencing of BRAF in A375-R1 cells leads to downregulation of both full-length and truncated BRAF proteins, suppression of MEK1/2 phosphorylation, and decrease in cell viability. (F) Whole-exome sequencing comparative analysis of A375-R1 versus A375 cells revealed acquired mutations in the four indicated genes at frequency > 35%. SIFT prediction of mutation impact on protein function is shown. (G) Sanger validation of BCORL1 heterozygous genomic mutation in A375-R1 cells.
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    Cell Signaling Technology Inc a raf
    Endogenous <t>RanBPM</t> and <t>c-Raf</t> interaction in HeLa cells using PLA. Duolink II proximity ligation assay (PLA) was performed in: ( A ) control shRNA HeLa cells, without the addition of primary antibodies (negative control); ( B ) control shRNA HeLa cells, with Hsp90 and c-Raf primary antibodies (positive control); ( C ) control shRNA HeLa cells, using c-Raf and RanBPM primary antibodies; ( D ). HeLa RanBPM shRNA cells, with c-Raf and RanBPM primary antibodies (negative control); ( E ) control shRNA HeLa cells, using MAEA (Macrophage erythroblast attacher) and RanBPM primary antibodies (positive control). ( F ) Control shRNA HeLa cells, using c-Raf and MAEA primary antibodies. The DAPI filter was used to visualize the nuclei, while the Cyanine 3 (Cy3) filter was used to visualize the PLA dots representing protein–protein interactions. Representative images from one of three independent experiments are shown. Scale bars, 10 μm.
    A Raf, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    TNF α induces ERK1/2 activation in a Ras-independent manner and induces Raf-1 kinase activity in a FLIP-L-dependent manner. ( a ) Serum-deprived PC12 cells were treated with 100 ng/ml of TNF α or NGF for 5 min, and activated Ras was pulled down using Raf-RBD conjugated agarose beads. GTP-bound Ras was detected by western blot using an anti-pan-Ras antibody. ( b ) Endogenous Raf-1 was immunoprecipitated from PC12 cells treated with TNF α or NGF and immunoprecipitates were incubated with recombinant MEK and ATP in vitro in order to detect Raf-1 kinase activity. Western blot analysis was performed for Raf-1 and P-MEK1. Inputs were blotted using anti-Raf-1, anti-P-ERK1/2, and anti-ERK1/2 antibody as a loading control. ( c ) Raf-1 kinase activity was assessed as in b , in PC12 cells transfected with Neo or SR-I κ B α , ( d ) or after 3 days of PC12 transduction with scrambled sequence (shRNA Scr) or shRNA against FLIP-L (shRNA FLIP-L) lentiviruses. Black lines indicate that intervening lanes have been spliced. ( e ) Serum-deprived PC12 cells were treated with TNF α for the indicated times prior harvesting and subcellular fractionation. Lysates corresponding to cytosolic (C) and membrane fractions (M) were resolved by SDS-PAGE and Raf-1 subcellular localization was assessed by western blot using an anti-Raf-1 antibody. ERK1/2 phosphorylation was also detected to control MAPK/ERK activation following TNF α stimulation. ( f ) PC12 cells were transfected with siRNA targeting Raf-1 or a scrambled sequence. Three days after transfection, cells were treated with TNF α for the indicated time points and western blot was performed to detect P-ERK1/2, Raf-1 and total ERK1/2 as loading control. ( g ) PC12 cells were transfected with pcDNA3-HA-FLIP-L, pcDNA3-Raf-1 or both plasmids. Cells were harvested 24 h later and FLIP-L was immunoprecipitated using a specific anti-FLIP antibody prior western blot using anti-Raf-1 antibody. Transfection efficiency of both plasmids was checked in the inputs. The asterisk indicates nonspecific bands

    Journal: Cell Death & Disease

    Article Title: TNFα induces survival through the FLIP-L-dependent activation of the MAPK/ERK pathway

    doi: 10.1038/cddis.2013.25

    Figure Lengend Snippet: TNF α induces ERK1/2 activation in a Ras-independent manner and induces Raf-1 kinase activity in a FLIP-L-dependent manner. ( a ) Serum-deprived PC12 cells were treated with 100 ng/ml of TNF α or NGF for 5 min, and activated Ras was pulled down using Raf-RBD conjugated agarose beads. GTP-bound Ras was detected by western blot using an anti-pan-Ras antibody. ( b ) Endogenous Raf-1 was immunoprecipitated from PC12 cells treated with TNF α or NGF and immunoprecipitates were incubated with recombinant MEK and ATP in vitro in order to detect Raf-1 kinase activity. Western blot analysis was performed for Raf-1 and P-MEK1. Inputs were blotted using anti-Raf-1, anti-P-ERK1/2, and anti-ERK1/2 antibody as a loading control. ( c ) Raf-1 kinase activity was assessed as in b , in PC12 cells transfected with Neo or SR-I κ B α , ( d ) or after 3 days of PC12 transduction with scrambled sequence (shRNA Scr) or shRNA against FLIP-L (shRNA FLIP-L) lentiviruses. Black lines indicate that intervening lanes have been spliced. ( e ) Serum-deprived PC12 cells were treated with TNF α for the indicated times prior harvesting and subcellular fractionation. Lysates corresponding to cytosolic (C) and membrane fractions (M) were resolved by SDS-PAGE and Raf-1 subcellular localization was assessed by western blot using an anti-Raf-1 antibody. ERK1/2 phosphorylation was also detected to control MAPK/ERK activation following TNF α stimulation. ( f ) PC12 cells were transfected with siRNA targeting Raf-1 or a scrambled sequence. Three days after transfection, cells were treated with TNF α for the indicated time points and western blot was performed to detect P-ERK1/2, Raf-1 and total ERK1/2 as loading control. ( g ) PC12 cells were transfected with pcDNA3-HA-FLIP-L, pcDNA3-Raf-1 or both plasmids. Cells were harvested 24 h later and FLIP-L was immunoprecipitated using a specific anti-FLIP antibody prior western blot using anti-Raf-1 antibody. Transfection efficiency of both plasmids was checked in the inputs. The asterisk indicates nonspecific bands

    Article Snippet: Determination of Raf-1 kinase activity In order to determine Raf-1 activity, we used a Raf-1 kinase assay kit with chemiluminescence detection (Millipore).

    Techniques: Activation Assay, Activity Assay, Western Blot, Immunoprecipitation, Incubation, Recombinant, In Vitro, Transfection, Transduction, Sequencing, shRNA, Fractionation, SDS Page

    RRD-251 inhibits Rb-Raf-1 interaction in AoSMCs. (A and B) Treatment with TNFα or PDGF in the presence of RRD-251 inhibits Raf-1 levels in AoSMCs (A) and A10s (B). (C) TNFα and PDGF treatment induced Rb-Raf-1 binding in AoSMCs. (D) RRD-251 inhibits TNFα-induced Rb-Raf-1 interaction.

    Journal: Cell Cycle

    Article Title: TNF?-mediated proliferation of vascular smooth muscle cells involves Raf-1-mediated inactivation of Rb and transcription of E2F1-regulated genes

    doi: 10.4161/cc.11.1.18473

    Figure Lengend Snippet: RRD-251 inhibits Rb-Raf-1 interaction in AoSMCs. (A and B) Treatment with TNFα or PDGF in the presence of RRD-251 inhibits Raf-1 levels in AoSMCs (A) and A10s (B). (C) TNFα and PDGF treatment induced Rb-Raf-1 binding in AoSMCs. (D) RRD-251 inhibits TNFα-induced Rb-Raf-1 interaction.

    Article Snippet: siRNA oligonucleotides targeting human E2F1, E2F2, E2F3 and Raf-1 were purchased from Santa Cruz biotechnology.

    Techniques: Binding Assay

    Whereas in AoSMCs, TNFα stimulation led to Raf-1-dependent Rb inactivation, elevated E2F1 activity, leading to the expression of proliferative promoters, resulting in increased cell proliferation.

    Journal: Cell Cycle

    Article Title: TNF?-mediated proliferation of vascular smooth muscle cells involves Raf-1-mediated inactivation of Rb and transcription of E2F1-regulated genes

    doi: 10.4161/cc.11.1.18473

    Figure Lengend Snippet: Whereas in AoSMCs, TNFα stimulation led to Raf-1-dependent Rb inactivation, elevated E2F1 activity, leading to the expression of proliferative promoters, resulting in increased cell proliferation.

    Article Snippet: siRNA oligonucleotides targeting human E2F1, E2F2, E2F3 and Raf-1 were purchased from Santa Cruz biotechnology.

    Techniques: Activity Assay, Expressing

    TNFα activates the Raf/MAPK pathway in VSMCs. (A) Time-course stimulation of AoSMCs results in Raf-1 activation, highest at 1 h, and ERK1/2 activation peaks at 30 min. (B) Activation of ERK1/2 coincides with JNK1 activation from 30 min of TNFα treatment. (C) PDGF and TNFα time-course stimulation shows ERK1/2 and JNK activation occurs simultaneously from the different stimuli.

    Journal: Cell Cycle

    Article Title: TNF?-mediated proliferation of vascular smooth muscle cells involves Raf-1-mediated inactivation of Rb and transcription of E2F1-regulated genes

    doi: 10.4161/cc.11.1.18473

    Figure Lengend Snippet: TNFα activates the Raf/MAPK pathway in VSMCs. (A) Time-course stimulation of AoSMCs results in Raf-1 activation, highest at 1 h, and ERK1/2 activation peaks at 30 min. (B) Activation of ERK1/2 coincides with JNK1 activation from 30 min of TNFα treatment. (C) PDGF and TNFα time-course stimulation shows ERK1/2 and JNK activation occurs simultaneously from the different stimuli.

    Article Snippet: siRNA oligonucleotides targeting human E2F1, E2F2, E2F3 and Raf-1 were purchased from Santa Cruz biotechnology.

    Techniques: Activation Assay

    Targeting Raf-1 activation blocks AoSMC proliferation. (A) Src inhibitor (PP2) and PKC inhibitor (Ro-31-8220) block TNFα- and PDGF-induced proliferation. (B) Multi-kinase inhibitor BAY-43-9006, which targets Raf-1, can completely inhibit PDGF- and TNFα-induced proliferation, while the JNK inhibitor (SP600125) does not significantly affect TNFα-induced proliferation. (C) MEK inhibitor PD98059 could block TNFα- and PDGF-induced S-phase entry as seen by BrdU incorporation assays. (D) Raf-1 depletion using Raf-1 siRNAs could inhibit TNFα- and PDGF-induced AoSMC proliferation.

    Journal: Cell Cycle

    Article Title: TNF?-mediated proliferation of vascular smooth muscle cells involves Raf-1-mediated inactivation of Rb and transcription of E2F1-regulated genes

    doi: 10.4161/cc.11.1.18473

    Figure Lengend Snippet: Targeting Raf-1 activation blocks AoSMC proliferation. (A) Src inhibitor (PP2) and PKC inhibitor (Ro-31-8220) block TNFα- and PDGF-induced proliferation. (B) Multi-kinase inhibitor BAY-43-9006, which targets Raf-1, can completely inhibit PDGF- and TNFα-induced proliferation, while the JNK inhibitor (SP600125) does not significantly affect TNFα-induced proliferation. (C) MEK inhibitor PD98059 could block TNFα- and PDGF-induced S-phase entry as seen by BrdU incorporation assays. (D) Raf-1 depletion using Raf-1 siRNAs could inhibit TNFα- and PDGF-induced AoSMC proliferation.

    Article Snippet: siRNA oligonucleotides targeting human E2F1, E2F2, E2F3 and Raf-1 were purchased from Santa Cruz biotechnology.

    Techniques: Activation Assay, Blocking Assay, BrdU Incorporation Assay

    TNFα and PDGF induce E2F regulated genes in AoSMCs. (A) Treatment with TNFα and PDGF for 18 h led to 3.5- and 4-fold increase, respectively, in cdc25A gene expression in real-time PCR assays. (B) Treatment with TNFα and PDGF for 18 h led to 3.5- and 7-fold increase, respectively in cdc6 gene expression in real-time PCR assays. (C) Treatment with TNFα and PDGF for 18 h led to 1.8 and 3.8 fold increase, respectively in TS gene expression in real time PCR assays. (D) Treatment with TNFα or PDGF led to an increase in E2F1 and dissociation of Rb on the proliferative promoters cdc25A, cdc6 and TS in ChIP assays; c-fos was used as the negative control. (E) Western blots for cdc 6, cdc 25A and TS after Raf-1 and E2F1 depletion. (F) Densitometric analysis of the bands to quantify the western blot data.

    Journal: Cell Cycle

    Article Title: TNF?-mediated proliferation of vascular smooth muscle cells involves Raf-1-mediated inactivation of Rb and transcription of E2F1-regulated genes

    doi: 10.4161/cc.11.1.18473

    Figure Lengend Snippet: TNFα and PDGF induce E2F regulated genes in AoSMCs. (A) Treatment with TNFα and PDGF for 18 h led to 3.5- and 4-fold increase, respectively, in cdc25A gene expression in real-time PCR assays. (B) Treatment with TNFα and PDGF for 18 h led to 3.5- and 7-fold increase, respectively in cdc6 gene expression in real-time PCR assays. (C) Treatment with TNFα and PDGF for 18 h led to 1.8 and 3.8 fold increase, respectively in TS gene expression in real time PCR assays. (D) Treatment with TNFα or PDGF led to an increase in E2F1 and dissociation of Rb on the proliferative promoters cdc25A, cdc6 and TS in ChIP assays; c-fos was used as the negative control. (E) Western blots for cdc 6, cdc 25A and TS after Raf-1 and E2F1 depletion. (F) Densitometric analysis of the bands to quantify the western blot data.

    Article Snippet: siRNA oligonucleotides targeting human E2F1, E2F2, E2F3 and Raf-1 were purchased from Santa Cruz biotechnology.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Western Blot

    Raf-1 is essential for ERK activation by EGF and for PAK165-dependent rescue of ERK activation in cells treated with forskolin and IBMX. (A) Effects of Raf-1 siRNA. HEK293 cells were transfected with GFPERK, vector (−), or PAK165 (+) and

    Journal: Molecular and Cellular Biology

    Article Title: Raf-1 Serine 338 Phosphorylation Plays a Key Role in Adhesion-Dependent Activation of Extracellular Signal-Regulated Kinase by Epidermal Growth Factor †

    doi: 10.1128/MCB.25.11.4466-4475.2005

    Figure Lengend Snippet: Raf-1 is essential for ERK activation by EGF and for PAK165-dependent rescue of ERK activation in cells treated with forskolin and IBMX. (A) Effects of Raf-1 siRNA. HEK293 cells were transfected with GFPERK, vector (−), or PAK165 (+) and

    Article Snippet: Raf-1 small inhibitory RNA (siRNA) was obtained from Ambion (catalog number 51197; Huntingdon, United Kingdom).

    Techniques: Activation Assay, Transfection, Plasmid Preparation

    cells were placed in suspension, or adherent cultures were serum-starved for two hours seventy-two hours post transfection with control or Raf1 siRNA. Adherent cultures were then stimulated with EGF (1 ng/mL for 5 min) or plated on fibronectin (FN)-coated dishes for the indicated times. Cell lysates were analyzed by western blot with the indicated antisera.

    Journal: Cellular signalling

    Article Title: MEK1 activation by PAK: A novel mechanism

    doi: 10.1016/j.cellsig.2007.01.018

    Figure Lengend Snippet: cells were placed in suspension, or adherent cultures were serum-starved for two hours seventy-two hours post transfection with control or Raf1 siRNA. Adherent cultures were then stimulated with EGF (1 ng/mL for 5 min) or plated on fibronectin (FN)-coated dishes for the indicated times. Cell lysates were analyzed by western blot with the indicated antisera.

    Article Snippet: Cells were transfected with 100 nM control or Raf1 siRNA (SMARTpool, Dharmacon) using 30 μL Lipofectamine 2000 (Invitrogen).

    Techniques: Transfection, Western Blot

    Intrathecal Raf-1-selective siRNA pretreatment attenuates sustained morphine-mediated tactile allodynia. For description of the animal groups see . Paw withdrawal thresholds in response to a series of von Frey filaments applied to the plantar surface

    Journal: British Journal of Pharmacology

    Article Title: Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA

    doi: 10.1111/j.1476-5381.2010.00869.x

    Figure Lengend Snippet: Intrathecal Raf-1-selective siRNA pretreatment attenuates sustained morphine-mediated tactile allodynia. For description of the animal groups see . Paw withdrawal thresholds in response to a series of von Frey filaments applied to the plantar surface

    Article Snippet: The rat Raf-1-selective siRNA mixture (Smart pool Dharmacon Inc., Chicago, IL, USA # L-087699-00) and the control, non-targeting double-stranded RNA (dsRNA) construct (Dharmacon Inc., Chicago, IL, USA #D-001810-01-20) were reconstituted in double distilled RNAse-free water to a stock concentration of 100 µM and stored in aliquots at −80°C.

    Techniques:

    Intrathecal (i.th) Raf-1-selective siRNA pretreatment attenuates sustained morphine-mediated antinociceptive tolerance. The groups of animals have been described in detail in . After sustained morphine (or saline) treatment, on day 6 the animals

    Journal: British Journal of Pharmacology

    Article Title: Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA

    doi: 10.1111/j.1476-5381.2010.00869.x

    Figure Lengend Snippet: Intrathecal (i.th) Raf-1-selective siRNA pretreatment attenuates sustained morphine-mediated antinociceptive tolerance. The groups of animals have been described in detail in . After sustained morphine (or saline) treatment, on day 6 the animals

    Article Snippet: The rat Raf-1-selective siRNA mixture (Smart pool Dharmacon Inc., Chicago, IL, USA # L-087699-00) and the control, non-targeting double-stranded RNA (dsRNA) construct (Dharmacon Inc., Chicago, IL, USA #D-001810-01-20) were reconstituted in double distilled RNAse-free water to a stock concentration of 100 µM and stored in aliquots at −80°C.

    Techniques:

    Intrathecal (i.th) Raf-1-selective siRNA treatment attenuates sustained morphine-mediated augmentation of calcitonin gene-related peptide (CGRP) immunoreactivity in the lumbar spinal cord of rats. Male Sprague-Dawley rats received i.th vehicle (A, B)

    Journal: British Journal of Pharmacology

    Article Title: Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA

    doi: 10.1111/j.1476-5381.2010.00869.x

    Figure Lengend Snippet: Intrathecal (i.th) Raf-1-selective siRNA treatment attenuates sustained morphine-mediated augmentation of calcitonin gene-related peptide (CGRP) immunoreactivity in the lumbar spinal cord of rats. Male Sprague-Dawley rats received i.th vehicle (A, B)

    Article Snippet: The rat Raf-1-selective siRNA mixture (Smart pool Dharmacon Inc., Chicago, IL, USA # L-087699-00) and the control, non-targeting double-stranded RNA (dsRNA) construct (Dharmacon Inc., Chicago, IL, USA #D-001810-01-20) were reconstituted in double distilled RNAse-free water to a stock concentration of 100 µM and stored in aliquots at −80°C.

    Techniques:

    Intrathecal Raf-1-selective siRNA treatment attenuates sustained morphine-mediated thermal hyperalgesia. The groups of rats included in the study were explained in . Paw withdrawal latencies in response to radiant heat applied to the plantar surface

    Journal: British Journal of Pharmacology

    Article Title: Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA

    doi: 10.1111/j.1476-5381.2010.00869.x

    Figure Lengend Snippet: Intrathecal Raf-1-selective siRNA treatment attenuates sustained morphine-mediated thermal hyperalgesia. The groups of rats included in the study were explained in . Paw withdrawal latencies in response to radiant heat applied to the plantar surface

    Article Snippet: The rat Raf-1-selective siRNA mixture (Smart pool Dharmacon Inc., Chicago, IL, USA # L-087699-00) and the control, non-targeting double-stranded RNA (dsRNA) construct (Dharmacon Inc., Chicago, IL, USA #D-001810-01-20) were reconstituted in double distilled RNAse-free water to a stock concentration of 100 µM and stored in aliquots at −80°C.

    Techniques:

    Intrathecal Raf-1-selective siRNA pretreatment attenuates sustained morphine-mediated augmentation of calcitonin gene-related peptide (CGRP) levels in lumbar spinal cord homogenates of rats. Male Sprague-Dawley rats were subcutaneously implanted with

    Journal: British Journal of Pharmacology

    Article Title: Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA

    doi: 10.1111/j.1476-5381.2010.00869.x

    Figure Lengend Snippet: Intrathecal Raf-1-selective siRNA pretreatment attenuates sustained morphine-mediated augmentation of calcitonin gene-related peptide (CGRP) levels in lumbar spinal cord homogenates of rats. Male Sprague-Dawley rats were subcutaneously implanted with

    Article Snippet: The rat Raf-1-selective siRNA mixture (Smart pool Dharmacon Inc., Chicago, IL, USA # L-087699-00) and the control, non-targeting double-stranded RNA (dsRNA) construct (Dharmacon Inc., Chicago, IL, USA #D-001810-01-20) were reconstituted in double distilled RNAse-free water to a stock concentration of 100 µM and stored in aliquots at −80°C.

    Techniques:

    (A) Intrathecal Raf-1-selective siRNA pretreatment reduces Raf-1 protein levels in rat lumbar spinal cord homogenates. Male Sprague-Dawley rats received intrathecal (i.th) injections of transfection reagent (lanes 1–2), 2 µg per 10 µL

    Journal: British Journal of Pharmacology

    Article Title: Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA

    doi: 10.1111/j.1476-5381.2010.00869.x

    Figure Lengend Snippet: (A) Intrathecal Raf-1-selective siRNA pretreatment reduces Raf-1 protein levels in rat lumbar spinal cord homogenates. Male Sprague-Dawley rats received intrathecal (i.th) injections of transfection reagent (lanes 1–2), 2 µg per 10 µL

    Article Snippet: The rat Raf-1-selective siRNA mixture (Smart pool Dharmacon Inc., Chicago, IL, USA # L-087699-00) and the control, non-targeting double-stranded RNA (dsRNA) construct (Dharmacon Inc., Chicago, IL, USA #D-001810-01-20) were reconstituted in double distilled RNAse-free water to a stock concentration of 100 µM and stored in aliquots at −80°C.

    Techniques: Transfection

    A. Experimental design. (Ith: Intrathecal; siRNA: Raf-1 siRNA or mismatch siRNA delivery via intrathecal catheter; BL: Baseline; D: Day; IR: Infrared heat). B. Raf-1 siRNA attenuates sustained morphine-mediated thermal hyperalgesia in rats: Male Sprague Dawley rats received intrathecal injections of i-Fect encapsulated Raf-1selective siRNA (Raf-1 siRNA groups); or non-targeting dsRNA (mismatch siRNA groups) or the transfection agent (i-Fect) alone (control) once daily, for 3 days. After pre-treatment, the rats received continous subcutaneous (osmotic minipump) saline (control group, Raf-1 siRNA group and mismatch siRNA group) or morphine (45μg/μl/h) (morphine group, Raf-1 siRNA+morphine group and mismatch siRNA+morphine group) infusions for 6 days. Six animals were included in each treatment group. Thermal hyperalgesia was measured as a decrease in paw withdrawal latencies in a radiant heat paw-withdrawal test.

    Journal: European journal of pharmacology

    Article Title: Intrathecal Raf-1-selective siRNA attenuates sustained morphinemediated thermal hyperalgesia

    doi: 10.1016/j.ejphar.2008.10.033

    Figure Lengend Snippet: A. Experimental design. (Ith: Intrathecal; siRNA: Raf-1 siRNA or mismatch siRNA delivery via intrathecal catheter; BL: Baseline; D: Day; IR: Infrared heat). B. Raf-1 siRNA attenuates sustained morphine-mediated thermal hyperalgesia in rats: Male Sprague Dawley rats received intrathecal injections of i-Fect encapsulated Raf-1selective siRNA (Raf-1 siRNA groups); or non-targeting dsRNA (mismatch siRNA groups) or the transfection agent (i-Fect) alone (control) once daily, for 3 days. After pre-treatment, the rats received continous subcutaneous (osmotic minipump) saline (control group, Raf-1 siRNA group and mismatch siRNA group) or morphine (45μg/μl/h) (morphine group, Raf-1 siRNA+morphine group and mismatch siRNA+morphine group) infusions for 6 days. Six animals were included in each treatment group. Thermal hyperalgesia was measured as a decrease in paw withdrawal latencies in a radiant heat paw-withdrawal test.

    Article Snippet: After recovery from the surgery (5–7 days), the animals received intrathecal injections (2ug siRNA/10 ul/rat) of either a lipid encapsulated Raf-1-selective siRNA mixture (Smart pool siRNA, Dharmacon Inc; Chicago, IL, Cat # L-087699-00 ) (Raf-1 siRNA groups) or i-Fect encapsulated non-targeting dsRNA (Dharmacon, # D-001810-01-20 ) (control mismatch siRNA groups) or the transfection lipid alone, once daily, for 3 days, as described earlier ( ).

    Techniques: Transfection

    Knockdown (KD) of Plk1 attenuates the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, but not Raf-1 phosphorylation. a Representative immunoblots illustrating the effects of Plk1shRNA on Plk1 expression. Blots of HASM cells infected with lentiviruses encoding control shRNA or Plk1 shRNA were probed with antibodies against Plk1 and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells producing Plk1 shRNA were normalized to ratios obtained from cells producing control shRNA. Values are mean ± SE ( n = 4). *Significantly lower Plk1/GAPDH ratios in cells producing Plk1 shRNA compared with cells producing control shRNA ( P

    Journal: Respiratory Research

    Article Title: Plk1 regulates MEK1/2 and proliferation in airway smooth muscle cells

    doi: 10.1186/s12931-015-0257-8

    Figure Lengend Snippet: Knockdown (KD) of Plk1 attenuates the PDGF-induced phosphorylation of MEK1/2 and ERK1/2, but not Raf-1 phosphorylation. a Representative immunoblots illustrating the effects of Plk1shRNA on Plk1 expression. Blots of HASM cells infected with lentiviruses encoding control shRNA or Plk1 shRNA were probed with antibodies against Plk1 and GAPDH. Duplicated samples of each treatment are shown. Ratios of Plk1/GAPDH protein in cells producing Plk1 shRNA were normalized to ratios obtained from cells producing control shRNA. Values are mean ± SE ( n = 4). *Significantly lower Plk1/GAPDH ratios in cells producing Plk1 shRNA compared with cells producing control shRNA ( P

    Article Snippet: Antibodies used were anti-phospho-Plk1 (Abcam), anti-Plk1 (EMD Millipore), anti-phospho-MEK1/2 (Santa Cruz Biotech.), anti-MEK1/2 (Santa Cruz Biotech.), anti-phospho-ERK1/2 (Cell signaling), anti-ERK1/2 (Cell signaling), anti-phospho-Raf-1 (Santa Cruz Biotech.), anti-Raf-1 (Santa Cruz Biotech.) and anti-GAPDH (Ambion).

    Techniques: Western Blot, Expressing, Infection, shRNA

    Effects of galectin-1 on the Ras-transmitted downstream signals. Scrambled RNA or galectin-1 shRNA were transfected in HeLa cells. GFP or galectin-1 cDNA were transfected in C33A cells. The cells were irradiated with 6 Gy and harvested 5 or 10 min later. Expressions of activated H-Ras, p-Raf-1, and p-ERK using western blots in HeLa and C33A cells were compared with or without galectin-1 modulation

    Journal: Cell Death & Disease

    Article Title: A novel radioresistant mechanism of galectin-1 mediated by H-Ras-dependent pathways in cervical cancer cells

    doi: 10.1038/cddis.2011.120

    Figure Lengend Snippet: Effects of galectin-1 on the Ras-transmitted downstream signals. Scrambled RNA or galectin-1 shRNA were transfected in HeLa cells. GFP or galectin-1 cDNA were transfected in C33A cells. The cells were irradiated with 6 Gy and harvested 5 or 10 min later. Expressions of activated H-Ras, p-Raf-1, and p-ERK using western blots in HeLa and C33A cells were compared with or without galectin-1 modulation

    Article Snippet: Antibodies to phospho-histone H2AX (Ser139), phospho-Raf-1, Raf-1, phospho-p42/44 ERK, and p42/44 ERK were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: shRNA, Transfection, Irradiation, Western Blot

    A proposed model of cooperation between H-Ras and galectin-1 for radioresistance. Galectin-1 enhances activation (GTP form) of H-Ras following irradiation. Galectin-1 potentiates downstream signals of H-Ras, such as Raf-1 and ERK, that may mediate DNA damage repair 33 and radioresistance. 31 , 32 Galectin-1 may regulate p21 expression 36 through Raf-1. 34 Hence, Raf-1 may have a significant role in galectin-1-mediated radioresistance

    Journal: Cell Death & Disease

    Article Title: A novel radioresistant mechanism of galectin-1 mediated by H-Ras-dependent pathways in cervical cancer cells

    doi: 10.1038/cddis.2011.120

    Figure Lengend Snippet: A proposed model of cooperation between H-Ras and galectin-1 for radioresistance. Galectin-1 enhances activation (GTP form) of H-Ras following irradiation. Galectin-1 potentiates downstream signals of H-Ras, such as Raf-1 and ERK, that may mediate DNA damage repair 33 and radioresistance. 31 , 32 Galectin-1 may regulate p21 expression 36 through Raf-1. 34 Hence, Raf-1 may have a significant role in galectin-1-mediated radioresistance

    Article Snippet: Antibodies to phospho-histone H2AX (Ser139), phospho-Raf-1, Raf-1, phospho-p42/44 ERK, and p42/44 ERK were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Activation Assay, Irradiation, Expressing

    RASAL2 knockdown activates the Ras-ERK pathway (A) The effect of RASAL2 knockdown on Ras activation in SK-OV-3, OVCAR3 and A2780 cells was evaluated by a Ras Activation ELISA Assay Kit. All the data were normalized to the results of cells transfected with scramble-shRNA. The data are shown as the means ± SD (n = 3). (B) Phosphorylation of Raf-1, MEK1/2 and ERK1/2 in SK-OV-3, OVCAR3 and A2780 cells infected with shRASAL2-1, shRASAL2-2 or scramble-shRNA was analyzed by Western blot. Xenograft tumor sections (SK-OV-3) were also examined for levels of p-ERK by IHC (C) and for p-Raf-1 and p-MEK by Western blot (D).

    Journal: Oncotarget

    Article Title: RASAL2 down-regulation in ovarian cancer promotes epithelial-mesenchymal transition and metastasis

    doi:

    Figure Lengend Snippet: RASAL2 knockdown activates the Ras-ERK pathway (A) The effect of RASAL2 knockdown on Ras activation in SK-OV-3, OVCAR3 and A2780 cells was evaluated by a Ras Activation ELISA Assay Kit. All the data were normalized to the results of cells transfected with scramble-shRNA. The data are shown as the means ± SD (n = 3). (B) Phosphorylation of Raf-1, MEK1/2 and ERK1/2 in SK-OV-3, OVCAR3 and A2780 cells infected with shRASAL2-1, shRASAL2-2 or scramble-shRNA was analyzed by Western blot. Xenograft tumor sections (SK-OV-3) were also examined for levels of p-ERK by IHC (C) and for p-Raf-1 and p-MEK by Western blot (D).

    Article Snippet: Antibodies were obtained from the following companies: anti-RASAL2 antibody from Abcam; anti-β-actin antibody from Sigma; anti-E-cadherin, anti-vimentin, anti-claudin-1, anti-N-cadherin, anti-ZO-1, anti-Snail, anti-Slug, anti-Raf-1, anti-p-Raf-1, anti-MEK1/2, anti-p-MEK1/2, anti-ERK1/2 and anti-p-ERK1/2 antibodies from Cell Signaling.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection, shRNA, Infection, Western Blot, Immunohistochemistry

    Raf-1 signaling is inhibited by RKIP in B-Raf deficient cells (A) Immortalized wild-type (RKIP+/+) and RKIP −/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1) or control siRNA (siCon). After 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP and Raf-1 were detected by western blot. (B) RKIP was stably depleted in WT and Raf-1−/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, and Raf-1 were detected by western blot. (C) Immortalized wild-type and RKIP−/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1), B-Raf siRNA (siB-Raf), double B-Raf and Raf-1 siRNA (siRaf-1+siB-Raf) or control siRNA (siCon); after 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP, Raf-1 and B-Raf were detected by western blot. *, p

    Journal: Cellular signalling

    Article Title: RKIP Regulates MAP Kinase Signaling in Cells with Defective B-Raf Activity

    doi: 10.1016/j.cellsig.2013.02.005

    Figure Lengend Snippet: Raf-1 signaling is inhibited by RKIP in B-Raf deficient cells (A) Immortalized wild-type (RKIP+/+) and RKIP −/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1) or control siRNA (siCon). After 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP and Raf-1 were detected by western blot. (B) RKIP was stably depleted in WT and Raf-1−/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, and Raf-1 were detected by western blot. (C) Immortalized wild-type and RKIP−/− MEFs were transiently electroporated with Raf-1 siRNA (siRaf-1), B-Raf siRNA (siB-Raf), double B-Raf and Raf-1 siRNA (siRaf-1+siB-Raf) or control siRNA (siCon); after 24 hour recovery at 33°C, these cells were serum-starved overnight at 39°C, and then were stimulated with EGF (10 ng/ml) at 39°C for 5 min. pERK1/2, ERK1/2, RKIP, Raf-1 and B-Raf were detected by western blot. *, p

    Article Snippet: Raf-1 antibody (Cat No: sc133) and B-Raf antibody (Cat No: sc-5284) and goat anti-mouse IgG-HRP (Cat No: sc-2005) were purchased from Santa Cruz.

    Techniques: Western Blot, Stable Transfection, shRNA

    Loss of RKIP rescues ERK activation in B-Raf knockout MEFs that express kinase-dead B-Raf mutants (A) RKIP was stably depleted in WT and B-Raf −/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, B-Raf and Raf-1 were detected by western blot. *, p

    Journal: Cellular signalling

    Article Title: RKIP Regulates MAP Kinase Signaling in Cells with Defective B-Raf Activity

    doi: 10.1016/j.cellsig.2013.02.005

    Figure Lengend Snippet: Loss of RKIP rescues ERK activation in B-Raf knockout MEFs that express kinase-dead B-Raf mutants (A) RKIP was stably depleted in WT and B-Raf −/− MEFs by shRNA. MEFs were serum-starved overnight at 37°C, and then were stimulated with EGF (10 ng/ml) for 5 min. pERK1/2, ERK1/2, RKIP, B-Raf and Raf-1 were detected by western blot. *, p

    Article Snippet: Raf-1 antibody (Cat No: sc133) and B-Raf antibody (Cat No: sc-5284) and goat anti-mouse IgG-HRP (Cat No: sc-2005) were purchased from Santa Cruz.

    Techniques: Activation Assay, Knock-Out, Stable Transfection, shRNA, Western Blot

    BRAF and BCORL1 mutations in resistant cells. (A) Whole-exome sequencing copy number variation analysis revealed focal imbalances of the distal region of chromosome 7 in A375-R1 cells compared to parental cells. BRAF locus (zoomed area) lies within the amplified region but shows loss of exons 2 to 8. (B) Sanger sequencing of the deleted BRAF allele showing in-frame junction of exon 1 with exon 9 in A375-R1 cells. (C) PCR amplification of the region across the break point reveals the presence of two BRAF transcripts in A375-R1 cells: full-length wild-type (1216-bp band) and truncated (213-bp band). (D) Anti-BRAF Western blotting shows the presence of a smaller band of approximately 47kDa in A375-R1 cells. Actin is shown as a loading control. (E) siRNA-mediated silencing of BRAF in A375-R1 cells leads to downregulation of both full-length and truncated BRAF proteins, suppression of MEK1/2 phosphorylation, and decrease in cell viability. (F) Whole-exome sequencing comparative analysis of A375-R1 versus A375 cells revealed acquired mutations in the four indicated genes at frequency > 35%. SIFT prediction of mutation impact on protein function is shown. (G) Sanger validation of BCORL1 heterozygous genomic mutation in A375-R1 cells.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells

    doi: 10.1016/j.neo.2018.02.009

    Figure Lengend Snippet: BRAF and BCORL1 mutations in resistant cells. (A) Whole-exome sequencing copy number variation analysis revealed focal imbalances of the distal region of chromosome 7 in A375-R1 cells compared to parental cells. BRAF locus (zoomed area) lies within the amplified region but shows loss of exons 2 to 8. (B) Sanger sequencing of the deleted BRAF allele showing in-frame junction of exon 1 with exon 9 in A375-R1 cells. (C) PCR amplification of the region across the break point reveals the presence of two BRAF transcripts in A375-R1 cells: full-length wild-type (1216-bp band) and truncated (213-bp band). (D) Anti-BRAF Western blotting shows the presence of a smaller band of approximately 47kDa in A375-R1 cells. Actin is shown as a loading control. (E) siRNA-mediated silencing of BRAF in A375-R1 cells leads to downregulation of both full-length and truncated BRAF proteins, suppression of MEK1/2 phosphorylation, and decrease in cell viability. (F) Whole-exome sequencing comparative analysis of A375-R1 versus A375 cells revealed acquired mutations in the four indicated genes at frequency > 35%. SIFT prediction of mutation impact on protein function is shown. (G) Sanger validation of BCORL1 heterozygous genomic mutation in A375-R1 cells.

    Article Snippet: Gene-specific siRNA pools (siGENOME SMARTpool; BCORL1, #M-019215-01; BRAF, #M-003460-03; RAF1, #M-003601-02) and control nontargeting siRNA (#D-001210-01) were purchased from Dharmacon and used for transient transfections following manufacturer's recommendations.

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Western Blot, Mutagenesis

    Stable knockdown of BCORL1 in A375 cells (A-C) and A375-p47BRAF V600E [clone E9] (D-F). (A, D) Efficiency of shRNA-mediated BCORL1 silencing as shown by quantitative PCR using GAPDH as a reference gene. (B, E) Dose-response curves obtained in the presence of increasing concentrations of vemurafenib, with cells expressing a nontargeting ( shNT , blue curves) or a BCORL1-specific ( shBCORL1 , red curves) shRNA. (C, F) CRISPR/Cas9 system was used to disrupt BCORL1 gene; vemurafenib dose-response curves are shown comparing knockout ( KO ) with parental ( WT ) cells. (G) CRISPR/Cas9-mediated gene editing was used to introduce the Q1076H substitution in the endogenous BCORL1 locus; vemurafenib dose-response curves are shown comparing two mutated clones ( C8 and D7 ) with a wild-type clone ( WT ) and with parental A375 cells ( Par ). All curves are representative of at least three experiments. For all panels, extra-sum-of-squares F test was run to compare the two curves; P values are indicated at the lower-left corner, where P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells

    doi: 10.1016/j.neo.2018.02.009

    Figure Lengend Snippet: Stable knockdown of BCORL1 in A375 cells (A-C) and A375-p47BRAF V600E [clone E9] (D-F). (A, D) Efficiency of shRNA-mediated BCORL1 silencing as shown by quantitative PCR using GAPDH as a reference gene. (B, E) Dose-response curves obtained in the presence of increasing concentrations of vemurafenib, with cells expressing a nontargeting ( shNT , blue curves) or a BCORL1-specific ( shBCORL1 , red curves) shRNA. (C, F) CRISPR/Cas9 system was used to disrupt BCORL1 gene; vemurafenib dose-response curves are shown comparing knockout ( KO ) with parental ( WT ) cells. (G) CRISPR/Cas9-mediated gene editing was used to introduce the Q1076H substitution in the endogenous BCORL1 locus; vemurafenib dose-response curves are shown comparing two mutated clones ( C8 and D7 ) with a wild-type clone ( WT ) and with parental A375 cells ( Par ). All curves are representative of at least three experiments. For all panels, extra-sum-of-squares F test was run to compare the two curves; P values are indicated at the lower-left corner, where P

    Article Snippet: Gene-specific siRNA pools (siGENOME SMARTpool; BCORL1, #M-019215-01; BRAF, #M-003460-03; RAF1, #M-003601-02) and control nontargeting siRNA (#D-001210-01) were purchased from Dharmacon and used for transient transfections following manufacturer's recommendations.

    Techniques: shRNA, Real-time Polymerase Chain Reaction, Expressing, CRISPR, Knock-Out, Introduce, Clone Assay

    Functional validation of mutations. (A-C) Transient co-transfection of p47BRAF V600E and BCORL1 Q1076H conferred partial resistance to vemurafenib-mediated inhibition of A375 cell growth (A) and MAPK signaling (C). Forty-eight hours after transfection, the cells were challenged for additional 48 (A) or 4 (C) hours with vemurafenib and harvested. Cell proliferation shown in A was detected by thymidine incorporation. (B) Histogram plot from two proliferation experiments (mean ± SEM). MAPK pathway activity shown in C was detected by Western blotting as MEK and ERK phosphorylation. (D-G) Stably transfected A375 clones expressing HA-tagged p47BRAF V600E (D, Western blot, clone E9) and wild-type or mutated BCORL1 (E, qPCR), singularly or combined, were isolated and tested in proliferation assays for vemurafenib sensitivity. Data from at least four independent experiments are reported as mean ± SEM IC50 values (F); the red bar represents A375-R1 cells, for comparison; ev, empty vector. (G) Representative Western blot showing MEK1/2 phosphorylation (pMEK) in transfected cells treated with the indicated vemurafenib doses for 4 hours. Actin is shown as a control. (H) Correlation between p47BRAF V600E expression ( x -axis, log scale) determined by p47-specific qPCR and vemurafenib IC50 ( y -axis, log scale). Clone E9, used for all experiments, is indicated. (I) Upper panel: siRNA-mediated silencing of BCORL1 ( siBCORL1 ) and BRAF ( siBRAF ) in A375-R1 cells; a nontargeting scrambled siRNA ( siNT ) was used as a control. Lysates were probed with the indicated antibodies. Lower panel: Parental A375 cells were transiently transfected with empty vector, wild-type ( WT ), or mutated ( Q1076H ) BCORL1 and checked for BRAF expression. Actin is shown for loading control.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells

    doi: 10.1016/j.neo.2018.02.009

    Figure Lengend Snippet: Functional validation of mutations. (A-C) Transient co-transfection of p47BRAF V600E and BCORL1 Q1076H conferred partial resistance to vemurafenib-mediated inhibition of A375 cell growth (A) and MAPK signaling (C). Forty-eight hours after transfection, the cells were challenged for additional 48 (A) or 4 (C) hours with vemurafenib and harvested. Cell proliferation shown in A was detected by thymidine incorporation. (B) Histogram plot from two proliferation experiments (mean ± SEM). MAPK pathway activity shown in C was detected by Western blotting as MEK and ERK phosphorylation. (D-G) Stably transfected A375 clones expressing HA-tagged p47BRAF V600E (D, Western blot, clone E9) and wild-type or mutated BCORL1 (E, qPCR), singularly or combined, were isolated and tested in proliferation assays for vemurafenib sensitivity. Data from at least four independent experiments are reported as mean ± SEM IC50 values (F); the red bar represents A375-R1 cells, for comparison; ev, empty vector. (G) Representative Western blot showing MEK1/2 phosphorylation (pMEK) in transfected cells treated with the indicated vemurafenib doses for 4 hours. Actin is shown as a control. (H) Correlation between p47BRAF V600E expression ( x -axis, log scale) determined by p47-specific qPCR and vemurafenib IC50 ( y -axis, log scale). Clone E9, used for all experiments, is indicated. (I) Upper panel: siRNA-mediated silencing of BCORL1 ( siBCORL1 ) and BRAF ( siBRAF ) in A375-R1 cells; a nontargeting scrambled siRNA ( siNT ) was used as a control. Lysates were probed with the indicated antibodies. Lower panel: Parental A375 cells were transiently transfected with empty vector, wild-type ( WT ), or mutated ( Q1076H ) BCORL1 and checked for BRAF expression. Actin is shown for loading control.

    Article Snippet: Gene-specific siRNA pools (siGENOME SMARTpool; BCORL1, #M-019215-01; BRAF, #M-003460-03; RAF1, #M-003601-02) and control nontargeting siRNA (#D-001210-01) were purchased from Dharmacon and used for transient transfections following manufacturer's recommendations.

    Techniques: Functional Assay, Cotransfection, Inhibition, Transfection, Activity Assay, Western Blot, Stable Transfection, Clone Assay, Expressing, Real-time Polymerase Chain Reaction, Isolation, Plasmid Preparation

    Endogenous RanBPM and c-Raf interaction in HeLa cells using PLA. Duolink II proximity ligation assay (PLA) was performed in: ( A ) control shRNA HeLa cells, without the addition of primary antibodies (negative control); ( B ) control shRNA HeLa cells, with Hsp90 and c-Raf primary antibodies (positive control); ( C ) control shRNA HeLa cells, using c-Raf and RanBPM primary antibodies; ( D ). HeLa RanBPM shRNA cells, with c-Raf and RanBPM primary antibodies (negative control); ( E ) control shRNA HeLa cells, using MAEA (Macrophage erythroblast attacher) and RanBPM primary antibodies (positive control). ( F ) Control shRNA HeLa cells, using c-Raf and MAEA primary antibodies. The DAPI filter was used to visualize the nuclei, while the Cyanine 3 (Cy3) filter was used to visualize the PLA dots representing protein–protein interactions. Representative images from one of three independent experiments are shown. Scale bars, 10 μm.

    Journal: International Journal of Molecular Sciences

    Article Title: Regulation of c-Raf Stability through the CTLH Complex

    doi: 10.3390/ijms20040934

    Figure Lengend Snippet: Endogenous RanBPM and c-Raf interaction in HeLa cells using PLA. Duolink II proximity ligation assay (PLA) was performed in: ( A ) control shRNA HeLa cells, without the addition of primary antibodies (negative control); ( B ) control shRNA HeLa cells, with Hsp90 and c-Raf primary antibodies (positive control); ( C ) control shRNA HeLa cells, using c-Raf and RanBPM primary antibodies; ( D ). HeLa RanBPM shRNA cells, with c-Raf and RanBPM primary antibodies (negative control); ( E ) control shRNA HeLa cells, using MAEA (Macrophage erythroblast attacher) and RanBPM primary antibodies (positive control). ( F ) Control shRNA HeLa cells, using c-Raf and MAEA primary antibodies. The DAPI filter was used to visualize the nuclei, while the Cyanine 3 (Cy3) filter was used to visualize the PLA dots representing protein–protein interactions. Representative images from one of three independent experiments are shown. Scale bars, 10 μm.

    Article Snippet: Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on either 8% or 10% acrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat dry milk in Tris Buffered Saline with Tween 20 (TBS-T) or in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA), and hybridized with the following antibodies: c-Raf (C-12, 1:500, Santa Cruz), HA (HA-7, 1:1000, Sigma–Aldrich, St. Louis, MO, USA), RMND5A (NBP1–92337, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (I-19, 1:2000, Santa Cruz), RanBPM (5M, 1:2000, Bioacademia, Osaka, Japan), GST (B-14, 1:500, Santa Cruz), A-Raf (1:500, Cell Signaling Technology, Danvers, MA, USA, #4432), B-Raf (1:1000, Cell Signaling Technology #9434), phospho-T202/Y204-ERK1/2 (1:2000, Cell Signaling #4370), ERK1/2 (1:1000, Cell Signaling #9102), phospho-S217/221-MEK1/2 (1:2000, Cell Signaling #9154), MEK1/2 (1:1000, Cell Signaling #9122).

    Techniques: Proximity Ligation Assay, shRNA, Negative Control, Positive Control

    Downregulation of RanBPM promotes tumour formation in NOD/SCID/gamma mice. ( A ) re-expression of RanBPM in HEK293 cells via Tet-off pBIG expression vector. HEK293 pool of cells stably expressing RanBPM shRNA were transfected with pBIG-RanBPM WT (si-mt) and maintained in media with 2 μg/mL Tetracycline and 250 μg/mL hygromycin to select for integration of the pBIG vector. Following selection, cells were either maintained (+) in Tetracyclin-containing media, or cultured in absence of Tetracyclin (−) for 24 h to allow induction of RanBPM. Tetracyclin removal leads to re-expression of RanBPM (lane 4); ( B ) ERK pathway activation in RanBPM shRNA cells. Samples shown in ( A ) were analyzed for ERK phosphorylation and A-Raf and B-Raf expression. The Western blot was analyzed with the indicated antibodies; ( C ) injections with HEK293 control and RanBPM shRNA pools were injected subcutaneously in the flank of 6–8 weeks old NOD/SCID/gamma. Tumour measurements were taken twice per week and a digital caliper was used to measure Length × Width × Depth of the tumour upon excision in order to calculate volume. n = 7, error bars represent SEM; ( D ) injections with HEK293 RanBPM shRNA pool of cells stably re-expressing RanBPM via pBIG Tet-off expression system (see C , lanes 3,4). Mice were fed chow containing Dox (purple line) or regular chow (green line). n = 6, error bars represent SEM. p

    Journal: International Journal of Molecular Sciences

    Article Title: Regulation of c-Raf Stability through the CTLH Complex

    doi: 10.3390/ijms20040934

    Figure Lengend Snippet: Downregulation of RanBPM promotes tumour formation in NOD/SCID/gamma mice. ( A ) re-expression of RanBPM in HEK293 cells via Tet-off pBIG expression vector. HEK293 pool of cells stably expressing RanBPM shRNA were transfected with pBIG-RanBPM WT (si-mt) and maintained in media with 2 μg/mL Tetracycline and 250 μg/mL hygromycin to select for integration of the pBIG vector. Following selection, cells were either maintained (+) in Tetracyclin-containing media, or cultured in absence of Tetracyclin (−) for 24 h to allow induction of RanBPM. Tetracyclin removal leads to re-expression of RanBPM (lane 4); ( B ) ERK pathway activation in RanBPM shRNA cells. Samples shown in ( A ) were analyzed for ERK phosphorylation and A-Raf and B-Raf expression. The Western blot was analyzed with the indicated antibodies; ( C ) injections with HEK293 control and RanBPM shRNA pools were injected subcutaneously in the flank of 6–8 weeks old NOD/SCID/gamma. Tumour measurements were taken twice per week and a digital caliper was used to measure Length × Width × Depth of the tumour upon excision in order to calculate volume. n = 7, error bars represent SEM; ( D ) injections with HEK293 RanBPM shRNA pool of cells stably re-expressing RanBPM via pBIG Tet-off expression system (see C , lanes 3,4). Mice were fed chow containing Dox (purple line) or regular chow (green line). n = 6, error bars represent SEM. p

    Article Snippet: Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on either 8% or 10% acrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat dry milk in Tris Buffered Saline with Tween 20 (TBS-T) or in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA), and hybridized with the following antibodies: c-Raf (C-12, 1:500, Santa Cruz), HA (HA-7, 1:1000, Sigma–Aldrich, St. Louis, MO, USA), RMND5A (NBP1–92337, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (I-19, 1:2000, Santa Cruz), RanBPM (5M, 1:2000, Bioacademia, Osaka, Japan), GST (B-14, 1:500, Santa Cruz), A-Raf (1:500, Cell Signaling Technology, Danvers, MA, USA, #4432), B-Raf (1:1000, Cell Signaling Technology #9434), phospho-T202/Y204-ERK1/2 (1:2000, Cell Signaling #4370), ERK1/2 (1:1000, Cell Signaling #9102), phospho-S217/221-MEK1/2 (1:2000, Cell Signaling #9154), MEK1/2 (1:1000, Cell Signaling #9122).

    Techniques: Mouse Assay, Expressing, Plasmid Preparation, Stable Transfection, shRNA, Transfection, Selection, Cell Culture, Activation Assay, Western Blot, Injection

    RanBPM C-terminal CRA domain directly interacts with ΔN-c-Raf and is necessary for C-Raf regulation. ( A ) diagram of WT RanBPM, N2 domain and C1 domain cloned into the bacterial expression vector pGEX-4T-1; ( B ) Left, Western blot analysis of GST pull-down assays for N c-Raf performed using GST, GST-WT-RanBPM, GST-N2-domain and GST-C1-domain E. coli extracts. A representative image is shown. Right, pull down assays experiments were quantified by normalizing ΔN-c-Raf levels to pulled-down GST, GST-WT-RanBPM, GST-N2 or GST-C1 and statistical analyses were performed ( n = 6, error bar indicates SEM) with different letters indicating statistical difference ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Regulation of c-Raf Stability through the CTLH Complex

    doi: 10.3390/ijms20040934

    Figure Lengend Snippet: RanBPM C-terminal CRA domain directly interacts with ΔN-c-Raf and is necessary for C-Raf regulation. ( A ) diagram of WT RanBPM, N2 domain and C1 domain cloned into the bacterial expression vector pGEX-4T-1; ( B ) Left, Western blot analysis of GST pull-down assays for N c-Raf performed using GST, GST-WT-RanBPM, GST-N2-domain and GST-C1-domain E. coli extracts. A representative image is shown. Right, pull down assays experiments were quantified by normalizing ΔN-c-Raf levels to pulled-down GST, GST-WT-RanBPM, GST-N2 or GST-C1 and statistical analyses were performed ( n = 6, error bar indicates SEM) with different letters indicating statistical difference ( p

    Article Snippet: Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on either 8% or 10% acrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat dry milk in Tris Buffered Saline with Tween 20 (TBS-T) or in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA), and hybridized with the following antibodies: c-Raf (C-12, 1:500, Santa Cruz), HA (HA-7, 1:1000, Sigma–Aldrich, St. Louis, MO, USA), RMND5A (NBP1–92337, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (I-19, 1:2000, Santa Cruz), RanBPM (5M, 1:2000, Bioacademia, Osaka, Japan), GST (B-14, 1:500, Santa Cruz), A-Raf (1:500, Cell Signaling Technology, Danvers, MA, USA, #4432), B-Raf (1:1000, Cell Signaling Technology #9434), phospho-T202/Y204-ERK1/2 (1:2000, Cell Signaling #4370), ERK1/2 (1:1000, Cell Signaling #9102), phospho-S217/221-MEK1/2 (1:2000, Cell Signaling #9154), MEK1/2 (1:1000, Cell Signaling #9122).

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Western Blot

    Analysis of RanBPM domains that control C-Raf stability. ( A ) schematic representation of RanBPM mutants. ( B ) and ( C ) Western blot analyses of HeLa RanBPM shRNA cells transfected with pEBG-GST-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs as indicated. c-Raf and HA antibodies were used to detect the levels of ΔN-c-Raf and RanBPM, respectively. β-actin was used as a loading control. A representative Western blot is shown (top) and quantifications of c-Raf levels are shown (bottom graph) with error bars indicating SEM ( n = 5). Deletion of RanBPM C-terminal domain (ΔC1) impairs RanBPM interaction with GST-ΔN-c-Raf. ( D ) and ( E ) GST-Pull-down assays. HeLa RanBPM shRNA cells were transfected with pEBG-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs. ΔN-c-Raf was pulled down through binding to glutathione-sepharose beads and interaction of RanBPM WT and mutants with GST-ΔN-c-Raf assessed by Western blot with an HA antibody. Below: Quantifications were performed by normalizing RanBPM mutant levels to pulled-down GST or GST-ΔN-c-Raf and statistical analyses were performed ( n = 4–7, SEM shown). Different letters are statistically different ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Regulation of c-Raf Stability through the CTLH Complex

    doi: 10.3390/ijms20040934

    Figure Lengend Snippet: Analysis of RanBPM domains that control C-Raf stability. ( A ) schematic representation of RanBPM mutants. ( B ) and ( C ) Western blot analyses of HeLa RanBPM shRNA cells transfected with pEBG-GST-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs as indicated. c-Raf and HA antibodies were used to detect the levels of ΔN-c-Raf and RanBPM, respectively. β-actin was used as a loading control. A representative Western blot is shown (top) and quantifications of c-Raf levels are shown (bottom graph) with error bars indicating SEM ( n = 5). Deletion of RanBPM C-terminal domain (ΔC1) impairs RanBPM interaction with GST-ΔN-c-Raf. ( D ) and ( E ) GST-Pull-down assays. HeLa RanBPM shRNA cells were transfected with pEBG-ΔN-c-Raf and either pCMV-HA (empty vector), pCMV-HA-WT-RanBPM or pCMV-HA RanBPM mutant constructs. ΔN-c-Raf was pulled down through binding to glutathione-sepharose beads and interaction of RanBPM WT and mutants with GST-ΔN-c-Raf assessed by Western blot with an HA antibody. Below: Quantifications were performed by normalizing RanBPM mutant levels to pulled-down GST or GST-ΔN-c-Raf and statistical analyses were performed ( n = 4–7, SEM shown). Different letters are statistically different ( p

    Article Snippet: Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on either 8% or 10% acrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat dry milk in Tris Buffered Saline with Tween 20 (TBS-T) or in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA), and hybridized with the following antibodies: c-Raf (C-12, 1:500, Santa Cruz), HA (HA-7, 1:1000, Sigma–Aldrich, St. Louis, MO, USA), RMND5A (NBP1–92337, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (I-19, 1:2000, Santa Cruz), RanBPM (5M, 1:2000, Bioacademia, Osaka, Japan), GST (B-14, 1:500, Santa Cruz), A-Raf (1:500, Cell Signaling Technology, Danvers, MA, USA, #4432), B-Raf (1:1000, Cell Signaling Technology #9434), phospho-T202/Y204-ERK1/2 (1:2000, Cell Signaling #4370), ERK1/2 (1:1000, Cell Signaling #9102), phospho-S217/221-MEK1/2 (1:2000, Cell Signaling #9154), MEK1/2 (1:1000, Cell Signaling #9122).

    Techniques: Western Blot, shRNA, Transfection, Plasmid Preparation, Mutagenesis, Construct, Binding Assay

    C-Raf is regulated by the proteasome through the CTLH complex. Non-targeting shRNA control and shRNA RanBPM cells were treated with 10 μM MG132 or DMSO, as vehicle, for 24 h. RIPA buffered whole cell extracts of HeLa ( A ), and HCT116 ( B ) were analyzed by Western blot with RanBPM, c-Raf and β-actin antibodies to detect RanBPM, c-Raf and β-actin proteins, respectively. c-Raf protein levels were normalized to β-actin levels. Quantifications of relative c-Raf protein levels are shown with error bars indicating SD ( n = 4). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Regulation of c-Raf Stability through the CTLH Complex

    doi: 10.3390/ijms20040934

    Figure Lengend Snippet: C-Raf is regulated by the proteasome through the CTLH complex. Non-targeting shRNA control and shRNA RanBPM cells were treated with 10 μM MG132 or DMSO, as vehicle, for 24 h. RIPA buffered whole cell extracts of HeLa ( A ), and HCT116 ( B ) were analyzed by Western blot with RanBPM, c-Raf and β-actin antibodies to detect RanBPM, c-Raf and β-actin proteins, respectively. c-Raf protein levels were normalized to β-actin levels. Quantifications of relative c-Raf protein levels are shown with error bars indicating SD ( n = 4). * p

    Article Snippet: Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on either 8% or 10% acrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat dry milk in Tris Buffered Saline with Tween 20 (TBS-T) or in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA), and hybridized with the following antibodies: c-Raf (C-12, 1:500, Santa Cruz), HA (HA-7, 1:1000, Sigma–Aldrich, St. Louis, MO, USA), RMND5A (NBP1–92337, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (I-19, 1:2000, Santa Cruz), RanBPM (5M, 1:2000, Bioacademia, Osaka, Japan), GST (B-14, 1:500, Santa Cruz), A-Raf (1:500, Cell Signaling Technology, Danvers, MA, USA, #4432), B-Raf (1:1000, Cell Signaling Technology #9434), phospho-T202/Y204-ERK1/2 (1:2000, Cell Signaling #4370), ERK1/2 (1:1000, Cell Signaling #9102), phospho-S217/221-MEK1/2 (1:2000, Cell Signaling #9154), MEK1/2 (1:1000, Cell Signaling #9122).

    Techniques: shRNA, Western Blot

    Model of regulation of c-Raf by the RanBPM/CTLH complex. Three RanBPM regions, N-terminal (1–102), LisH/CTLH (360–460) and C-terminal CRA (615–729) are necessary to regulate c-Raf expression/stability, but only the CRA domain is able to directly interact with c-Raf in vitro. Our data suggest that RanBPM interacts with c-Raf through the CRA domain and recruits c-Raf to the CTLH complex to which RanBPM is associated through its LisH/CTLH domain. The CTLH complex promotes c-Raf ubiquitination and degradation. The role of RanBPM N-terminal domain is unclear, but it may be involved in RanBPM stability and folding and potentially stabilizes c-Raf interaction (dashed line). The minimum region of c-Raf defined so far as necessary for interaction with RanBPM is ∆N-c-Raf, which is comprised of conserved region CR3 and short flanking sequences. The position of the CR1, CR2 and CR3 conserved regions are shown. The location of the c-Raf catalytic domain (KD, kinase domain) is indicated. The thick double-head arrow indicates interaction. The dashed arrow indicates a regulation of c-Raf by the RanBPM N-terminal domain. Ubiquitination of c-Raf by the CTLH complex is indicated by the pink arrow. The bracket indicates that the LiSH/CTLH domain mediates interaction with CTLH complex members.

    Journal: International Journal of Molecular Sciences

    Article Title: Regulation of c-Raf Stability through the CTLH Complex

    doi: 10.3390/ijms20040934

    Figure Lengend Snippet: Model of regulation of c-Raf by the RanBPM/CTLH complex. Three RanBPM regions, N-terminal (1–102), LisH/CTLH (360–460) and C-terminal CRA (615–729) are necessary to regulate c-Raf expression/stability, but only the CRA domain is able to directly interact with c-Raf in vitro. Our data suggest that RanBPM interacts with c-Raf through the CRA domain and recruits c-Raf to the CTLH complex to which RanBPM is associated through its LisH/CTLH domain. The CTLH complex promotes c-Raf ubiquitination and degradation. The role of RanBPM N-terminal domain is unclear, but it may be involved in RanBPM stability and folding and potentially stabilizes c-Raf interaction (dashed line). The minimum region of c-Raf defined so far as necessary for interaction with RanBPM is ∆N-c-Raf, which is comprised of conserved region CR3 and short flanking sequences. The position of the CR1, CR2 and CR3 conserved regions are shown. The location of the c-Raf catalytic domain (KD, kinase domain) is indicated. The thick double-head arrow indicates interaction. The dashed arrow indicates a regulation of c-Raf by the RanBPM N-terminal domain. Ubiquitination of c-Raf by the CTLH complex is indicated by the pink arrow. The bracket indicates that the LiSH/CTLH domain mediates interaction with CTLH complex members.

    Article Snippet: Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on either 8% or 10% acrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat dry milk in Tris Buffered Saline with Tween 20 (TBS-T) or in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA), and hybridized with the following antibodies: c-Raf (C-12, 1:500, Santa Cruz), HA (HA-7, 1:1000, Sigma–Aldrich, St. Louis, MO, USA), RMND5A (NBP1–92337, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (I-19, 1:2000, Santa Cruz), RanBPM (5M, 1:2000, Bioacademia, Osaka, Japan), GST (B-14, 1:500, Santa Cruz), A-Raf (1:500, Cell Signaling Technology, Danvers, MA, USA, #4432), B-Raf (1:1000, Cell Signaling Technology #9434), phospho-T202/Y204-ERK1/2 (1:2000, Cell Signaling #4370), ERK1/2 (1:1000, Cell Signaling #9102), phospho-S217/221-MEK1/2 (1:2000, Cell Signaling #9154), MEK1/2 (1:1000, Cell Signaling #9122).

    Techniques: Expressing, In Vitro

    CTLH complex members RMND5A and RanBPM regulate c-Raf levels and cell proliferation. ( A ) RMND5A regulates endogenous c-Raf protein levels. Whole cell extracts from wild-type (WT) HEK293 cells and CRISPR KO RMND5A HEK293 cells untransfected (−) or transfected with pCGN-HA-RMND5A (+) were prepared and analyzed by Western blot. The top shows a representative analysis using c-Raf, HA (hemagglutinin), RMND5A, and β-actin antibodies to detect endogenous c-Raf, exogenous HA-RMND5A, endogenous RMND5A, and β-actin, respectively. Below, relative endogenous c-Raf protein levels were quantified by normalizing c-Raf to β-actin, and comparing values to wild-type HEK293 when set to a value of 1. Quantifications are shown with error bars indicating SD. p

    Journal: International Journal of Molecular Sciences

    Article Title: Regulation of c-Raf Stability through the CTLH Complex

    doi: 10.3390/ijms20040934

    Figure Lengend Snippet: CTLH complex members RMND5A and RanBPM regulate c-Raf levels and cell proliferation. ( A ) RMND5A regulates endogenous c-Raf protein levels. Whole cell extracts from wild-type (WT) HEK293 cells and CRISPR KO RMND5A HEK293 cells untransfected (−) or transfected with pCGN-HA-RMND5A (+) were prepared and analyzed by Western blot. The top shows a representative analysis using c-Raf, HA (hemagglutinin), RMND5A, and β-actin antibodies to detect endogenous c-Raf, exogenous HA-RMND5A, endogenous RMND5A, and β-actin, respectively. Below, relative endogenous c-Raf protein levels were quantified by normalizing c-Raf to β-actin, and comparing values to wild-type HEK293 when set to a value of 1. Quantifications are shown with error bars indicating SD. p

    Article Snippet: Samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on either 8% or 10% acrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% non-fat dry milk in Tris Buffered Saline with Tween 20 (TBS-T) or in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE, USA), and hybridized with the following antibodies: c-Raf (C-12, 1:500, Santa Cruz), HA (HA-7, 1:1000, Sigma–Aldrich, St. Louis, MO, USA), RMND5A (NBP1–92337, 1:300, Novus Biologicals, Littleton, CO, USA), β-actin (I-19, 1:2000, Santa Cruz), RanBPM (5M, 1:2000, Bioacademia, Osaka, Japan), GST (B-14, 1:500, Santa Cruz), A-Raf (1:500, Cell Signaling Technology, Danvers, MA, USA, #4432), B-Raf (1:1000, Cell Signaling Technology #9434), phospho-T202/Y204-ERK1/2 (1:2000, Cell Signaling #4370), ERK1/2 (1:1000, Cell Signaling #9102), phospho-S217/221-MEK1/2 (1:2000, Cell Signaling #9154), MEK1/2 (1:1000, Cell Signaling #9122).

    Techniques: CRISPR, Transfection, Western Blot