rabbit anti ksr1  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: praja2 regulates KSR1 stability and mitogenic signaling

doi: 10.1038/cddis.2016.109

Figure Lengend Snippet: praja2 interacts with KSR1. ( a ) Isolation of endogenous KSR1 and praja2 complex from lysates (2 mg) of HEK293 cells. ( b ) Schematic representation of the praja2 constructs used (upper diagram). HEK293 cells were transiently transfected with flag-praja2 (either wild-type, ring mutant (RM) or deletion mutants). Cells were treated for 12 h with MG132 (10 μ M) before harvesting. Twenty-four hours following transfection, cells were harvested and lysed. Lysates were subjected to immunoprecipitation with anti-KSR1 antibody. Precipitates were immunoblotted with anti-KSR1 and anti-flag antibodies (lower panels). ( c ) In vitro translated, [ 35 S]-labeled KSR1 was subjected to pull-down assays with purified GST or GST–praja2 fusion. ( d ) Spotted peptides (25 mers, 15mer overlap) of the human KSR1 sequence were overlaid with recombinant GST-praja2 (1–531) followed by immunoblotting with anti-GST antibody. The sequences (in red) refer to the praja2-binding domain of KSR1. The amino acids methionine and cysteine have been substituted with alanine or serine, respectively. ( e ) Schematic representation of the peptides used for pull-down experiments (upper panel). Lysates from flag-praja2-transfected cells were subjected to pull-down experiment with GST or GST-KSR1pep carrying the praja2-binding domain fused to GST (lower panels). ( f ) Predicted structure of KSR1 kinase domain modeled on KSR2 template PDB 2Y4I. The position of the potential praja2-binding domain is indicated. ( g ) HEK293 cells were subjected to double immunostaining with monoclonal anti-KSR1 and polyclonal anti-praja2 antibodies. Images were collected and analyzed by confocal microscopy. Pearson's coefficients between praja2 and KSR1

Article Snippet: The following primary antibodies were used: rabbit Praja2 (Bethyl Laboratories, Montgomery, TX, USA), mouse GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit KSR1 (Cell Signalling, Danvers, MA, USA), rabbit Phospho ERK (Thr202/Tyr204) (Cell Signalling), rabbit ERK2 (Santa Cruz) and rabbit ERK1 (Santa Cruz).

Techniques: Isolation, Construct, Transfection, Mutagenesis, Immunoprecipitation, In Vitro, Labeling, Purification, Sequencing, Recombinant, Western Blot, Binding Assay, Double Immunostaining, Confocal Microscopy

Journal: Cell Death & Disease

Article Title: praja2 regulates KSR1 stability and mitogenic signaling

doi: 10.1038/cddis.2016.109

Figure Lengend Snippet: praja2 ubiquitylates KSR1. ( a ) HEK293 cells were transfected with HA-ubiquitin and flag-praja2 or flag-praja2RM. Twenty-four hours after transfection, cells were treated with MG132 (20 μ M) for 8 h. Lysates were subjected to immunoprecipitations with anti-KSR1 and immunoblotted with anti-HA and anti-flag antibodies. ( b ) Cells were transfected with HA-ubiquitin, serum-deprived overnight and stimulated with EGF (100 ng/ml) for the indicated time points. Lysates were subjected to immunoprecipitations with anti-KSR1 and immunoblotted with anti-HA and anti-KSR1 antibodies. ( c ) Cells were transfected with HA-ubiquitin and flag-praja2 or flag-praja2RM and processed as in panel ( b ). ( d ) HEK293 cells were transfected with HA-ubiquitin and control (siRNAc) or SMARTpool siRNApraja2. Twenty-four hours after transfection, cells were either left untreated or stimulated with isoproterenol in the presence of MG132. Lysates were subjected to immunoprecipitations with anti-KSR1 and immunoblotted with anti-HA and anti-KSR1 antibodies. ( e ) Same as in panel ( d ), with the exception that the cells were stimulated with Fsk (40 μ M, 30 min). ( f ) In vitro -translated, 35 S-labeled KSR1 was incubated with anti-flag precipitates (flag-praja2 or flag-praja2RM) isolated from growing cells and his 6 -tagged ubiquitin, in the presence of E1 and UbcH5c (E2). The reaction mix was denatured, size-fractionated on SDS-PAGE and analyzed by autoradiography. A fraction of the reaction mixture was immunoblotted with anti-flag antibody (lower panel)

Article Snippet: The following primary antibodies were used: rabbit Praja2 (Bethyl Laboratories, Montgomery, TX, USA), mouse GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit KSR1 (Cell Signalling, Danvers, MA, USA), rabbit Phospho ERK (Thr202/Tyr204) (Cell Signalling), rabbit ERK2 (Santa Cruz) and rabbit ERK1 (Santa Cruz).

Techniques: Transfection, Ubiquitin Proteomics, Control, In Vitro, Labeling, Incubation, Isolation, SDS Page, Autoradiography

Journal: Cell Death & Disease

Article Title: praja2 regulates KSR1 stability and mitogenic signaling

doi: 10.1038/cddis.2016.109

Figure Lengend Snippet: praja2 decreases KSR1 levels. ( a ) Immunoblot of lysates from cells transiently transfected with Flag-praja2 vector (either wild type or RING mutant). ( b ) Quantitative analysis (mean±S.E.M) of three independent experiments shown in panel ( a ) ** P <0.01. ( c ) Cells were transfected with control (siRNAc) or SMARTpool siRNApraja2. Twenty-four hours after transfection, cells were either left untreated or stimulated with Fsk (40 μ M, 30 min). To prevent effects of Fsk on protein synthesis, cells were pretreated with cycloheximide. ( d ) Quantitative analysis of three independent experiments shown in panel ( e ). * P <0.05 versus control (siRNAc), untreated cells. ( e ) Immunoblot of lysates from cells transfected with flag-praja2 vector (either wild type or praja2 S342A,T389A ). Twenty-four hours after transfection, cells were either left untreated or stimulated with Fsk (40 μ M) and harvested at the indicated time points

Article Snippet: The following primary antibodies were used: rabbit Praja2 (Bethyl Laboratories, Montgomery, TX, USA), mouse GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit KSR1 (Cell Signalling, Danvers, MA, USA), rabbit Phospho ERK (Thr202/Tyr204) (Cell Signalling), rabbit ERK2 (Santa Cruz) and rabbit ERK1 (Santa Cruz).

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

Journal: Cell Death & Disease

Article Title: praja2 regulates KSR1 stability and mitogenic signaling

doi: 10.1038/cddis.2016.109

Figure Lengend Snippet: praja2 regulates ERK1/2 signaling. ( a ) HEK293 cells were transiently transfected with control (CMV) or Flag-praja2RM, serum deprived and then treated with EGF (100 ng/ml). Lysates were immunoblotted with the indicated antibodies. ( b ) Quantitative analysis of the experiments shown in panel ( a ). ( c ) HEK293 were transfected with Flag praja2RM mutant or Flag-praja2 RM Δ402–531 and treated with EGF for 5 and 15 min. Lysates were immunoblotted with the indicated antibodies. ( d ) Cells were transfected with control (siRNAc) or SMARTpool siRNApraja2, serum deprived and stimulated with EGF. Lysates were immunoblotted with the indicated antibodies. ( e ) Cells were transfected with control (siRNAc), SMARTpool siRNApraja2 or SMARTpool siRNApraja2 and siRNA-KSR1 and stimulated with EGF. Lysates were immunoblotted with the indicated antibodies. ( f ) Cells were transfected with pCMV vector or Flag-praja2 S342A,T389A . Twenty-four hours after transfection, cells were stimulated with Fsk (40 μ M) and harvested at the indicated time points. ( g ) Quantitative analysis of the experiments shown in panel ( e )

Article Snippet: The following primary antibodies were used: rabbit Praja2 (Bethyl Laboratories, Montgomery, TX, USA), mouse GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit KSR1 (Cell Signalling, Danvers, MA, USA), rabbit Phospho ERK (Thr202/Tyr204) (Cell Signalling), rabbit ERK2 (Santa Cruz) and rabbit ERK1 (Santa Cruz).

Techniques: Transfection, Control, Mutagenesis, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: praja2 regulates KSR1 stability and mitogenic signaling

doi: 10.1038/cddis.2016.109

Figure Lengend Snippet: Interfering with praja2-KSR1 complex enhances ERK signaling in ES and cancer cells. ( a ) Cells were pretreated for 8 h with the following synthetic peptides (1 μ M): scrambled-Flag and KSR1pep-Flag, renamed as go-onERK-Flag, given its role in ERK signaling (see below). Lysates were immunoprecipitated for KSR1. The precipitates were immunoblotted for praja2 and KSR1 (upper panel). Schematic representation of the scrambled and go-onERK peptides (lower panel). ( b ) Quantitative analysis of the experiments shown in panel ( a ). The data represent a mean of two independent experiments. ( c ) Cells pretreated (7 h) with the synthetic peptides (1 μ M) (scrambled-Flag and go-onERK-Flag) were left untreated or stimulated with EGF. Lysates were immunoblotted for phosphoERK and ERK. ( d ) Quantitative analysis of the experiments shown in panel ( c ). ( e ) MCF-7 cells were treated for 48 h with the scrambled-Flag peptide or with go-onERK-Flag peptide, harvested and counted. A mean of three independent experiments±S.E.M. is shown. ( f ) E14Tg2a mouse ESCs were grown on feeder-free, gelatin-coated plates as described and were induced to differentiate into EpiSCs in fibronectin-coated dishes at a density of 2.5 × 10 5 cells/cm 2 in the presence of 20 ng/ml Activin A and 12 ng/ml bFGF as described. Lysates from cells were immunoblotted with the indicated antibodies. ( g ) Undifferentiated ESCs were transfected with control (siRNAc) or SMARTpool siRNApraja2. Lysates were immunoblotted with the indicated antibodies. ( h ) Cells treated (1 and 7 h) with the synthetic peptides (scrambled-Flag and go-onERK-Flag-Flag) or with 12 ng/ml bFGF. Lysates were immunoblotted for the indicated antibodies. ( i ) ESCs were grown in undifferentiated conditions in the presence of LIF and serum (ESCs) or in EpiSC medium (containing Activin) for 48 h with bFGF (EpiSCs+bFGF) or without it (EpiSCs), in the presence of scrambled-Flag or go-onERK-Flag. Q-PCR analysis of Tbx3 and Nanog mRNAs, relative to GAPDH mRNA. The results are expressed as mean±S.E.M of three independent experiments (* P <0.05)

Article Snippet: The following primary antibodies were used: rabbit Praja2 (Bethyl Laboratories, Montgomery, TX, USA), mouse GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit KSR1 (Cell Signalling, Danvers, MA, USA), rabbit Phospho ERK (Thr202/Tyr204) (Cell Signalling), rabbit ERK2 (Santa Cruz) and rabbit ERK1 (Santa Cruz).

Techniques: Immunoprecipitation, Transfection, Control

Journal: British journal of cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1.

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Figure 1. Identification of KSR1-regulated phosphoproteome in breast cancer cells. (A) Experimental schematic outline of SILAC experiment. (B) Scatter plot comparison of phosphosite ratios quantified from control vs KSR1-overexpressed MCF7 cells. (C) Gene ontology (GO) Classification of the KSR1-regulated phosphoproteome in MCF7 cells according to molecular functions, biological processes and cellular compartmentalisation.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phosphoDBC1 Thr454 rabbit polyclonal (Cell Signaling) and b-actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Multiplex sample analysis, Comparison, Phospho-proteomics, Control

Journal: British journal of cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1.

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Figure 2. Effects of KSR1 on p53 transcriptional activity in the presence or absence of etoposide by luciferase assays. (A) MCF7 cells were transiently co-transfected with either pCMV6 (vector) or pCMV6-KSR1 plasmids in the presence of four individual p53-dependent promoter constructs expressing firefly luciferase genes (p53-R2, p53-AIP1, p53-CYCLIN G1 and p53-IGFBP3) following dimethylsulphoxide (DMSO) or etoposide (40 mM) treatment for 3 h. (B) MCF7 cells were transfected with control siRNA (siCT) or siKSR1 for 48 h, followed by transfection of three p53-dependent promoter constructs expressing firefly luciferase genes (p53-R2, p53-AIP1 and p53-CYCLIN G1) for additional 24 h. DMSO or etoposide (40 mM) were subsequently added as described above. Firefly luciferase activity was measured (renilla luciferase activity was used to normalise transfection efficiency). The normalised luciferase activity of empty vector is set as 1. Results shown are the average of at least three independent experiments and error bars represent s.d. Student’s t-test was performed using SPSS 16.0 statistical software (SPSS Inc.). (*Po0.05, **Po0.01).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phosphoDBC1 Thr454 rabbit polyclonal (Cell Signaling) and b-actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Construct, Expressing, Control, Software

Journal: British journal of cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1.

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Figure 3. Effects of KSR1 on p53 mRNA, total protein and neddylation levels and on p53 subcellular localisation. (A) Effects on p53 mRNA and total protein levels after KSR1 overexpression. MCF7 cells were transiently transfected with pCMV6 or pCMV6-KSR1 plasmids for 24 h. Subsequently, relative mRNA levels of TP53 and p53 total protein were measured by RT-qPCR and western blotting, respectively. Gene expression level from cells transfected with pCMV6 was set as 1. Results shown are the average of at least three independent experiments. Similarly, in MCF7 stably overexpressing KSR1 cells, p53 total protein was evaluated by western blot. Blots shown are representatives of at least three independent experiments. (B) Immunofluorescence staining of p53 cells after 24-h transfection with either pCMV6 or pCMV6-KSR1 plasmids in MCF7. p53 was detected with an anti-p53 antibody while the nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI). Representative pictures of three independent experiments are shown. Subcellular fractionation assays were performed after 24-h transfection with either pCMV6 or pCMV6-KSR1 plasmids in MCF7. Tubulin and histone deacetylase 1 (HDAC1) expression served as positive normalising control for cytoplasmic and nuclear proteins respectively. Blots shown are representatives of at least three independent experiments. (C) Neddylation assay on p53 after KSR1 overexpression. MCF7 cells were co-transfected with HA-NEDD8 and pCMV6 or pCMV6-KSR1 plasmids as indicated. p53 was immunoprecipitated using a p53-specific antibody (DO-1) and the neddylated-p53 was detected by immunoblotting using anti-NEDD8 and anti- p53-specific antibodies. Blots shown are representatives of at least three independent experiments. Abbreviations: IgG ¼ immunoglobulin G; IP ¼ immunoprecipitation.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phosphoDBC1 Thr454 rabbit polyclonal (Cell Signaling) and b-actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Western Blot, Gene Expression, Stable Transfection, Immunofluorescence, Staining, Fractionation, Histone Deacetylase Assay, Expressing, Control, Immunoprecipitation

Journal: British journal of cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1.

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Figure 4. Mechanisms of KSR1-regulated p53 transcriptional activity. (A) Effects on p53 acetylation and phosphorylation of DBC1 after KSR1 overexpression followed by etoposide treatment. MCF7 cells were transiently transfected with pCMV6 (vector) or pCMV6-KSR1 plasmids for 24 h. Subsequently, cells were treated with various concentrations of etoposide (20, 40, 80 mM, 3 h). p53 acetylation and DBC1 phosphorylation at Thr454 were assessed by immunoblotting with specific antibodies as indicated. (B) Effects on p53 acetylation and phosphorylation of DBC1 after KSR1 silencing followed by a titration of etoposide treatment. MCF7 cells were transfected with control siRNA (siCT) or siKSR1 for 72 h followed by etoposide treatment (20, 40, 80 mM, 3 h). p53 acetylation and DBC1 phosphorylation at Thr454 were assessed by immunoblotting with specific antibodies as indicated. (C) Effect of KSR1 on p53 acetylation is through DBC1. MCF7 cells were transfected with control siRNA (siCT) or siKSR1 in concordance with siCT or siDBC1 for 72h followed by etoposide treatment (40mM, 3 h). Acetylated p53, DBC1 and KSR1 protein levels were assessed by immunoblotting with specific antibodies as indicated. (D) Effect of KSR1 on DBC1 phosphorylation is dependent on its intact kinase domain. MCF7 cells were transiently transfected with vector, wild-type KSR1 or mutant KSR1 (R502M) plasmids for 24 h followed by etoposide treatment (40 mM, 3 h). DBC1 phosphorylation was measured by immunoblotting with specific antibody. (E) Interaction of DBC1 and SIRT1 after KSR1 overexpression with etoposide treatment by immunoprecipitation (IP). MCF7 cells were transiently transfected with pCMV6 or pCMV6-KSR1 plasmids for 24 h. Subsequently, cells were treated with etoposide (40 mM, 3 h). The interactions between SIRT1 and DBC1 were detected by IP of SIRT1 or DBC1 followed by immunoblotting with DBC1 and SIRT1 antibodies respectively. Blots shown are representatives of at least three independent experiments. Quantification of blots was analysed by ImageJ software (NIH, Bethesda, MD, USA). (F) Schematic model illustrating the role of KSR1 on p53 transcriptional activity in breast cancer cells with (i) basal or (ii) up-regulated levels of KSR1. Abbreviation: IgG ¼ immunoglobulin G.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phosphoDBC1 Thr454 rabbit polyclonal (Cell Signaling) and b-actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Activity Assay, Phospho-proteomics, Over Expression, Transfection, Plasmid Preparation, Western Blot, Titration, Control, Mutagenesis, Immunoprecipitation, Software

Journal: British journal of cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1.

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Figure 5. Effects of KSR1 silencing on breast cancer cell proliferation in vitro. SRB assays of MCF7, ZR75-1, SKBR3 and MDA231 cells after transfection with 20 nM of either siKSR1 or ‘non-targeting’ siRNA (control siRNA) or vehicle (Hiperfect) for 6 days. Error bars represent s.d. of three experiements each in quintuplicates (*Po0.05, compared with control siRNA at day 6; Student’s t-test).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phosphoDBC1 Thr454 rabbit polyclonal (Cell Signaling) and b-actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: In Vitro, Transfection, Control

Journal: British journal of cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1.

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Figure 6. KSR1 expression is altered in breast cancer tissues. Oncomine analysis was performed to examine KSR1 expression in breast normal and cancer tissues using online TCGA microarray data (www.oncomine.org).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phosphoDBC1 Thr454 rabbit polyclonal (Cell Signaling) and b-actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Expressing, Microarray