Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Raf-1 (E-10) , Santa Cruz Biotechnology , Cat# sc-7267; RRID: AB_628196.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Saline, Concentration Assay, Over Expression, Protein Extraction, Membrane, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Reverse Transcription, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

doi: 10.1111/j.1582-4934.2011.01520.x

Figure Lengend Snippet: Effects of Raf-1 knockdown on hESC cell morphology. (A) Immunofluorescence analysis of cytoskeleton by vimentin (green), phalloidin (red) and merge (green and red), 48 hrs after transfection of Co-hESC, Eu-hESC and Ec-hESC cells with siRNA are shown, scale bar line = 100 μm. (B) Average number of cells with contracted phenotype (estimated by two independent investigators) out of three biological replicates are given as% of cells with contracted phenotype relative to the total cell number (set to 100%), ** P < 0.005.

Article Snippet: Cells plated on fibronectin (Roche, Mannheim, Germany) were fixed in 4% Paraformaldehyde (PFA), permeabilized (0.02% Triton-X-100) and blocked with 0.2% gelatine before incubation with the primary antibodies: vimentin (rabbit; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Raf-1 (mouse monoclonal; BD Transduction Laboratories) and staining with appropriate Alexa Fluor-conjugated secondary antibody.

Techniques: Immunofluorescence, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

doi: 10.1111/j.1582-4934.2011.01520.x

Figure Lengend Snippet: Raf-1 regulates hESC morphology and migration. (A) Immunofluorescence analysis of cytoskeleton by phalloidin (red), Raf-1 cellular levels (green) and merge (green and red) 48 and 144 hrs after transfection of Eu-hESC cells with siRNA are shown on the left. Average number of cells with contracted phenotype (estimated by two independent investigators) out of three biological triplicates are given on the right as % of cells with contracted phenotype relative to the total cell number (set to 100%, ± S.D.). ** P < 0.005; scale bar line = 100 μm. (B) Effects of Raf-1 knockdown on cell migration in Eu-hESC 48 and 144 hrs after initial transfection with either control (transfection reagent only) or control siRNA or Raf-1 siRNA are given. Average values of biological triplicates are shown as percentage of migrating cells relative to the controls (mean value set to 100%), ** P < 0.005. (C) Raf-1 knockdown does not change the migration rate of Ec-hESC. A migration assay, 48 hrs after Raf-1 knockdown in Ec-hESC, was performed. Average values of biological triplicates are shown as percentage of migrating cells relative to the siRNA controls (mean value set to 100%).

Article Snippet: Cells plated on fibronectin (Roche, Mannheim, Germany) were fixed in 4% Paraformaldehyde (PFA), permeabilized (0.02% Triton-X-100) and blocked with 0.2% gelatine before incubation with the primary antibodies: vimentin (rabbit; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Raf-1 (mouse monoclonal; BD Transduction Laboratories) and staining with appropriate Alexa Fluor-conjugated secondary antibody.

Techniques: Migration, Immunofluorescence, Transfection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

doi: 10.1111/j.1582-4934.2011.01520.x

Figure Lengend Snippet: Cellular Raf-1 levels determine hESC motility. (A) Western blot analysis of Raf-1 expression levels in individual hESC cultures (numbered at the top of the immunoblot and corresponding to the sample ID number given in (for Ec-hESC; n = 7) and in for Eu-hESC; n = 7) is shown on the left and the graphical representation of the analysis on the right. The level of the protein is shown as optical density (OD) of the Western blot lanes normalized to the OD of α-tubulin. (B) Box plots of the data obtained using Western blot analysis pE/R/M (left panel; n = 8 per group) and total E/R/M levels (right panel; n = 8 per group) in Co-, Eu- and Ec-hESC are given. The levels of the proteins are shown as OD of the Western blot lanes normalized to the OD of β-actin. The corresponding P -values obtained after anova and Post hoc analysis are additionally inserted on the top of the box plots. (C) The ROCK II activity in Co-, Eu- and Ec-hESC is shown as average values of absorbance (OD 450 nm) determined in six biological replicates (mean ± S.D.) and normalized to the absorbance in Co-hESC. The statistical analysis of the data was performed with anova followed by Post-hoc test, * P < 0.05. (D) The analysis of cell migration in Co-, Eu- and Ec-hESC is given in per cent as average values of migrating cells from biological triplicates relative to that in Co-hESC (mean value set to 100%).

Article Snippet: Cells plated on fibronectin (Roche, Mannheim, Germany) were fixed in 4% Paraformaldehyde (PFA), permeabilized (0.02% Triton-X-100) and blocked with 0.2% gelatine before incubation with the primary antibodies: vimentin (rabbit; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Raf-1 (mouse monoclonal; BD Transduction Laboratories) and staining with appropriate Alexa Fluor-conjugated secondary antibody.

Techniques: Western Blot, Expressing, Activity Assay, Migration

Journal: Journal of Cellular and Molecular Medicine

Article Title: Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

doi: 10.1111/j.1582-4934.2011.01520.x

Figure Lengend Snippet: Raf-1 knockdown affects phosphorylation levels of downstream target proteins. (A) Total cell lysates (TCL) from Co- and Eu-hESC cells were analysed for Raf-1, pE/R/M, phospho-paxillin (pPax), E/R/M, pMYPT1, MYPT1 and α-tubulin, 48 hrs after their transfection with either Raf-1 or control siRNA. The Raf-1 and pPax levels were normalized by total α-tubulin. The pE/R/M and pMYPT1 levels were normalized by E/R/M and MYPT1 respectively. Representative blots from biological triplicates (left panel) and graphical representation of Raf-1, pE/R/M, pPax and pMYPT1 (right panel) are shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 100%) in cells transfected with control siRNA, ** P < 0.0001. (B) TCLs from Ec-hESC were analysed for Raf-1, pE/R/M, E/R/M, pPax, pMYPT1, MYPT1 and α-tubulin 48 hrs after their transfection with Raf-1 siRNA or control siRNA. The levels of the analysed proteins were normalized as described in (A). Representative blots from biological triplicates and graphical representation of Raf-1, pE/R/M, pMYPT1 and pPax as described in (A) are shown. ** P < 0.0001. (C) ROCK II activity in Raf-1 knockdown Ec-hESC is shown as average values of absorbance (OD 450 nm) determined in four biological replicates (mean ± S.D.) and normalized to the absorbance in siRNA controls (mean value set to 1), * P < 0.05. (D) TCL from Eu- and Ec-hESC were analysed for pMLC, 48 hrs after their transfection with Raf-1 siRNA or control siRNA. The pMLC levels were normalized by GAPDH. Representative blots from biological triplicates and graphical representation of pMLC are shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 100%) in cells transfected with control siRNA, * P < 0.05.

Article Snippet: Cells plated on fibronectin (Roche, Mannheim, Germany) were fixed in 4% Paraformaldehyde (PFA), permeabilized (0.02% Triton-X-100) and blocked with 0.2% gelatine before incubation with the primary antibodies: vimentin (rabbit; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Raf-1 (mouse monoclonal; BD Transduction Laboratories) and staining with appropriate Alexa Fluor-conjugated secondary antibody.

Techniques: Transfection, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis

doi: 10.1111/j.1582-4934.2011.01520.x

Figure Lengend Snippet: Western blot analysis of MYPT1 and E/R/M phosphorylation analysed 24 hrs after treatment of hESC with specific Raf-1 inhibitors (ZM336372 and GW5074 – 1 μM) is shown on the left. Representative blots from three independent experiments are given. Graphical representation of pMYPT1 (right panels) is shown as protein levels in% (mean value ± S.D.) relative to their normalized levels (mean value set to 1) in the non-treated controls, * P < 0.05.

Article Snippet: Cells plated on fibronectin (Roche, Mannheim, Germany) were fixed in 4% Paraformaldehyde (PFA), permeabilized (0.02% Triton-X-100) and blocked with 0.2% gelatine before incubation with the primary antibodies: vimentin (rabbit; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Raf-1 (mouse monoclonal; BD Transduction Laboratories) and staining with appropriate Alexa Fluor-conjugated secondary antibody.

Techniques: Western Blot