Structured Review

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Activation of Dictyostelium Ras. The FLAG-RasG or endogenous Ras activation level in response to cAMP was assayed by a GST-RBD pull-down assay (see Materials and methods). Cells were stimulated with cAMP for the indicated duration, and the amount of Ras protein bound to GST-RBD was determined by Western blotting with the indicated antibody. The activated Ras was quantified by densitometry and normalized with total Ras. Cells were treated with 50 μM <t>LY294002</t> or DMSO as a control for 20 min before the addition of cAMP (C). Similar results were observed over at least three independent experiments.
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Images

1) Product Images from "Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement"

Article Title: Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200406177

Activation of Dictyostelium Ras. The FLAG-RasG or endogenous Ras activation level in response to cAMP was assayed by a GST-RBD pull-down assay (see Materials and methods). Cells were stimulated with cAMP for the indicated duration, and the amount of Ras protein bound to GST-RBD was determined by Western blotting with the indicated antibody. The activated Ras was quantified by densitometry and normalized with total Ras. Cells were treated with 50 μM LY294002 or DMSO as a control for 20 min before the addition of cAMP (C). Similar results were observed over at least three independent experiments.
Figure Legend Snippet: Activation of Dictyostelium Ras. The FLAG-RasG or endogenous Ras activation level in response to cAMP was assayed by a GST-RBD pull-down assay (see Materials and methods). Cells were stimulated with cAMP for the indicated duration, and the amount of Ras protein bound to GST-RBD was determined by Western blotting with the indicated antibody. The activated Ras was quantified by densitometry and normalized with total Ras. Cells were treated with 50 μM LY294002 or DMSO as a control for 20 min before the addition of cAMP (C). Similar results were observed over at least three independent experiments.

Techniques Used: Activation Assay, Pull Down Assay, Western Blot

Feedback loop–mediated Ras/PI3K activation. (A) Fluorescent images of indicated GFP-protein expressing pten null cells before or 5 min after 50-μM LY294002 treatment or 25 min after 5 μM LatA treatment, and 20 min after the removal of LatA. (B) Fluorescent images of GFP-PhdA– and RasG Q61L -expressing rasG null cells.
Figure Legend Snippet: Feedback loop–mediated Ras/PI3K activation. (A) Fluorescent images of indicated GFP-protein expressing pten null cells before or 5 min after 50-μM LY294002 treatment or 25 min after 5 μM LatA treatment, and 20 min after the removal of LatA. (B) Fluorescent images of GFP-PhdA– and RasG Q61L -expressing rasG null cells.

Techniques Used: Activation Assay, Expressing

Spatial-temporal activation of Ras during chemotaxis. (A and B) The localization of GFP-RBD in wild-type vegetative cells (A) or aggregation-competent cells (B) was imaged. Translocation of GFP-RBD was imaged after stimulation with cAMP as described previously ( Funamoto et al., 2001 ). (C) Translocation kinetics of GFP-tagged PhdA, CRAC-PH, N-PI3K1, and RBD in wild-type cells were obtained from time-lapse recordings. The graphs represent an average of data of movies taken from at least three separate experiments. The fluorescence intensity of membrane-localized GFP fusion protein was quantitated as E t as defined in Materials and methods. (D) Translocation of indicated GFP proteins in pi3k1/2 null cells or wild-type cells treated with 50 μM LY294002 for 20 min before cAMP stimulation. (E–I) Fluorescent images of GFP-RBD expressing wild-type cells (E and F), pi3k1/2 null cells (G), or pten null and myr-PI3K expressing cells (I) chemotaxing in a gradient of chemoattractant. An asterisk indicates the position of the micropipette. Fluorescent images of GFP-RBD and myr-PI3K1–expressing pi3k1/2 null cells (H, left) or GFP-RBD-expressing pten null cells (H, right) are shown. The sites producing multiple pseudopodia are marked with arrowheads.
Figure Legend Snippet: Spatial-temporal activation of Ras during chemotaxis. (A and B) The localization of GFP-RBD in wild-type vegetative cells (A) or aggregation-competent cells (B) was imaged. Translocation of GFP-RBD was imaged after stimulation with cAMP as described previously ( Funamoto et al., 2001 ). (C) Translocation kinetics of GFP-tagged PhdA, CRAC-PH, N-PI3K1, and RBD in wild-type cells were obtained from time-lapse recordings. The graphs represent an average of data of movies taken from at least three separate experiments. The fluorescence intensity of membrane-localized GFP fusion protein was quantitated as E t as defined in Materials and methods. (D) Translocation of indicated GFP proteins in pi3k1/2 null cells or wild-type cells treated with 50 μM LY294002 for 20 min before cAMP stimulation. (E–I) Fluorescent images of GFP-RBD expressing wild-type cells (E and F), pi3k1/2 null cells (G), or pten null and myr-PI3K expressing cells (I) chemotaxing in a gradient of chemoattractant. An asterisk indicates the position of the micropipette. Fluorescent images of GFP-RBD and myr-PI3K1–expressing pi3k1/2 null cells (H, left) or GFP-RBD-expressing pten null cells (H, right) are shown. The sites producing multiple pseudopodia are marked with arrowheads.

Techniques Used: Activation Assay, Chemotaxis Assay, Translocation Assay, Fluorescence, Expressing

2) Product Images from "Novel Histone Deacetylase Inhibitors with Enhanced Enzymatic Inhibition Effects and Potent in vitro and in vivo Anti-tumor Activities"

Article Title: Novel Histone Deacetylase Inhibitors with Enhanced Enzymatic Inhibition Effects and Potent in vitro and in vivo Anti-tumor Activities

Journal: ChemMedChem

doi: 10.1002/cmdc.201300297

Western blot analysis of c-Raf, p-Erk, Erk, p-Akt, Akt and histone H3 in U937 and MDA-MB-231 cell line after 24h of treatment with compounds ( D3 and D17 ) at 1 µM. Histone H3 was used as a loading control.
Figure Legend Snippet: Western blot analysis of c-Raf, p-Erk, Erk, p-Akt, Akt and histone H3 in U937 and MDA-MB-231 cell line after 24h of treatment with compounds ( D3 and D17 ) at 1 µM. Histone H3 was used as a loading control.

Techniques Used: Western Blot, Multiple Displacement Amplification

3) Product Images from "Raf Kinase Inhibitory Protein is Required for Cerebellar Long-Term Synaptic Depression by Mediating PKC-Dependent MAPK Activation"

Article Title: Raf Kinase Inhibitory Protein is Required for Cerebellar Long-Term Synaptic Depression by Mediating PKC-Dependent MAPK Activation

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.2812-12.2012

Interaction of Raf-1 and MEK with RKIP, and localization of Raf-1 and MEK in cerebellar slices. A , Immunoblot analysis of coimmunoprecipitated Raf-1, coimmunoprecipitated MEK, and precipitated RKIP in lysates from control cerebellar slices. B–E , Confocal images of cerebellar slices double stained with antibodies against calbindin (green) and Raf-1 (red; B , D ), or with antibodies against calbindin (green) and MEK (red; C , E ). Areas within the white squares shown in B and C are magnified in D and E , respectively.
Figure Legend Snippet: Interaction of Raf-1 and MEK with RKIP, and localization of Raf-1 and MEK in cerebellar slices. A , Immunoblot analysis of coimmunoprecipitated Raf-1, coimmunoprecipitated MEK, and precipitated RKIP in lysates from control cerebellar slices. B–E , Confocal images of cerebellar slices double stained with antibodies against calbindin (green) and Raf-1 (red; B , D ), or with antibodies against calbindin (green) and MEK (red; C , E ). Areas within the white squares shown in B and C are magnified in D and E , respectively.

Techniques Used: Staining

4) Product Images from "Synthetic Lethality of Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic Efficacy"

Article Title: Synthetic Lethality of Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic Efficacy

Journal: Cancer cell

doi: 10.1016/j.ccell.2017.11.003

Bcl-2 Inhibition Reciprocally Overcomes Resistance to p53 Activation by Shifting Cellular Response from G 1 Arrest to Apoptosis (A) Apoptosis percentage of MV-4-11, MOLM-13, and OCI-AML3 cells after treatment with indicated concentrations of RG for 12, 24, or 48 h. (B) Relative mRNA expression of CDKN1A (p21-encoding gene) in OCI-AML3 cells after 12 hr treatment with 1 μM RG. (C) Immunoblots of p53 and p21 in OCI-AML3 cells after 24 hr treatment with indicated concentrations of RG. (D) The cell numbers and viability of OCI-AML3 cells treated with 1 μM RG as indicated. Cells were analyzed with Vi-CELL viability analyzer (Trypan blue exclusion assay). (E) Immunoblots showing stable p21 knockdown in OCI-AML3 cells. Due to the low basal expression of p21, the control (shC) or knockdown cells were treated with 1 μM RG for 12 hr to induce p21 prior to immunoblot analysis. (F) Apoptosis of the control and p21 knockdown OCI-AML3 cells after 48 hr treatment with indicated concentrations of RG. (G) Flow cytometry plots showing the gating strategy to simultaneously analyze apoptosis and cell cycle distribution. The absolute cell numbers were enumerated using CountBright counting beads. (H) Representative flow cytometry plots of OCI-AML3 cells after 24 hr treatment. The gating strategy was the same as in (G). (I) Apoptosis and cell cycle analysis of OCI-AML3 cells treated with RG in the absence or presence of ABT for 24 hr. The gating strategy was the same as in (G). (J) Cleavage of caspase-3, -9 and PARP-1 in response to 24 hr treatment with DMSO (vehicle), 1 μM RG, 1 μM ABT, or the ABT/RG combination. c-, cleaved. .
Figure Legend Snippet: Bcl-2 Inhibition Reciprocally Overcomes Resistance to p53 Activation by Shifting Cellular Response from G 1 Arrest to Apoptosis (A) Apoptosis percentage of MV-4-11, MOLM-13, and OCI-AML3 cells after treatment with indicated concentrations of RG for 12, 24, or 48 h. (B) Relative mRNA expression of CDKN1A (p21-encoding gene) in OCI-AML3 cells after 12 hr treatment with 1 μM RG. (C) Immunoblots of p53 and p21 in OCI-AML3 cells after 24 hr treatment with indicated concentrations of RG. (D) The cell numbers and viability of OCI-AML3 cells treated with 1 μM RG as indicated. Cells were analyzed with Vi-CELL viability analyzer (Trypan blue exclusion assay). (E) Immunoblots showing stable p21 knockdown in OCI-AML3 cells. Due to the low basal expression of p21, the control (shC) or knockdown cells were treated with 1 μM RG for 12 hr to induce p21 prior to immunoblot analysis. (F) Apoptosis of the control and p21 knockdown OCI-AML3 cells after 48 hr treatment with indicated concentrations of RG. (G) Flow cytometry plots showing the gating strategy to simultaneously analyze apoptosis and cell cycle distribution. The absolute cell numbers were enumerated using CountBright counting beads. (H) Representative flow cytometry plots of OCI-AML3 cells after 24 hr treatment. The gating strategy was the same as in (G). (I) Apoptosis and cell cycle analysis of OCI-AML3 cells treated with RG in the absence or presence of ABT for 24 hr. The gating strategy was the same as in (G). (J) Cleavage of caspase-3, -9 and PARP-1 in response to 24 hr treatment with DMSO (vehicle), 1 μM RG, 1 μM ABT, or the ABT/RG combination. c-, cleaved. .

Techniques Used: Inhibition, Activation Assay, Expressing, Western Blot, Trypan Blue Exclusion Assay, Flow Cytometry, Cytometry, Cell Cycle Assay

5) Product Images from "A G1‐like state allows HIV‐1 to bypass SAMHD1 restriction in macrophages"

Article Title: A G1‐like state allows HIV‐1 to bypass SAMHD1 restriction in macrophages

Journal: The EMBO Journal

doi: 10.15252/embj.201696025

Bidirectional transitions shape SAMHD 1‐mediated restriction of HIV ‐1 MDM from three donors (D1, D2, D3) were used for immunoblotting to detect SAMHD1 and CDK proteins. The blot for D1 is the same as that in Fig 1 G in order to facilitate comparison of different cell cycle‐associated proteins and SAMHD1. MDM were co‐infected with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing vpx (VLP‐vpx). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). MDM were transfected with control or pool of SAMHD1 siRNAs and infected 3 days later with VSV‐G‐pseudotyped HIV‐1 GFP. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). Experimental approach used to model MDM bidirectional G0–G1‐like transitions. MDM were as follows: (unstim) cultured in HS or (stim) cultured in FCS as described in Materials and Methods ; [stim (day 3)] grown in HS conditions for 3 days and changed to stimulating FCS conditions for 3 days; [unstim (3 days)] grown in stimulating FCS condition for the 3 days and changed to non‐stimulating HS for the remaining 3 days. Single round of infection of MDM with full‐length HIV‐1 BaL. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins. Graph is a representative example of n ≥ 3, mean ± s.e.m. Proposed signalling pathway leading to SAMHD1 phosphorylation in stimulated MDM. Unstimulated (G) and stimulated (H) MDM were treated with inhibitors of RAF (2 μM), B‐RAF (3 μM), MEK1/2 (AS‐703026, 1 μM), JAK 1–3 (1 μM), GSK3 (2 μM), PIM 1–3 (3 μM) and CDK4/6 (1 μM) for 18 h before infection and infected with VSV‐G HIV‐1 GFP. The percentage of infected cells was quantified by FACS 48 h post‐infection. Graphs are representative example of n ≥ 3, mean ± s.e.m., *P‐ value ≤ 0.05; **P‐ value ≤ 0.01; calculated from triplicates, unpaired t ‐test). MDM were treated with a MEK/ERK inhibitor (U0126, 10 μM) for 18 h before infection and where indicated VLP‐vpx was added at the time of infection. Percentage of infected cells were detected by FACS 48 h post‐infection. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins ( n = 3, mean ± s.e.m.; (ns) non‐significant; ***P‐ value ≤ 0.001, unpaired t ‐test). Source data are available online for this figure.
Figure Legend Snippet: Bidirectional transitions shape SAMHD 1‐mediated restriction of HIV ‐1 MDM from three donors (D1, D2, D3) were used for immunoblotting to detect SAMHD1 and CDK proteins. The blot for D1 is the same as that in Fig 1 G in order to facilitate comparison of different cell cycle‐associated proteins and SAMHD1. MDM were co‐infected with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing vpx (VLP‐vpx). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). MDM were transfected with control or pool of SAMHD1 siRNAs and infected 3 days later with VSV‐G‐pseudotyped HIV‐1 GFP. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). Experimental approach used to model MDM bidirectional G0–G1‐like transitions. MDM were as follows: (unstim) cultured in HS or (stim) cultured in FCS as described in Materials and Methods ; [stim (day 3)] grown in HS conditions for 3 days and changed to stimulating FCS conditions for 3 days; [unstim (3 days)] grown in stimulating FCS condition for the 3 days and changed to non‐stimulating HS for the remaining 3 days. Single round of infection of MDM with full‐length HIV‐1 BaL. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins. Graph is a representative example of n ≥ 3, mean ± s.e.m. Proposed signalling pathway leading to SAMHD1 phosphorylation in stimulated MDM. Unstimulated (G) and stimulated (H) MDM were treated with inhibitors of RAF (2 μM), B‐RAF (3 μM), MEK1/2 (AS‐703026, 1 μM), JAK 1–3 (1 μM), GSK3 (2 μM), PIM 1–3 (3 μM) and CDK4/6 (1 μM) for 18 h before infection and infected with VSV‐G HIV‐1 GFP. The percentage of infected cells was quantified by FACS 48 h post‐infection. Graphs are representative example of n ≥ 3, mean ± s.e.m., *P‐ value ≤ 0.05; **P‐ value ≤ 0.01; calculated from triplicates, unpaired t ‐test). MDM were treated with a MEK/ERK inhibitor (U0126, 10 μM) for 18 h before infection and where indicated VLP‐vpx was added at the time of infection. Percentage of infected cells were detected by FACS 48 h post‐infection. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins ( n = 3, mean ± s.e.m.; (ns) non‐significant; ***P‐ value ≤ 0.001, unpaired t ‐test). Source data are available online for this figure.

Techniques Used: Infection, FACS, Transfection, Cell Culture

6) Product Images from "PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer"

Article Title: PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.10427

Schematic diagram illustrates the roles of PATZ-1 and PP4R2 in regulating the effects of growth factors and PGE2 on IKK/NF-κB signaling, COX-2, Snail and migration/invasion of lung cancer
Figure Legend Snippet: Schematic diagram illustrates the roles of PATZ-1 and PP4R2 in regulating the effects of growth factors and PGE2 on IKK/NF-κB signaling, COX-2, Snail and migration/invasion of lung cancer

Techniques Used: Migration

Knockdown of PP4R2 decreases the association of PP4C to phospho-IKK and enhances IKK/NF-κB signaling, COX-2 and Snail Lung cancer cells (A549) were transfected with PP4R2 siRNA ( siPP4R2 ) or PP4R1 siRNA ( siPP4R1 ) for 48 h and then treated with PGE2 (20 μg/mL) for various durations as indicated. A and B. Cells were examined by immunoblotting for protein expression. C. The cell extracts were immunoprecipitated with anti-phospho-IKKα/β S176/180 or control IgGs antibodies, and the pull-down proteins were detected by immunoblotting (top panel).
Figure Legend Snippet: Knockdown of PP4R2 decreases the association of PP4C to phospho-IKK and enhances IKK/NF-κB signaling, COX-2 and Snail Lung cancer cells (A549) were transfected with PP4R2 siRNA ( siPP4R2 ) or PP4R1 siRNA ( siPP4R1 ) for 48 h and then treated with PGE2 (20 μg/mL) for various durations as indicated. A and B. Cells were examined by immunoblotting for protein expression. C. The cell extracts were immunoprecipitated with anti-phospho-IKKα/β S176/180 or control IgGs antibodies, and the pull-down proteins were detected by immunoblotting (top panel).

Techniques Used: Transfection, Expressing, Immunoprecipitation

Knockdown of PATZ1 decreases PP4R2 but not PP4C and increases IKK/IkB/NF-kB signaling in the late phase of PGE2 stimulation Lung cancer cells (A549) were transfected with A. Control scrambled siRNA ( siCont ), B. Empty vector, wild type IKK, dominant negative IKK and C. PATZ1 siRNA ( siPATZ1 ) for 48 h and then treated with PGE2 (20 μg/mL) for various durations as indicated. Cells were examined by immunoblotting for protein expression.
Figure Legend Snippet: Knockdown of PATZ1 decreases PP4R2 but not PP4C and increases IKK/IkB/NF-kB signaling in the late phase of PGE2 stimulation Lung cancer cells (A549) were transfected with A. Control scrambled siRNA ( siCont ), B. Empty vector, wild type IKK, dominant negative IKK and C. PATZ1 siRNA ( siPATZ1 ) for 48 h and then treated with PGE2 (20 μg/mL) for various durations as indicated. Cells were examined by immunoblotting for protein expression.

Techniques Used: Transfection, Plasmid Preparation, Dominant Negative Mutation, Expressing

Overexpression of PATZ1 attenuates the effects of growth factors and PGE2 on phospho-NF-κB, COX-2, Snail, EMT and MMP-2 activity as well as migration/invasion of the lung cancer cells Lung cancer cells (A549 and CL1-5) were transfected either with control empty vector (EV) or pCMV6- PATZ1 ( pPATZ1 ) for 24 h and then treated for 24 h with 20 μg/ml of PGE2 or 100 ng/ml growth factors as indicated. A. The cell lysates were examined by immunoblotting for protein expression. PATZ1, phospho-IKKα/β S176/180 , IKKα/β, phospho-NF-κB p65 S536 and NF-κB were measured at 1 h, all the others were determined at 24 h after PGE2 and growth factor treatment. B and C. The migration and invasion abilities of cells were examined by transwell assays after 20 h incubation. The activities of MMP-2 in cell-conditioned media were analyzed by gelatin zymography after culturing cells in serum-free medium for 24 h. Blots are representative of three independent experiments. Data represent means ± s.d. of three independent experiments; * P
Figure Legend Snippet: Overexpression of PATZ1 attenuates the effects of growth factors and PGE2 on phospho-NF-κB, COX-2, Snail, EMT and MMP-2 activity as well as migration/invasion of the lung cancer cells Lung cancer cells (A549 and CL1-5) were transfected either with control empty vector (EV) or pCMV6- PATZ1 ( pPATZ1 ) for 24 h and then treated for 24 h with 20 μg/ml of PGE2 or 100 ng/ml growth factors as indicated. A. The cell lysates were examined by immunoblotting for protein expression. PATZ1, phospho-IKKα/β S176/180 , IKKα/β, phospho-NF-κB p65 S536 and NF-κB were measured at 1 h, all the others were determined at 24 h after PGE2 and growth factor treatment. B and C. The migration and invasion abilities of cells were examined by transwell assays after 20 h incubation. The activities of MMP-2 in cell-conditioned media were analyzed by gelatin zymography after culturing cells in serum-free medium for 24 h. Blots are representative of three independent experiments. Data represent means ± s.d. of three independent experiments; * P

Techniques Used: Over Expression, Activity Assay, Migration, Transfection, Plasmid Preparation, Expressing, Incubation, Zymography

Association of PP4R2 and PP4C with phospho-IKK correlates with decline in IKK/NF-κB signaling, COX-2 and Snail in the late phase of PGE2 stimulation A and B. Lung cancer cells (A549) were treated with PGE2 (20 μg/ml) for various durations and the proteins of the treated cells were examined by immunoblotting as indicated. C. The cell extracts were immunoprecipitated with anti-phospho-IKKα/β S176/180 or control IgGs antibodies and the pull-down proteins were detected by immunoblotting. β–actin was used as a loading control. Blots are representative of three independent experiments.
Figure Legend Snippet: Association of PP4R2 and PP4C with phospho-IKK correlates with decline in IKK/NF-κB signaling, COX-2 and Snail in the late phase of PGE2 stimulation A and B. Lung cancer cells (A549) were treated with PGE2 (20 μg/ml) for various durations and the proteins of the treated cells were examined by immunoblotting as indicated. C. The cell extracts were immunoprecipitated with anti-phospho-IKKα/β S176/180 or control IgGs antibodies and the pull-down proteins were detected by immunoblotting. β–actin was used as a loading control. Blots are representative of three independent experiments.

Techniques Used: Immunoprecipitation

7) Product Images from "Xentry, a new class of cell-penetrating peptide uniquely equipped for delivery of drugs"

Article Title: Xentry, a new class of cell-penetrating peptide uniquely equipped for delivery of drugs

Journal: Scientific Reports

doi: 10.1038/srep01661

Xentry-mediated delivery of antibody and siRNA cargoes into cells. (a) Uptake of Xentry-conjugated and unconjugated FITC-labelled rabbit IgG by HepG2 cells. Cell nuclei were stained blue with DAPI. (b) Representative cell showing staining of tubulin by a Cy3-labelled anti-β-tubulin antibody delivered to HepG2 cells by Xentry. HepG2 cells were incubated with the Xentry- anti-β-tubulin antibody conjugate for 1 h, washed, then incubated and photographed 24 h later. For comparison, a representative fixed and permeabilized cell is shown which has been stained by the free anti-β-tubulin antibody. The unconjugated anti-β-tubulin antibody could not penetrate and stain non-permeabilized HepG2 cells. (c) The abilities of a Xentry-conjugated and unconjugated anti-B-raf antibody to induce the apoptosis of WM-266-4 melanoma cells, as evidenced by staining of cells with annexin-V fluos (green). (d) A Xentry-KALA fusion peptide delivers an siRNA directed against B-raf transcripts into WM-266-4 melanoma cells, causing cell apoptosis as evidenced by staining of cells with annexin-V fluos (green). For comparison, the anti-B-raf siRNA was transfected into cells using the PolyMag agent. The siRNA was omitted from controls. Scale bar, 50 μm.
Figure Legend Snippet: Xentry-mediated delivery of antibody and siRNA cargoes into cells. (a) Uptake of Xentry-conjugated and unconjugated FITC-labelled rabbit IgG by HepG2 cells. Cell nuclei were stained blue with DAPI. (b) Representative cell showing staining of tubulin by a Cy3-labelled anti-β-tubulin antibody delivered to HepG2 cells by Xentry. HepG2 cells were incubated with the Xentry- anti-β-tubulin antibody conjugate for 1 h, washed, then incubated and photographed 24 h later. For comparison, a representative fixed and permeabilized cell is shown which has been stained by the free anti-β-tubulin antibody. The unconjugated anti-β-tubulin antibody could not penetrate and stain non-permeabilized HepG2 cells. (c) The abilities of a Xentry-conjugated and unconjugated anti-B-raf antibody to induce the apoptosis of WM-266-4 melanoma cells, as evidenced by staining of cells with annexin-V fluos (green). (d) A Xentry-KALA fusion peptide delivers an siRNA directed against B-raf transcripts into WM-266-4 melanoma cells, causing cell apoptosis as evidenced by staining of cells with annexin-V fluos (green). For comparison, the anti-B-raf siRNA was transfected into cells using the PolyMag agent. The siRNA was omitted from controls. Scale bar, 50 μm.

Techniques Used: Staining, Incubation, Transfection

8) Product Images from "Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation"

Article Title: Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01633-13

KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.
Figure Legend Snippet: KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.

Techniques Used: Expressing, Binding Assay, Plasmid Preparation, Immunoprecipitation, Proximity Ligation Assay, Negative Control, Generated, Fluorescence

The KSR1–caveolin-1 interaction promotes EGF-stimulated ERK1/2 activation. (A) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated for caveolin-1, and the immunoprecipitates were probed for MEK1/2 and ERK1/2 to assess the KSR1-MEK1/2 and KSR1-ERK1/2 interaction. (B) WCLs from 293T cells transfected with vector, FLAG-tagged KSR1, or FLAG-tagged CBM were immunoprecipitated with anti-FLAG antibodies and subjected to Western blotting for B-Raf and c-Raf to assess KSR1–B-Raf and KSR1–c-Raf interactions. The arrow denotes the band specific for c-Raf. The asterisk indicates s a nonspecific band. (C) Triplicate wells of immortalized KSR1 −/− MEFs expressing either KSR1 or KSR1.CBM were treated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation levels were determined in situ for ERK1 and phospho-ERK1/2 with a Li-Cor Odyssey system. Data are expressed as the ratio of phospho-ERK1/2 to ERK1. Data are expressed as means ± standard deviations from three independent experiments. ****, P
Figure Legend Snippet: The KSR1–caveolin-1 interaction promotes EGF-stimulated ERK1/2 activation. (A) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated for caveolin-1, and the immunoprecipitates were probed for MEK1/2 and ERK1/2 to assess the KSR1-MEK1/2 and KSR1-ERK1/2 interaction. (B) WCLs from 293T cells transfected with vector, FLAG-tagged KSR1, or FLAG-tagged CBM were immunoprecipitated with anti-FLAG antibodies and subjected to Western blotting for B-Raf and c-Raf to assess KSR1–B-Raf and KSR1–c-Raf interactions. The arrow denotes the band specific for c-Raf. The asterisk indicates s a nonspecific band. (C) Triplicate wells of immortalized KSR1 −/− MEFs expressing either KSR1 or KSR1.CBM were treated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation levels were determined in situ for ERK1 and phospho-ERK1/2 with a Li-Cor Odyssey system. Data are expressed as the ratio of phospho-ERK1/2 to ERK1. Data are expressed as means ± standard deviations from three independent experiments. ****, P

Techniques Used: Activation Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection, Western Blot, In Situ

9) Product Images from "A G1‐like state allows HIV‐1 to bypass SAMHD1 restriction in macrophages"

Article Title: A G1‐like state allows HIV‐1 to bypass SAMHD1 restriction in macrophages

Journal: The EMBO Journal

doi: 10.15252/embj.201696025

MDM can be manipulated to transition between G0‐ and a G1‐like phase Ingenuity pathway interaction analysis of nuclear proteins that show significant transcriptional upregulation in stimulated MDM compared to unstimulated MDM. The figure demonstrates a single dominant cluster of interacting proteins. Example of acquisition by Hermes WiScan cell‐imaging system, an automated microscopic platform. MDM were labelled for nuclei, MCM2 and active DNA synthesis using Click‐iT® EdU Alexa Fluor® 488 Imaging Kit. Scale bars: 10 μm. MDM were treated for 2, 4 and 50 h with 10 μM EdU to detect active DNA synthesis using Click‐iT® EdU Alexa Fluor® 488 Imaging Kit. The percentage of EdU‐positive cells was quantified by FACS. Quantification of cell cycle phases from PI labelling. dNTP levels in stimulated and unstimulated MDM. MDM differentiated and cultured in RPMI complemented with MCSF and 10% human serum for 7 days were changed into stimulatory medium (10% FCS) and cultured for additional 3 days. Cells were infected with VSV‐G‐pseudotyped HIV‐1 GFP and the percentage of infected cells detected 48 h post‐infection by FACS. MDM were treated with the CDK4/6 inhibitor Palbociclib (1 μM) 18 h before infection and infected with VSV‐G‐pseudotyped GFP virus; VLP‐vpx was added at the time of infection. Cells from this experiment were lysed and used for immunoblotting. Data information: Cells in each experiment were recorded and analysed using Hermes WiScan cell‐imaging system, ImageJ or FACS ( n ≥ 3, mean ± s.e.m.).
Figure Legend Snippet: MDM can be manipulated to transition between G0‐ and a G1‐like phase Ingenuity pathway interaction analysis of nuclear proteins that show significant transcriptional upregulation in stimulated MDM compared to unstimulated MDM. The figure demonstrates a single dominant cluster of interacting proteins. Example of acquisition by Hermes WiScan cell‐imaging system, an automated microscopic platform. MDM were labelled for nuclei, MCM2 and active DNA synthesis using Click‐iT® EdU Alexa Fluor® 488 Imaging Kit. Scale bars: 10 μm. MDM were treated for 2, 4 and 50 h with 10 μM EdU to detect active DNA synthesis using Click‐iT® EdU Alexa Fluor® 488 Imaging Kit. The percentage of EdU‐positive cells was quantified by FACS. Quantification of cell cycle phases from PI labelling. dNTP levels in stimulated and unstimulated MDM. MDM differentiated and cultured in RPMI complemented with MCSF and 10% human serum for 7 days were changed into stimulatory medium (10% FCS) and cultured for additional 3 days. Cells were infected with VSV‐G‐pseudotyped HIV‐1 GFP and the percentage of infected cells detected 48 h post‐infection by FACS. MDM were treated with the CDK4/6 inhibitor Palbociclib (1 μM) 18 h before infection and infected with VSV‐G‐pseudotyped GFP virus; VLP‐vpx was added at the time of infection. Cells from this experiment were lysed and used for immunoblotting. Data information: Cells in each experiment were recorded and analysed using Hermes WiScan cell‐imaging system, ImageJ or FACS ( n ≥ 3, mean ± s.e.m.).

Techniques Used: Imaging, DNA Synthesis, FACS, Cell Culture, Infection

Bidirectional transitions shape SAMHD 1‐mediated restriction of HIV ‐1 MDM from three donors (D1, D2, D3) were used for immunoblotting to detect SAMHD1 and CDK proteins. The blot for D1 is the same as that in Fig 1 G in order to facilitate comparison of different cell cycle‐associated proteins and SAMHD1. MDM were co‐infected with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing vpx (VLP‐vpx). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). MDM were transfected with control or pool of SAMHD1 siRNAs and infected 3 days later with VSV‐G‐pseudotyped HIV‐1 GFP. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). Experimental approach used to model MDM bidirectional G0–G1‐like transitions. MDM were as follows: (unstim) cultured in HS or (stim) cultured in FCS as described in Materials and Methods ; [stim (day 3)] grown in HS conditions for 3 days and changed to stimulating FCS conditions for 3 days; [unstim (3 days)] grown in stimulating FCS condition for the 3 days and changed to non‐stimulating HS for the remaining 3 days. Single round of infection of MDM with full‐length HIV‐1 BaL. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins. Graph is a representative example of n ≥ 3, mean ± s.e.m. Proposed signalling pathway leading to SAMHD1 phosphorylation in stimulated MDM. Unstimulated (G) and stimulated (H) MDM were treated with inhibitors of RAF (2 μM), B‐RAF (3 μM), MEK1/2 (AS‐703026, 1 μM), JAK 1–3 (1 μM), GSK3 (2 μM), PIM 1–3 (3 μM) and CDK4/6 (1 μM) for 18 h before infection and infected with VSV‐G HIV‐1 GFP. The percentage of infected cells was quantified by FACS 48 h post‐infection. Graphs are representative example of n ≥ 3, mean ± s.e.m., *P‐ value ≤ 0.05; **P‐ value ≤ 0.01; calculated from triplicates, unpaired t ‐test). MDM were treated with a MEK/ERK inhibitor (U0126, 10 μM) for 18 h before infection and where indicated VLP‐vpx was added at the time of infection. Percentage of infected cells were detected by FACS 48 h post‐infection. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins ( n = 3, mean ± s.e.m.; (ns) non‐significant; ***P‐ value ≤ 0.001, unpaired t ‐test). Source data are available online for this figure.
Figure Legend Snippet: Bidirectional transitions shape SAMHD 1‐mediated restriction of HIV ‐1 MDM from three donors (D1, D2, D3) were used for immunoblotting to detect SAMHD1 and CDK proteins. The blot for D1 is the same as that in Fig 1 G in order to facilitate comparison of different cell cycle‐associated proteins and SAMHD1. MDM were co‐infected with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing vpx (VLP‐vpx). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). MDM were transfected with control or pool of SAMHD1 siRNAs and infected 3 days later with VSV‐G‐pseudotyped HIV‐1 GFP. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by FACS 48 h post‐infection ( n = 2, mean ± s.e.m.; **P‐ value ≤ 0.01; (ns) non‐significant, unpaired t ‐test). Experimental approach used to model MDM bidirectional G0–G1‐like transitions. MDM were as follows: (unstim) cultured in HS or (stim) cultured in FCS as described in Materials and Methods ; [stim (day 3)] grown in HS conditions for 3 days and changed to stimulating FCS conditions for 3 days; [unstim (3 days)] grown in stimulating FCS condition for the 3 days and changed to non‐stimulating HS for the remaining 3 days. Single round of infection of MDM with full‐length HIV‐1 BaL. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins. Graph is a representative example of n ≥ 3, mean ± s.e.m. Proposed signalling pathway leading to SAMHD1 phosphorylation in stimulated MDM. Unstimulated (G) and stimulated (H) MDM were treated with inhibitors of RAF (2 μM), B‐RAF (3 μM), MEK1/2 (AS‐703026, 1 μM), JAK 1–3 (1 μM), GSK3 (2 μM), PIM 1–3 (3 μM) and CDK4/6 (1 μM) for 18 h before infection and infected with VSV‐G HIV‐1 GFP. The percentage of infected cells was quantified by FACS 48 h post‐infection. Graphs are representative example of n ≥ 3, mean ± s.e.m., *P‐ value ≤ 0.05; **P‐ value ≤ 0.01; calculated from triplicates, unpaired t ‐test). MDM were treated with a MEK/ERK inhibitor (U0126, 10 μM) for 18 h before infection and where indicated VLP‐vpx was added at the time of infection. Percentage of infected cells were detected by FACS 48 h post‐infection. Cells were used for immunoblotting to detect CDKs, SAMHD1 and MCM2 proteins ( n = 3, mean ± s.e.m.; (ns) non‐significant; ***P‐ value ≤ 0.001, unpaired t ‐test). Source data are available online for this figure.

Techniques Used: Infection, FACS, Transfection, Cell Culture

10) Product Images from "Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation"

Article Title: Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01633-13

KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.
Figure Legend Snippet: KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.

Techniques Used: Expressing, Binding Assay, Plasmid Preparation, Immunoprecipitation, Proximity Ligation Assay, Negative Control, Generated, Fluorescence

The KSR1–caveolin-1 interaction promotes EGF-stimulated ERK1/2 activation. (A) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated for caveolin-1, and the immunoprecipitates were probed for MEK1/2 and ERK1/2 to assess the KSR1-MEK1/2 and KSR1-ERK1/2 interaction. (B) WCLs from 293T cells transfected with vector, FLAG-tagged KSR1, or FLAG-tagged CBM were immunoprecipitated with anti-FLAG antibodies and subjected to Western blotting for B-Raf and c-Raf to assess KSR1–B-Raf and KSR1–c-Raf interactions. The arrow denotes the band specific for c-Raf. The asterisk indicates s a nonspecific band. (C) Triplicate wells of immortalized KSR1 −/− MEFs expressing either KSR1 or KSR1.CBM were treated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation levels were determined in situ for ERK1 and phospho-ERK1/2 with a Li-Cor Odyssey system. Data are expressed as the ratio of phospho-ERK1/2 to ERK1. Data are expressed as means ± standard deviations from three independent experiments. ****, P
Figure Legend Snippet: The KSR1–caveolin-1 interaction promotes EGF-stimulated ERK1/2 activation. (A) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated for caveolin-1, and the immunoprecipitates were probed for MEK1/2 and ERK1/2 to assess the KSR1-MEK1/2 and KSR1-ERK1/2 interaction. (B) WCLs from 293T cells transfected with vector, FLAG-tagged KSR1, or FLAG-tagged CBM were immunoprecipitated with anti-FLAG antibodies and subjected to Western blotting for B-Raf and c-Raf to assess KSR1–B-Raf and KSR1–c-Raf interactions. The arrow denotes the band specific for c-Raf. The asterisk indicates s a nonspecific band. (C) Triplicate wells of immortalized KSR1 −/− MEFs expressing either KSR1 or KSR1.CBM were treated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation levels were determined in situ for ERK1 and phospho-ERK1/2 with a Li-Cor Odyssey system. Data are expressed as the ratio of phospho-ERK1/2 to ERK1. Data are expressed as means ± standard deviations from three independent experiments. ****, P

Techniques Used: Activation Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection, Western Blot, In Situ

11) Product Images from "Quercetin and Sorafenib as a Novel and Effective Couple in Programmed Cell Death Induction in Human Gliomas"

Article Title: Quercetin and Sorafenib as a Novel and Effective Couple in Programmed Cell Death Induction in Human Gliomas

Journal: Neurotoxicity Research

doi: 10.1007/s12640-013-9452-x

The level of cytochrome c ( a cytoplasmic, b mitochondrial fraction), Ras ( c ), Raf ( d ), LC3 ( e ) and beclin 1 ( f ) expression with representative blots and the activity of caspase 3, 8, 9 ( g ) after sorafenib (S) and quercetin (Q) treatment for 48 h in T98G cells transfected with specific siRNA anti-Hsp27 (si27) and anti-Hsp72 (si72). The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, si27 or si72 specific siRNA blocking Hsp27 or Hsp72 expression, TR transfection reagent, TRsi transfection reagent with specific siRNA, * P
Figure Legend Snippet: The level of cytochrome c ( a cytoplasmic, b mitochondrial fraction), Ras ( c ), Raf ( d ), LC3 ( e ) and beclin 1 ( f ) expression with representative blots and the activity of caspase 3, 8, 9 ( g ) after sorafenib (S) and quercetin (Q) treatment for 48 h in T98G cells transfected with specific siRNA anti-Hsp27 (si27) and anti-Hsp72 (si72). The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, si27 or si72 specific siRNA blocking Hsp27 or Hsp72 expression, TR transfection reagent, TRsi transfection reagent with specific siRNA, * P

Techniques Used: Expressing, Activity Assay, Transfection, Blocking Assay

The level of cytochrome c ( a cytoplasmic, b mitochondrial fraction), Ras ( c ), Raf ( d ), LC3 ( e ) and beclin 1 ( f ) expression with representative blots and the activity of caspase 3, 8, 9 ( f ) after sorafenib (S) and quercetin (Q) treatment for 24 h in MOGGCCM cells transfected with specific siRNA anti-Hsp27 (si27) and anti-Hsp72 (si72). The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, si27 or si72 specific siRNA blocking Hsp27 or Hsp72 expression, TR transfection reagent, TRsi transfection reagent with specific siRNA * P
Figure Legend Snippet: The level of cytochrome c ( a cytoplasmic, b mitochondrial fraction), Ras ( c ), Raf ( d ), LC3 ( e ) and beclin 1 ( f ) expression with representative blots and the activity of caspase 3, 8, 9 ( f ) after sorafenib (S) and quercetin (Q) treatment for 24 h in MOGGCCM cells transfected with specific siRNA anti-Hsp27 (si27) and anti-Hsp72 (si72). The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, si27 or si72 specific siRNA blocking Hsp27 or Hsp72 expression, TR transfection reagent, TRsi transfection reagent with specific siRNA * P

Techniques Used: Expressing, Activity Assay, Transfection, Blocking Assay

The level of Hsp27 ( a ), Hsp72 ( b ), cytochrome c ( c cytoplasmic, d mitochondrial fraction), beclin 1 ( e ), LC3 ( f ), Ras ( g ) and Raf ( h ) expression with representative blots and the activity of caspase 3, 8, 9 ( i ) after sorafenib (S) and quercetin (Q) treatment for 48 h in T98G. The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, * P
Figure Legend Snippet: The level of Hsp27 ( a ), Hsp72 ( b ), cytochrome c ( c cytoplasmic, d mitochondrial fraction), beclin 1 ( e ), LC3 ( f ), Ras ( g ) and Raf ( h ) expression with representative blots and the activity of caspase 3, 8, 9 ( i ) after sorafenib (S) and quercetin (Q) treatment for 48 h in T98G. The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, * P

Techniques Used: Expressing, Activity Assay

The level of Hsp27 ( a ), Hsp72 ( b ), cytochrome c ( c cytoplasmic, d mitochondrial fraction), beclin 1 ( e ), LC3 ( f ), Ras ( g ) and Raf ( h ) expression with representative blots and the activity of caspase 3, 8, 9 (i) after sorafenib (S) and quercetin (Q) treatment for 24 h in MOGGCCM. The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, * P
Figure Legend Snippet: The level of Hsp27 ( a ), Hsp72 ( b ), cytochrome c ( c cytoplasmic, d mitochondrial fraction), beclin 1 ( e ), LC3 ( f ), Ras ( g ) and Raf ( h ) expression with representative blots and the activity of caspase 3, 8, 9 (i) after sorafenib (S) and quercetin (Q) treatment for 24 h in MOGGCCM. The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, * P

Techniques Used: Expressing, Activity Assay

12) Product Images from "Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells"

Article Title: Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0125903

The cSrc/Kras/Raf-1/AKT/ERK/p38-mediated pathway is involved in P4-induced NFκB (p65) nuclear translocation. (A) Pre-treatment of RASMCs with PP2 (200 nM) for 1 h prevented the P4-induced NFκB nuclear translocation. Pre-treatment of RASMCs with 200 nM PP2 (B), 1 μM SB 203580 (C), or 100 nM wortmannin (D) prevented P4-induced increases in the levels of p-IκBα. (E) Pre-transfection with dn-AKT cDNA prevented P4-induced increases the levels of p53, p21 cip1 and p27 kip1 protein. (F) Pre-treatment with BAY (10 nM) prevented P4-induced increases in the levels of p53, p21 cip1 and p27 kip1 protein. (A-F) Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with their own total protein level, RARP (for nuclear protein) or G3PDH (for cytosolic protein) and expressed as ratio over control. (G) Pre-transfection with dn-p53 cDNA prevented P4-induced increases in the levels of p21 cip1 and p27 kip1 protein. Values shown in parentheses represent the quantified results adjusted with α-tubulin protein level and expressed as ratio over control. Values represent the means±s.e.mean. (n = 3). * P
Figure Legend Snippet: The cSrc/Kras/Raf-1/AKT/ERK/p38-mediated pathway is involved in P4-induced NFκB (p65) nuclear translocation. (A) Pre-treatment of RASMCs with PP2 (200 nM) for 1 h prevented the P4-induced NFκB nuclear translocation. Pre-treatment of RASMCs with 200 nM PP2 (B), 1 μM SB 203580 (C), or 100 nM wortmannin (D) prevented P4-induced increases in the levels of p-IκBα. (E) Pre-transfection with dn-AKT cDNA prevented P4-induced increases the levels of p53, p21 cip1 and p27 kip1 protein. (F) Pre-treatment with BAY (10 nM) prevented P4-induced increases in the levels of p53, p21 cip1 and p27 kip1 protein. (A-F) Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with their own total protein level, RARP (for nuclear protein) or G3PDH (for cytosolic protein) and expressed as ratio over control. (G) Pre-transfection with dn-p53 cDNA prevented P4-induced increases in the levels of p21 cip1 and p27 kip1 protein. Values shown in parentheses represent the quantified results adjusted with α-tubulin protein level and expressed as ratio over control. Values represent the means±s.e.mean. (n = 3). * P

Techniques Used: Translocation Assay, Transfection

cSrc is the most upstream molecule involved in P4-induced increases in the levels of p21 cip1 and p27 kip1 . Pre-treatment with PP2 (200 nM) for 1 h prevented P4-induced membrane translocation of Kras (A), increases in the levels of p-Raf-1, p-ERK1/2, p-AKT and p-p38 protein (B), and p21 cip1 , p27 kip1 and p53 protein (C). Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with G3PDH and cadherin for cytosol and membrane, respectively (A), with their own total protein level (B), or with α-tubulin (C) and expressed as ratio over control. Con, control.
Figure Legend Snippet: cSrc is the most upstream molecule involved in P4-induced increases in the levels of p21 cip1 and p27 kip1 . Pre-treatment with PP2 (200 nM) for 1 h prevented P4-induced membrane translocation of Kras (A), increases in the levels of p-Raf-1, p-ERK1/2, p-AKT and p-p38 protein (B), and p21 cip1 , p27 kip1 and p53 protein (C). Data are representative of 2 independent experiments with similar results. Values shown in parentheses represent the quantified results adjusted with G3PDH and cadherin for cytosol and membrane, respectively (A), with their own total protein level (B), or with α-tubulin (C) and expressed as ratio over control. Con, control.

Techniques Used: Translocation Assay

13) Product Images from "Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis"

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

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2011.01520.x

(A) Western blot analysis of ROCKII knockdown in Co- and Eu-hESC, showing down-regulation of E/R/M, MYPT1, paxillin and MLC phosphorylation in knockdown cells versus respective siRNA controls. Representative blots of six independent experiments (left panel) and graphical representation of the levels of phosphorylated proteins (right panel) normalized by α-tubulin are shown as protein level in % (mean ± SD) relative to their normalized levels (set to 100%) in cells transfected with control siRNA, * P
Figure Legend Snippet: (A) Western blot analysis of ROCKII knockdown in Co- and Eu-hESC, showing down-regulation of E/R/M, MYPT1, paxillin and MLC phosphorylation in knockdown cells versus respective siRNA controls. Representative blots of six independent experiments (left panel) and graphical representation of the levels of phosphorylated proteins (right panel) normalized by α-tubulin are shown as protein level in % (mean ± SD) relative to their normalized levels (set to 100%) in cells transfected with control siRNA, * P

Techniques Used: Western Blot, Transfection

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
Figure Legend 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

Techniques Used: Transfection

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 Table S1 (for Ec-hESC; n = 7) and in [ 25 ] 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
Figure Legend 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 Table S1 (for Ec-hESC; n = 7) and in [ 25 ] 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

Techniques Used: Western Blot, Expressing, Activity Assay

14) Product Images from "Synergistic interaction between the fibroblast growth factor and bone morphogenetic protein signaling pathways in lens cells"

Article Title: Synergistic interaction between the fibroblast growth factor and bone morphogenetic protein signaling pathways in lens cells

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E15-02-0117

Up-regulation of the BMP signaling reporter BRE-Luc by BMPs is specifically blocked by noggin and dorsomorphin. DCDMLs were transfected with the BRE-luciferase reporter construct BRE-Luc and cultured for 2 d with no additions (control), noggin, 5 ng/ml BMP4, 10 ng/ml BMP6, or the indicated BMP plus either noggin or dorsomorphin. Fixed cultures were immunostained with anti-luciferase antibodies, and confluent regions of the monolayer were imaged. See Figure 8 C for Western blot quantification of the effect of noggin and dorsomorphin on BRE-luciferase protein levels.
Figure Legend Snippet: Up-regulation of the BMP signaling reporter BRE-Luc by BMPs is specifically blocked by noggin and dorsomorphin. DCDMLs were transfected with the BRE-luciferase reporter construct BRE-Luc and cultured for 2 d with no additions (control), noggin, 5 ng/ml BMP4, 10 ng/ml BMP6, or the indicated BMP plus either noggin or dorsomorphin. Fixed cultures were immunostained with anti-luciferase antibodies, and confluent regions of the monolayer were imaged. See Figure 8 C for Western blot quantification of the effect of noggin and dorsomorphin on BRE-luciferase protein levels.

Techniques Used: Transfection, Luciferase, Construct, Cell Culture, Western Blot

Up-regulation of BRE-Luc expression by FGF requires signaling from endogenously expressed, noggin-sensitive BMPs. DCDMLs were transfected with BRE-Luc and cultured with no added growth factor (control), 10 ng/ml FGF2, 20 ng/ml FGF9, or 5 ng/ml BMP4. Incubations were conducted in either the absence or presence of noggin (nog), the anti-BMP antibody MAB3552 (MAB), or dorsomorphin (DM). After 22 h, cultures were either fixed and immunostained with anti-luciferase antibodies (A) or lysed and equal amount of total cell protein analyzed by Western blot using the same antibody (B). (C) Summary of experiments conducted as in B, compared with results obtained in the same experiments from cells cultured for 22 h with 5 ng/ml BMP4. Results graphed as fold increase in luciferase levels relative to untreated control transfectants ( n = 3). The percentage inhibition relative to growth factor alone is included. The low level of anti-luciferase immunoreactivity in untreated control transfectants is due to endogenous BMP4/7 signaling, as demonstrated by its near absence in cells treated with noggin, MAB3552, or dorsomorphin alone. Immunofluorescence images showing the effect of noggin and dorsomorphin on BMP4-induced BRE-Luc expression are presented in Figure 6 .
Figure Legend Snippet: Up-regulation of BRE-Luc expression by FGF requires signaling from endogenously expressed, noggin-sensitive BMPs. DCDMLs were transfected with BRE-Luc and cultured with no added growth factor (control), 10 ng/ml FGF2, 20 ng/ml FGF9, or 5 ng/ml BMP4. Incubations were conducted in either the absence or presence of noggin (nog), the anti-BMP antibody MAB3552 (MAB), or dorsomorphin (DM). After 22 h, cultures were either fixed and immunostained with anti-luciferase antibodies (A) or lysed and equal amount of total cell protein analyzed by Western blot using the same antibody (B). (C) Summary of experiments conducted as in B, compared with results obtained in the same experiments from cells cultured for 22 h with 5 ng/ml BMP4. Results graphed as fold increase in luciferase levels relative to untreated control transfectants ( n = 3). The percentage inhibition relative to growth factor alone is included. The low level of anti-luciferase immunoreactivity in untreated control transfectants is due to endogenous BMP4/7 signaling, as demonstrated by its near absence in cells treated with noggin, MAB3552, or dorsomorphin alone. Immunofluorescence images showing the effect of noggin and dorsomorphin on BMP4-induced BRE-Luc expression are presented in Figure 6 .

Techniques Used: Expressing, Transfection, Cell Culture, Luciferase, Western Blot, Inhibition, Immunofluorescence

BMP-dependent FGF signaling in lens cells requires active BMP receptors. (A) Dorsomorphin is a specific inhibitor of BMP signaling in DCDMLs. Cultures were treated for 45 min without factors (–) or with 5 ng/ml BMP4, 4 ng/ml TGFβ1, or 10 ng/ml FGF2. Where indicated, cells were pretreated with 5 μM dorsomorphin (DM) before addition of the factor. Whole-cell lysates were probed with antibodies specific for the phosphorylated (activated) forms of Smad1/5, Smad3, or ERK. Representative of three independent experiments; inhibition of either Smad3 or ERK activation never exceeded 20%. (B, C) Dorsomorphin blocks both BMP- and FGF-induced processes in DCDMLs. DCDMLs were incubated without growth factor or with 10 ng/ml BMP4 or FGF2 in either the absence or presence of dorsomorphin as indicated. (B) After 6 d of culture, cells were assayed for synthesis of the fiber differentiation markers δ-crystallin (by [ 35 S]methionine labeling) and CP49 (by quantitative anti-CP49 Western blotting). Fold increase over control ± SD given for each condition ( n = 5). (C) Cultures were assessed for their ability to activate ERK in response to an 8-h incubation with FGF2 as in Figure 1 A. Percentage decrease in activation of ERK in response to FGF in dorsomorphin-treated cells relative to cells not cultured with dorsomorphin was 65 ± 7.7% ( n = 3).
Figure Legend Snippet: BMP-dependent FGF signaling in lens cells requires active BMP receptors. (A) Dorsomorphin is a specific inhibitor of BMP signaling in DCDMLs. Cultures were treated for 45 min without factors (–) or with 5 ng/ml BMP4, 4 ng/ml TGFβ1, or 10 ng/ml FGF2. Where indicated, cells were pretreated with 5 μM dorsomorphin (DM) before addition of the factor. Whole-cell lysates were probed with antibodies specific for the phosphorylated (activated) forms of Smad1/5, Smad3, or ERK. Representative of three independent experiments; inhibition of either Smad3 or ERK activation never exceeded 20%. (B, C) Dorsomorphin blocks both BMP- and FGF-induced processes in DCDMLs. DCDMLs were incubated without growth factor or with 10 ng/ml BMP4 or FGF2 in either the absence or presence of dorsomorphin as indicated. (B) After 6 d of culture, cells were assayed for synthesis of the fiber differentiation markers δ-crystallin (by [ 35 S]methionine labeling) and CP49 (by quantitative anti-CP49 Western blotting). Fold increase over control ± SD given for each condition ( n = 5). (C) Cultures were assessed for their ability to activate ERK in response to an 8-h incubation with FGF2 as in Figure 1 A. Percentage decrease in activation of ERK in response to FGF in dorsomorphin-treated cells relative to cells not cultured with dorsomorphin was 65 ± 7.7% ( n = 3).

Techniques Used: Inhibition, Activation Assay, Incubation, Labeling, Western Blot, Cell Culture

Up-regulation of gap junctional communication, but not of cell proliferation, by FGF requires signaling from endogenously expressed BMPs. (A) DCDMLs were incubated without growth factor (control) or with 10 ng/ml BMP4, 10 ng/ml FGF2, or 20 ng/ml FGF9 in either the absence or presence of noggin or dorsomorphin as indicated. After 2 d of culture, cells were assayed for gap junction–mediated intercellular spread of Lucifer yellow (LY) using the scrape-load dye transfer assay. Typical of three independent experiments. (B) DCDMLs were plated at low density (0.7 × 10 5 cells/well) and incubated with no additions (cont), 2 ng/ml FGF2, 2 ng/ml FGF2 + noggin, 0.2 ng/ml BMP4, or 20 ng/ml BMP4 (10X). After 2 d, the MTT assay was used to colorimetrically assess cell proliferation. Data expressed as fold OD 570 nm experimental/OD 570 nm untreated control. n = 3.
Figure Legend Snippet: Up-regulation of gap junctional communication, but not of cell proliferation, by FGF requires signaling from endogenously expressed BMPs. (A) DCDMLs were incubated without growth factor (control) or with 10 ng/ml BMP4, 10 ng/ml FGF2, or 20 ng/ml FGF9 in either the absence or presence of noggin or dorsomorphin as indicated. After 2 d of culture, cells were assayed for gap junction–mediated intercellular spread of Lucifer yellow (LY) using the scrape-load dye transfer assay. Typical of three independent experiments. (B) DCDMLs were plated at low density (0.7 × 10 5 cells/well) and incubated with no additions (cont), 2 ng/ml FGF2, 2 ng/ml FGF2 + noggin, 0.2 ng/ml BMP4, or 20 ng/ml BMP4 (10X). After 2 d, the MTT assay was used to colorimetrically assess cell proliferation. Data expressed as fold OD 570 nm experimental/OD 570 nm untreated control. n = 3.

Techniques Used: Incubation, MTT Assay

15) Product Images from "Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide"

Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

Journal: PLoS ONE

doi: 10.1371/journal.pone.0033287

Knockdown of AP-1 subunits decreases Blimp1 expression in lung cancer cells. (A) The immunoblot of nuclear extracts from lung cancer cells in Fig. 1A was stripped and re-probed to assess expression of the AP-1 subunits c-Jun, Fra-1, Fra-2 and c-Fos. (B) A549, H441 and H1299 cells were transfected with 20 nM of JUN siRNA alone or 10 nM of JUN siRNA in combination with 10 nM of FRA-1 or FRA-2 siRNA or with 20 nM of a negative control siRNA (Qiagen) for 24 h. WCE (30 µg) were subjected to immunoblotting for Blimp1, c-Jun, Fra-1, Fra-2 and α-tubulin, as a loading control. The Blimp1 bands were quantified and normalized to α-tubulin expression, and average values from two independent experiments presented relative to control siRNA, set to 1.0.
Figure Legend Snippet: Knockdown of AP-1 subunits decreases Blimp1 expression in lung cancer cells. (A) The immunoblot of nuclear extracts from lung cancer cells in Fig. 1A was stripped and re-probed to assess expression of the AP-1 subunits c-Jun, Fra-1, Fra-2 and c-Fos. (B) A549, H441 and H1299 cells were transfected with 20 nM of JUN siRNA alone or 10 nM of JUN siRNA in combination with 10 nM of FRA-1 or FRA-2 siRNA or with 20 nM of a negative control siRNA (Qiagen) for 24 h. WCE (30 µg) were subjected to immunoblotting for Blimp1, c-Jun, Fra-1, Fra-2 and α-tubulin, as a loading control. The Blimp1 bands were quantified and normalized to α-tubulin expression, and average values from two independent experiments presented relative to control siRNA, set to 1.0.

Techniques Used: Expressing, Transfection, Negative Control

A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).
Figure Legend Snippet: A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Software, Isolation, Polymerase Chain Reaction, Luciferase, Negative Control, Incubation, Concentration Assay, Western Blot, Construct

AP-1 subunits bind to the BLIMP1 promoter. (A) Schematic of the localization of the two TRE sites on the human BLIMP1 promoter. Two primer sets encompassing these sites are shown: 1F/1R amplifies the −1647 bp TRE and 2F/2R amplifies the −1813 bp TRE. (B) H441 cells at 90% confluence in P100 plates were transfected with 4 µg of c-Jun expression vector, and after 48 h subjected to a ChIP assay using a control IgG or c-Jun antibody, as described in the Materials and Methods . Input, 1% of the WCE. Positive (Pos.) control: a genomic region of the JUN promoter containing two TREs (−1 and −120 bp). Negative (Neg.) control: region upstream of BLIMP1 transcription start site (∼−5.4 kB) that does not contain any known TRE sites. (C) A549 lung cancer cells were incubated in serum free DMEM for 48 h. FBS was added back to 10%. Samples were harvested at 0, 15, 30 minutes or 1, 2, 4 h and RNA and WCE prepared. Upper panel: RNA was subjected to Q-PCR, in triplicate, and values for BLIMP1 normalized to GAPDH RNA levels presented relative to the 0 time point which was set to 1.0. Data for the mean ± SD from three independent experiments are presented. Lower panels: WCE (25 µg) were subjected to immunoblot analysis for phospho-c-Jun (p-c-Jun), c-Jun, Fra-1, Fra-2, c-Fos AP-1 subunits, and α-tubulin, which confirmed essentially equal loading control. Data shown is a representative of two independent experiments with similar results. (D) A549 cells were incubated in serum free DMEM for 48 h, and stimulated with addition of FBS (final 10%) for 30 min. Whole cell lysates were subjected to ChIP analysis using antibodies against c-Jun, Fra-1, Fra-2 or normal rabbit IgG, as described in part B. Data shown is a representative of two independent experiments with similar results.
Figure Legend Snippet: AP-1 subunits bind to the BLIMP1 promoter. (A) Schematic of the localization of the two TRE sites on the human BLIMP1 promoter. Two primer sets encompassing these sites are shown: 1F/1R amplifies the −1647 bp TRE and 2F/2R amplifies the −1813 bp TRE. (B) H441 cells at 90% confluence in P100 plates were transfected with 4 µg of c-Jun expression vector, and after 48 h subjected to a ChIP assay using a control IgG or c-Jun antibody, as described in the Materials and Methods . Input, 1% of the WCE. Positive (Pos.) control: a genomic region of the JUN promoter containing two TREs (−1 and −120 bp). Negative (Neg.) control: region upstream of BLIMP1 transcription start site (∼−5.4 kB) that does not contain any known TRE sites. (C) A549 lung cancer cells were incubated in serum free DMEM for 48 h. FBS was added back to 10%. Samples were harvested at 0, 15, 30 minutes or 1, 2, 4 h and RNA and WCE prepared. Upper panel: RNA was subjected to Q-PCR, in triplicate, and values for BLIMP1 normalized to GAPDH RNA levels presented relative to the 0 time point which was set to 1.0. Data for the mean ± SD from three independent experiments are presented. Lower panels: WCE (25 µg) were subjected to immunoblot analysis for phospho-c-Jun (p-c-Jun), c-Jun, Fra-1, Fra-2, c-Fos AP-1 subunits, and α-tubulin, which confirmed essentially equal loading control. Data shown is a representative of two independent experiments with similar results. (D) A549 cells were incubated in serum free DMEM for 48 h, and stimulated with addition of FBS (final 10%) for 30 min. Whole cell lysates were subjected to ChIP analysis using antibodies against c-Jun, Fra-1, Fra-2 or normal rabbit IgG, as described in part B. Data shown is a representative of two independent experiments with similar results.

Techniques Used: Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Incubation, Polymerase Chain Reaction

Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7 (PP7) clones, isolated as described previously [25] , were treated in triplicate with 2 µg/ml dox for 48 h. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). (B) A549-EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 µg/ml dox for 48 h in DMEM supplemented with 0.5% FBS. FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 µl) were subjected to immunoblotting for V5. Samples of WCE (25 µg) were probed for Blimp1 and β-actin, and average normalized Blimp1 values from two independent experiments presented relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 µg/ml, respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and α-tubulin, as a loading control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.
Figure Legend Snippet: Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7 (PP7) clones, isolated as described previously [25] , were treated in triplicate with 2 µg/ml dox for 48 h. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). (B) A549-EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 µg/ml dox for 48 h in DMEM supplemented with 0.5% FBS. FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 µl) were subjected to immunoblotting for V5. Samples of WCE (25 µg) were probed for Blimp1 and β-actin, and average normalized Blimp1 values from two independent experiments presented relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 µg/ml, respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and α-tubulin, as a loading control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.

Techniques Used: Expressing, Isolation, Polymerase Chain Reaction, Incubation, Immunoprecipitation, Transfection, Purification, Recombinant, Concentration Assay

LOX-PP represses the migratory phenotype of lung cancer cells via inhibiting Blimp1. (A) A549 and (B) H441 cells were transfected with 2 µg of EV or human LOX-PP cDNA. Upper panels: After 24 h, 1×10 5 transfected cells were subjected to migration assay. Lower panels: After 48 h, culture media was isolated and samples (50 µl of 2 ml total) subjected to immunoblotting using an anti-V5 antibody for LOX-PP. (C and D) H441 were transiently transfected with 1 µg of LOX-PP or Blimp1 DNA alone or in combination, or EV DNA (2 µg total DNA). (C) After 24 h. cells were subjected to migration assays, in triplicate, for 16 h. The average migration from two independent experiments ± SEM is presented relative to the EV (set at 1.0). (D) WCE and media were isolated. WCE samples (25 µg) were subjected to immunoblotting for Blimp1 and α-tubulin. Media samples (50 µl) were subjected to immunoblotting for LOX-PP-V5. Immunoblots from one of two independent experiments with similar results are presented.
Figure Legend Snippet: LOX-PP represses the migratory phenotype of lung cancer cells via inhibiting Blimp1. (A) A549 and (B) H441 cells were transfected with 2 µg of EV or human LOX-PP cDNA. Upper panels: After 24 h, 1×10 5 transfected cells were subjected to migration assay. Lower panels: After 48 h, culture media was isolated and samples (50 µl of 2 ml total) subjected to immunoblotting using an anti-V5 antibody for LOX-PP. (C and D) H441 were transiently transfected with 1 µg of LOX-PP or Blimp1 DNA alone or in combination, or EV DNA (2 µg total DNA). (C) After 24 h. cells were subjected to migration assays, in triplicate, for 16 h. The average migration from two independent experiments ± SEM is presented relative to the EV (set at 1.0). (D) WCE and media were isolated. WCE samples (25 µg) were subjected to immunoblotting for Blimp1 and α-tubulin. Media samples (50 µl) were subjected to immunoblotting for LOX-PP-V5. Immunoblots from one of two independent experiments with similar results are presented.

Techniques Used: Transfection, Migration, Isolation, Western Blot

16) Product Images from "Sulfur dioxide reduces lipopolysaccharide-induced acute lung injury in rats"

Article Title: Sulfur dioxide reduces lipopolysaccharide-induced acute lung injury in rats

Journal: Central-European Journal of Immunology

doi: 10.5114/ceji.2019.89593

Raf-1 and MEK-1 protein expression by Western blotting analysis ( A ) and densitometric analysis ( B ) in different groups. Data are presented as the mean ±SD ( n = 6 in each group); * p
Figure Legend Snippet: Raf-1 and MEK-1 protein expression by Western blotting analysis ( A ) and densitometric analysis ( B ) in different groups. Data are presented as the mean ±SD ( n = 6 in each group); * p

Techniques Used: Expressing, Western Blot

17) Product Images from "Splicing factor hnRNP A2 activates the Ras-MAPK-ERK pathway by controlling A-Raf splicing in hepatocellular carcinoma development"

Article Title: Splicing factor hnRNP A2 activates the Ras-MAPK-ERK pathway by controlling A-Raf splicing in hepatocellular carcinoma development

Journal: RNA

doi: 10.1261/rna.042259.113

hnRNP A1/A1b and A2, but not B1, can transform PHM-1 cells in vivo. ( A , B ) PHM-1 cells transduced with retroviruses encoding empty vector (pBABE) or hnRNP A1, A1b, A2, and B1 were analyzed by Western blotting for hnRNP A1/A1b, hnRNP A2/B1, and T7-Tag protein
Figure Legend Snippet: hnRNP A1/A1b and A2, but not B1, can transform PHM-1 cells in vivo. ( A , B ) PHM-1 cells transduced with retroviruses encoding empty vector (pBABE) or hnRNP A1, A1b, A2, and B1 were analyzed by Western blotting for hnRNP A1/A1b, hnRNP A2/B1, and T7-Tag protein

Techniques Used: In Vivo, Transduction, Plasmid Preparation, Western Blot

18) Product Images from "Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation"

Article Title: Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01633-13

KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.
Figure Legend Snippet: KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.

Techniques Used: Expressing, Binding Assay, Plasmid Preparation, Immunoprecipitation, Proximity Ligation Assay, Negative Control, Generated, Fluorescence

The KSR1–caveolin-1 interaction promotes EGF-stimulated ERK1/2 activation. (A) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated for caveolin-1, and the immunoprecipitates were probed for MEK1/2 and ERK1/2 to assess the KSR1-MEK1/2 and KSR1-ERK1/2 interaction. (B) WCLs from 293T cells transfected with vector, FLAG-tagged KSR1, or FLAG-tagged CBM were immunoprecipitated with anti-FLAG antibodies and subjected to Western blotting for B-Raf and c-Raf to assess KSR1–B-Raf and KSR1–c-Raf interactions. The arrow denotes the band specific for c-Raf. The asterisk indicates s a nonspecific band. (C) Triplicate wells of immortalized KSR1 −/− MEFs expressing either KSR1 or KSR1.CBM were treated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation levels were determined in situ for ERK1 and phospho-ERK1/2 with a Li-Cor Odyssey system. Data are expressed as the ratio of phospho-ERK1/2 to ERK1. Data are expressed as means ± standard deviations from three independent experiments. ****, P
Figure Legend Snippet: The KSR1–caveolin-1 interaction promotes EGF-stimulated ERK1/2 activation. (A) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated for caveolin-1, and the immunoprecipitates were probed for MEK1/2 and ERK1/2 to assess the KSR1-MEK1/2 and KSR1-ERK1/2 interaction. (B) WCLs from 293T cells transfected with vector, FLAG-tagged KSR1, or FLAG-tagged CBM were immunoprecipitated with anti-FLAG antibodies and subjected to Western blotting for B-Raf and c-Raf to assess KSR1–B-Raf and KSR1–c-Raf interactions. The arrow denotes the band specific for c-Raf. The asterisk indicates s a nonspecific band. (C) Triplicate wells of immortalized KSR1 −/− MEFs expressing either KSR1 or KSR1.CBM were treated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation levels were determined in situ for ERK1 and phospho-ERK1/2 with a Li-Cor Odyssey system. Data are expressed as the ratio of phospho-ERK1/2 to ERK1. Data are expressed as means ± standard deviations from three independent experiments. ****, P

Techniques Used: Activation Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection, Western Blot, In Situ

19) Product Images from "Apigenin inhibits proliferation and induces apoptosis in human multiple myeloma cells through targeting the trinity of CK2, Cdc37 and Hsp90"

Article Title: Apigenin inhibits proliferation and induces apoptosis in human multiple myeloma cells through targeting the trinity of CK2, Cdc37 and Hsp90

Journal: Molecular Cancer

doi: 10.1186/1476-4598-10-104

Apigenin-induced proteasome-dependent degradation of Hsp90/Cdc37 client proteins are correlated with inhibition of CK2 . (A) HeLa cells were treated with the indicated concentrations of apigenin or TBB for 24 h. The protein levels of CK2α, RIP1, Raf-1 and Cdk4 were detected by western blot analysis. (B) HeLa cells were transiently transfected with control siRNA or CK2α siRNA for 48 h. Whole-cell lysates were analyzed by western blotting using the indicated antibodies. (C) Next, siRNA was introduced intoU266 and RPMI 8226 cells using electroporation. After 48 h, the cells were harvested to detect the protein levels by western blot. β-actin, as well as α-tubulin, served as loading control. (D) U266 cells were pretreated with MG132 (1 μM) for 1 h, and the cells were subsequently treated with apigenin (90 μM) for an additional 12 h. Whole-cell lysates were subjected to western blot analysis using antibodies against Raf-1, Src, Cdk4 and β-actin. (E) and (F) U266 cells were incubated with or without the Hsp90 inhibitor GA (0.2 μM) or SAHA (1 μM) for 24 h in the presence or absence of 30 μM apigenin. Whole-cell lysates were subjected to western blotting to determine the levels of Raf-1, Src, Cdk4 and β-actin. The bar graphs on the right show the percentage of intensities of the protein band from each treatment relative to the controls, which were defined as 100%. Values represent the means ± SD. *Significant difference from the three groups was designed by ANOVA, ** p
Figure Legend Snippet: Apigenin-induced proteasome-dependent degradation of Hsp90/Cdc37 client proteins are correlated with inhibition of CK2 . (A) HeLa cells were treated with the indicated concentrations of apigenin or TBB for 24 h. The protein levels of CK2α, RIP1, Raf-1 and Cdk4 were detected by western blot analysis. (B) HeLa cells were transiently transfected with control siRNA or CK2α siRNA for 48 h. Whole-cell lysates were analyzed by western blotting using the indicated antibodies. (C) Next, siRNA was introduced intoU266 and RPMI 8226 cells using electroporation. After 48 h, the cells were harvested to detect the protein levels by western blot. β-actin, as well as α-tubulin, served as loading control. (D) U266 cells were pretreated with MG132 (1 μM) for 1 h, and the cells were subsequently treated with apigenin (90 μM) for an additional 12 h. Whole-cell lysates were subjected to western blot analysis using antibodies against Raf-1, Src, Cdk4 and β-actin. (E) and (F) U266 cells were incubated with or without the Hsp90 inhibitor GA (0.2 μM) or SAHA (1 μM) for 24 h in the presence or absence of 30 μM apigenin. Whole-cell lysates were subjected to western blotting to determine the levels of Raf-1, Src, Cdk4 and β-actin. The bar graphs on the right show the percentage of intensities of the protein band from each treatment relative to the controls, which were defined as 100%. Values represent the means ± SD. *Significant difference from the three groups was designed by ANOVA, ** p

Techniques Used: Inhibition, Western Blot, Transfection, Electroporation, Incubation

20) Product Images from "Epidermal growth factor receptor controls glycogen phosphorylase in T cells through small GTPases of the RAS family"

Article Title: Epidermal growth factor receptor controls glycogen phosphorylase in T cells through small GTPases of the RAS family

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.005997

Epidermal growth factor receptor requires adenylyl cyclase 6 expression to induce glycogen phosphorylase activation. A and B , T cells deprived of IL-2 for 24 h were transfected with EGFP ( esiRNA ) or ADCY6 ( esiRNA ). At 24 h post-transfection, the cells were stimulated (+) or not (−) with 10 ng ml −1 EGF for 10 min and lysed. Cell extracts were used to measure glycogen phosphorylase activity ( A ) and cAMP generation ( B ). Protein expression levels were analyzed by Western blotting using specific anti-ADCY6 and anti-γ-tubulin antibodies. The results show the mean ± S.D. ( error bars ) of three and independent experiments performed in triplicates ( A ) and duplicates ( B ). ***, p
Figure Legend Snippet: Epidermal growth factor receptor requires adenylyl cyclase 6 expression to induce glycogen phosphorylase activation. A and B , T cells deprived of IL-2 for 24 h were transfected with EGFP ( esiRNA ) or ADCY6 ( esiRNA ). At 24 h post-transfection, the cells were stimulated (+) or not (−) with 10 ng ml −1 EGF for 10 min and lysed. Cell extracts were used to measure glycogen phosphorylase activity ( A ) and cAMP generation ( B ). Protein expression levels were analyzed by Western blotting using specific anti-ADCY6 and anti-γ-tubulin antibodies. The results show the mean ± S.D. ( error bars ) of three and independent experiments performed in triplicates ( A ) and duplicates ( B ). ***, p

Techniques Used: Expressing, Activation Assay, Transfection, esiRNA, Activity Assay, Western Blot

21) Product Images from "Ginkgolide B inhibits renal cyst development in in vitro and in vivo cyst models"

Article Title: Ginkgolide B inhibits renal cyst development in in vitro and in vivo cyst models

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00356.2011

GB retards cyst development in embryonic kidney cyst model. A : representative light micrographs of embryonic kidneys cultured in Transwells and maintained from day 0 to day 4 . Embryonic day 13.5 ( E13.5 ) kidneys were exposed to 100 μM 8-bromo (Br)-cAMP as a positive control ( top ) or were treated with 2 μM GB from day 0 to day 4 ( middle ) or from day 0 to day 2 ( bottom ) in the presence of 100 μM 8-Br-cAMP. Each series of photographs shows the same kidney on successive days in culture. Scale bar = 1 mm. Thick solid lines indicate the culture time with GB. B : inhibition of 8-Br-cAMP-induced cyst growth by GB at indicated concentrations. Images show embryonic kidneys before ( day 0 ) and after ( day 4 ) continuous GB treatment. Scale bar = 1 mm. C : fractional cyst area (%) in positive control and GB-treated group (means ± SD; n = 6–10). * P
Figure Legend Snippet: GB retards cyst development in embryonic kidney cyst model. A : representative light micrographs of embryonic kidneys cultured in Transwells and maintained from day 0 to day 4 . Embryonic day 13.5 ( E13.5 ) kidneys were exposed to 100 μM 8-bromo (Br)-cAMP as a positive control ( top ) or were treated with 2 μM GB from day 0 to day 4 ( middle ) or from day 0 to day 2 ( bottom ) in the presence of 100 μM 8-Br-cAMP. Each series of photographs shows the same kidney on successive days in culture. Scale bar = 1 mm. Thick solid lines indicate the culture time with GB. B : inhibition of 8-Br-cAMP-induced cyst growth by GB at indicated concentrations. Images show embryonic kidneys before ( day 0 ) and after ( day 4 ) continuous GB treatment. Scale bar = 1 mm. C : fractional cyst area (%) in positive control and GB-treated group (means ± SD; n = 6–10). * P

Techniques Used: Cell Culture, Positive Control, Inhibition

22) Product Images from "Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies"

Article Title: Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20171960

USP28/FBW7 complex regulates BRAF stability. (A) Representative images of immunoblot analysis of 293T cells expressing BRAF, Flag-USP28, or Flag-USP28 DD. Whole cell extracts were probed with the indicated antibodies. (B) Immunoblot analysis of 293T cells overexpressing Flag-USP28 or Flag-USP28 DD. Whole cell extracts were probed with the indicated antibodies. (C) Immunoblot analysis of 293T cells expressing mutant BRAF (V600E) and Flag-USP28. (D) Immunoblot analysis of 293T cells overexpressing BRAF, Myc-FBW7, or Myc-FBW7(R505L). Whole cell extracts were probed with the indicated antibodies. (E) Immunoblot analysis of 293T cells expressing shRNA vectors against USP28 (C and D). Whole cell extracts were probed with the indicated antibodies. (F) Immunoblot analysis in 293T cells expressing shRNA vectors targeting FBW7. (G) Immunoprecipitation with anti-BRAF resin in 293T cells overexpressing BRAF, Myc-FBW7, and Myc-FBW7 (R505L). Immunoblot analysis of indicated proteins is shown. (H) Immunoprecipitation with anti-BRAF resin in 293T cells expressing BRAF and an shRNA targeting FBW7 treated with proteasome inhibitor, MG132. Immunoblot analysis of indicated proteins is shown. (I) Immunoprecipitation with anti-BRAF in 293T cells expressing BRAF, shRNA vector targeting USP28, and HA-Ub, treated with proteasome inhibitor, MG132. Immunoblot analysis of indicated proteins is shown. (J) Immunoblot analysis of WM164 melanoma cells treated with PLX4032 at indicated concentrations for 18 h. Immunoblot analysis of indicated proteins is shown. (K) Immunoblot analysis of WM164 melanoma cells stably expressing shRNA vectors against USP28 or FBW7 and treated with PLX4032 (5 µM) for 18 h. Whole cell extracts were probed with the indicated antibodies. (L) Graph representing the percentage of BRAF degradation from three independent experiments after vemurafenib treatment as in K. Data shown are representative of three independent and reproducible experiments. Figure G, H, and I were performed in duplicate. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls.
Figure Legend Snippet: USP28/FBW7 complex regulates BRAF stability. (A) Representative images of immunoblot analysis of 293T cells expressing BRAF, Flag-USP28, or Flag-USP28 DD. Whole cell extracts were probed with the indicated antibodies. (B) Immunoblot analysis of 293T cells overexpressing Flag-USP28 or Flag-USP28 DD. Whole cell extracts were probed with the indicated antibodies. (C) Immunoblot analysis of 293T cells expressing mutant BRAF (V600E) and Flag-USP28. (D) Immunoblot analysis of 293T cells overexpressing BRAF, Myc-FBW7, or Myc-FBW7(R505L). Whole cell extracts were probed with the indicated antibodies. (E) Immunoblot analysis of 293T cells expressing shRNA vectors against USP28 (C and D). Whole cell extracts were probed with the indicated antibodies. (F) Immunoblot analysis in 293T cells expressing shRNA vectors targeting FBW7. (G) Immunoprecipitation with anti-BRAF resin in 293T cells overexpressing BRAF, Myc-FBW7, and Myc-FBW7 (R505L). Immunoblot analysis of indicated proteins is shown. (H) Immunoprecipitation with anti-BRAF resin in 293T cells expressing BRAF and an shRNA targeting FBW7 treated with proteasome inhibitor, MG132. Immunoblot analysis of indicated proteins is shown. (I) Immunoprecipitation with anti-BRAF in 293T cells expressing BRAF, shRNA vector targeting USP28, and HA-Ub, treated with proteasome inhibitor, MG132. Immunoblot analysis of indicated proteins is shown. (J) Immunoblot analysis of WM164 melanoma cells treated with PLX4032 at indicated concentrations for 18 h. Immunoblot analysis of indicated proteins is shown. (K) Immunoblot analysis of WM164 melanoma cells stably expressing shRNA vectors against USP28 or FBW7 and treated with PLX4032 (5 µM) for 18 h. Whole cell extracts were probed with the indicated antibodies. (L) Graph representing the percentage of BRAF degradation from three independent experiments after vemurafenib treatment as in K. Data shown are representative of three independent and reproducible experiments. Figure G, H, and I were performed in duplicate. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls.

Techniques Used: Expressing, Mutagenesis, shRNA, Immunoprecipitation, Plasmid Preparation, Stable Transfection

USP28 is down-regulated in melanoma and confers poor prognosis. (A) Oncomine box plot of USP28 in melanoma. (B) Matrix heat map generated using cBioportal showing genetic alterations of BRAF, NRAS, NF1, USP28, and FBW7 in melanoma patients ( n = 287; TCGA). (C) Beeswarm plot demonstrating relative copy number variation of USP28 in melanoma patients, along with their respective mutational status of BRAF (blue), NRAS (red), NF1 (green), and USP28 (X) genes, respectively ( n = 118). (D) Kaplan-Meier curves showing probability of overall survival of melanoma patients with lower copy number of USP28 is significantly less than those with higher level of USP28 (P = 0.05; HR = 8.15). (E) Kaplan-Meier curves showing probability of tumor free survival of melanoma patients with low levels of USP28 is significantly less than those with high levels of USP28 (P = 0.0065; HR = 14.45). (F) Kaplan-Meier survival analysis of melanoma patients harboring BRAF V600E mutation in respect to expression of USP28. Lower expression of USP28 confers poorer overall survival to melanoma patients carrying BRAF 600E mutation (P = 0.046; HR = 3.8).
Figure Legend Snippet: USP28 is down-regulated in melanoma and confers poor prognosis. (A) Oncomine box plot of USP28 in melanoma. (B) Matrix heat map generated using cBioportal showing genetic alterations of BRAF, NRAS, NF1, USP28, and FBW7 in melanoma patients ( n = 287; TCGA). (C) Beeswarm plot demonstrating relative copy number variation of USP28 in melanoma patients, along with their respective mutational status of BRAF (blue), NRAS (red), NF1 (green), and USP28 (X) genes, respectively ( n = 118). (D) Kaplan-Meier curves showing probability of overall survival of melanoma patients with lower copy number of USP28 is significantly less than those with higher level of USP28 (P = 0.05; HR = 8.15). (E) Kaplan-Meier curves showing probability of tumor free survival of melanoma patients with low levels of USP28 is significantly less than those with high levels of USP28 (P = 0.0065; HR = 14.45). (F) Kaplan-Meier survival analysis of melanoma patients harboring BRAF V600E mutation in respect to expression of USP28. Lower expression of USP28 confers poorer overall survival to melanoma patients carrying BRAF 600E mutation (P = 0.046; HR = 3.8).

Techniques Used: Generated, Mutagenesis, Expressing

Down-regulation of USP28 impairs apoptosis induced by vemurafenib. (A) Representative images of immunoblot analysis of A373C.6 or A373C.6 USP28 knockdown cells treated with 2 µM vemurafenib (PLX4032) for indicated time points. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of two independent and reproducible experiments. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls. (B) Representative images of immunoblot analysis of A373C.6 or A373C.6 USP28 knockdown cells treated with different concentrations of vemurafenib (PLX4032) for 72 h. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of two independent and reproducible experiments. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls. (C) Representative images of cell-cycle analysis of A373C.6 or A373C.6 USP28 knockdown cells after 72 h of treatment with vemurafenib (2 µM). Data shown are representative of three independent and reproducible experiments. (D) Quantification of sub-G 1 population after treatment with vemurafenib as indicated, mean ± SEM of three independent experiments. A two-tailed Student’s t test compares the treated populations; **, P
Figure Legend Snippet: Down-regulation of USP28 impairs apoptosis induced by vemurafenib. (A) Representative images of immunoblot analysis of A373C.6 or A373C.6 USP28 knockdown cells treated with 2 µM vemurafenib (PLX4032) for indicated time points. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of two independent and reproducible experiments. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls. (B) Representative images of immunoblot analysis of A373C.6 or A373C.6 USP28 knockdown cells treated with different concentrations of vemurafenib (PLX4032) for 72 h. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of two independent and reproducible experiments. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls. (C) Representative images of cell-cycle analysis of A373C.6 or A373C.6 USP28 knockdown cells after 72 h of treatment with vemurafenib (2 µM). Data shown are representative of three independent and reproducible experiments. (D) Quantification of sub-G 1 population after treatment with vemurafenib as indicated, mean ± SEM of three independent experiments. A two-tailed Student’s t test compares the treated populations; **, P

Techniques Used: Cell Cycle Assay, Two Tailed Test

Down-regulation of USP28 leads to BRAF inhibitor resistance. (A–C) Representative images of immunoblot analysis of BRAF (V600E) mutant melanoma cell lines A373C.6 (A), WM164 (B), and SK-MEL-28 (C) infected with scrambled or USP28 shRNA lentivirus. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. (D) Immunoblot analysis of WM164 or WM164 USP28CRSP cells. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. (E) Correlation between USP28 and BRAF protein levels in melanoma patients ( n = 98). Statistical significance was determined by an χ 2 test (P = 0.023). R is the correlation coefficient (R=−0.18; top). Immunohistochemical staining of BRAF and USP28 on sequential sections of ME2082B (Biomax) melanoma tissue microarray. Red staining indicates positive immunoreactivity. Bars, 50 µm. Dashed boxes indicate zoomed area. (F) WM164 or WM164 USP28CRSP cells treated with escalating doses of vemurafenib (PLX4032) for 72 h. Viability was assessed using CellTiter Glo as described by the manufacturer. Data represent the mean of six replicates. (G) A373C.6 cells or A373C.6 USP28 knockdown cells treated with escalating doses of vemurafenib (PLX4032) for 72 h. Viability was assessed using CellTiter Glo as described by the manufacturer. Data represent the mean of six replicates. (H) Immunoblot analysis of WM164 or WM164 USP28 knockdown cells treated with different concentrations of vemurafenib (PLX4032) for 1 h. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. (I) Immunoblot analysis of A373C.6 or A373C.6 USP28 knockdown cells treated with different concentrations of vemurafenib (PLX4032) for 1 h. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. For respective immunoblots proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls.
Figure Legend Snippet: Down-regulation of USP28 leads to BRAF inhibitor resistance. (A–C) Representative images of immunoblot analysis of BRAF (V600E) mutant melanoma cell lines A373C.6 (A), WM164 (B), and SK-MEL-28 (C) infected with scrambled or USP28 shRNA lentivirus. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. (D) Immunoblot analysis of WM164 or WM164 USP28CRSP cells. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. (E) Correlation between USP28 and BRAF protein levels in melanoma patients ( n = 98). Statistical significance was determined by an χ 2 test (P = 0.023). R is the correlation coefficient (R=−0.18; top). Immunohistochemical staining of BRAF and USP28 on sequential sections of ME2082B (Biomax) melanoma tissue microarray. Red staining indicates positive immunoreactivity. Bars, 50 µm. Dashed boxes indicate zoomed area. (F) WM164 or WM164 USP28CRSP cells treated with escalating doses of vemurafenib (PLX4032) for 72 h. Viability was assessed using CellTiter Glo as described by the manufacturer. Data represent the mean of six replicates. (G) A373C.6 cells or A373C.6 USP28 knockdown cells treated with escalating doses of vemurafenib (PLX4032) for 72 h. Viability was assessed using CellTiter Glo as described by the manufacturer. Data represent the mean of six replicates. (H) Immunoblot analysis of WM164 or WM164 USP28 knockdown cells treated with different concentrations of vemurafenib (PLX4032) for 1 h. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. (I) Immunoblot analysis of A373C.6 or A373C.6 USP28 knockdown cells treated with different concentrations of vemurafenib (PLX4032) for 1 h. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. For respective immunoblots proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls.

Techniques Used: Mutagenesis, Infection, shRNA, Immunohistochemistry, Staining, Microarray, Western Blot

Identification of USP28 as negative regulator of ERK signaling. (A) Third round selection of DUB screen. Immunoblot analysis of 293T cells expressing shRNA vectors targeting the indicated DUBs. ( B) Immunoblot analysis of WM164 melanoma cells treated with indicated concentrations of PLX4032 and probed with the indicated antibodies. (C) Immunoblot analysis of 293T cells expressing shRNA vectors (A–D) targeting USP28 and probed with the indicated antibodies. (D) Immunoblot analysis of 293T cells expressing USP28 shRNA vectors (C and D). Whole cell extracts were probed with the indicated antibodies. (E) Immunoblot analysis in 293T cells expressing Flag-USP28 or Flag-USP28DD. Whole cell extracts were probed with the indicated antibodies. (F) Immunoblot analysis showing 293T cells expressing Myc-FBW7 and BRAF. Whole cell extracts were probed with the indicated antibodies. (G–I) Immunoprecipitation of endogenous USP28 in 293T cells and an immunoblot analysis of indicated proteins BRAF (G), ARAF (H), and CRAF (I). (J) Immunoblot analysis of 293T cells expressing BRAF (V600E), BRAF (V600E) CPD, and wild-type Myc-FBW7 and immunoprecipitated with an anti-BRAF antibody. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls.
Figure Legend Snippet: Identification of USP28 as negative regulator of ERK signaling. (A) Third round selection of DUB screen. Immunoblot analysis of 293T cells expressing shRNA vectors targeting the indicated DUBs. ( B) Immunoblot analysis of WM164 melanoma cells treated with indicated concentrations of PLX4032 and probed with the indicated antibodies. (C) Immunoblot analysis of 293T cells expressing shRNA vectors (A–D) targeting USP28 and probed with the indicated antibodies. (D) Immunoblot analysis of 293T cells expressing USP28 shRNA vectors (C and D). Whole cell extracts were probed with the indicated antibodies. (E) Immunoblot analysis in 293T cells expressing Flag-USP28 or Flag-USP28DD. Whole cell extracts were probed with the indicated antibodies. (F) Immunoblot analysis showing 293T cells expressing Myc-FBW7 and BRAF. Whole cell extracts were probed with the indicated antibodies. (G–I) Immunoprecipitation of endogenous USP28 in 293T cells and an immunoblot analysis of indicated proteins BRAF (G), ARAF (H), and CRAF (I). (J) Immunoblot analysis of 293T cells expressing BRAF (V600E), BRAF (V600E) CPD, and wild-type Myc-FBW7 and immunoprecipitated with an anti-BRAF antibody. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls.

Techniques Used: Selection, Expressing, shRNA, Immunoprecipitation

Down-regulation of USP28 impairs the effects of vemurafenib in vivo. (A) Waterfall plot showing the percentage change in tumor volume for the individual tumors at day 15 for untreated controls, day 30 for mice treated twice daily with 35 mg/kg and day 37 for mice treated with 75 mg/kg ( n = 12). (B) Quantification of nude mice bearing xenograft tumors of A373 C.6 or A373 C.6 USP28 knockdown cells ( n = 12). Mice were treated twice daily with vemurafenib (PLX4032; 35 mg/kg, light blue) for 30 d or (75 mg/kg, dark blue) for 37 d (end of experiment). Points indicate mean tumor volume; bars, SE. A two-tailed Student’s t test compares the two treated grouped populations of control cells versus USP28-depleted cells. ****, P
Figure Legend Snippet: Down-regulation of USP28 impairs the effects of vemurafenib in vivo. (A) Waterfall plot showing the percentage change in tumor volume for the individual tumors at day 15 for untreated controls, day 30 for mice treated twice daily with 35 mg/kg and day 37 for mice treated with 75 mg/kg ( n = 12). (B) Quantification of nude mice bearing xenograft tumors of A373 C.6 or A373 C.6 USP28 knockdown cells ( n = 12). Mice were treated twice daily with vemurafenib (PLX4032; 35 mg/kg, light blue) for 30 d or (75 mg/kg, dark blue) for 37 d (end of experiment). Points indicate mean tumor volume; bars, SE. A two-tailed Student’s t test compares the two treated grouped populations of control cells versus USP28-depleted cells. ****, P

Techniques Used: In Vivo, Mouse Assay, Two Tailed Test

23) Product Images from "Ras isoforms: signaling specificities in CD40 pathway"

Article Title: Ras isoforms: signaling specificities in CD40 pathway

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/s12964-019-0497-1

Kinases Lyn and Syk selectively regulate Ras isoform’s activation through Ras-GEFs. a CD40 activates the upstream kinases Lyn and Syk in a dose-dependent manner, with lower doses activating Syk and higher doses activating Lyn. b Immunoblot analysis of total Ras and activated Ras isoforms in the lysates of un-transfected or Syk- or Lyn-specific siRNA transfected, anti-CD40 antibody (3 μg/ml) treated P388D1 cells. c Immunoblot analyses of phospho-tyrosine (p-tyr-Vav)), and Vav translocated Sos-1/2 (Tr-Sos-1/2), translocated Ras-GRP (Tr-RasGRP), CD71, syk, lyn and β-actin in the lysates of untreated or Syk siRNA or Lyn siRNA or anti-CD40 antibody (3 μg/ml) treated P388D1 cells. d Immunoblot analysis of phospho-tyrosine (p-tyr Vav) and Vav translocated Sos-1/2, translocated Ras GRP, CD71, Lyn and phospho-Lyn, Syk and phospho-Syk (p-syk) in the lysates of untreated or Syk inhibitor (Syk Inh, 3 μM; Calbiochem, San Diego, CA) or PP-1 (340 nM; BIOMOL International, PA) treated and/or anti-CD40 antibody (3 μg/ml) treated macrophages. e Analysis of the association of Syk or Lyn with Sos-1/2, Vav, and Ras-GRP in the co-immunoprecipitates from the lysates of anti-CD40 antibody (1 μg/ml, 3 μg/ml and 6 μg/ml) treated macrophages.
Figure Legend Snippet: Kinases Lyn and Syk selectively regulate Ras isoform’s activation through Ras-GEFs. a CD40 activates the upstream kinases Lyn and Syk in a dose-dependent manner, with lower doses activating Syk and higher doses activating Lyn. b Immunoblot analysis of total Ras and activated Ras isoforms in the lysates of un-transfected or Syk- or Lyn-specific siRNA transfected, anti-CD40 antibody (3 μg/ml) treated P388D1 cells. c Immunoblot analyses of phospho-tyrosine (p-tyr-Vav)), and Vav translocated Sos-1/2 (Tr-Sos-1/2), translocated Ras-GRP (Tr-RasGRP), CD71, syk, lyn and β-actin in the lysates of untreated or Syk siRNA or Lyn siRNA or anti-CD40 antibody (3 μg/ml) treated P388D1 cells. d Immunoblot analysis of phospho-tyrosine (p-tyr Vav) and Vav translocated Sos-1/2, translocated Ras GRP, CD71, Lyn and phospho-Lyn, Syk and phospho-Syk (p-syk) in the lysates of untreated or Syk inhibitor (Syk Inh, 3 μM; Calbiochem, San Diego, CA) or PP-1 (340 nM; BIOMOL International, PA) treated and/or anti-CD40 antibody (3 μg/ml) treated macrophages. e Analysis of the association of Syk or Lyn with Sos-1/2, Vav, and Ras-GRP in the co-immunoprecipitates from the lysates of anti-CD40 antibody (1 μg/ml, 3 μg/ml and 6 μg/ml) treated macrophages.

Techniques Used: Activation Assay, Transfection

Ras isoforms differ in their effector specificity and Ras isoforms exhibit a difference in symmetry in functional site surface roughness. a H-Ras, K-Ras and N-Ras specific siRNAs modulated the anti-CD40 antibody (3 μg/ml)-induced downstream Ras effector proteins Raf-1 and PI-3 K in P388D1 cells, as assessed by Western blot. siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 10 min. b Analysis of association of H-Ras, K-Ras or N-Ras with PI3K or Raf in the co-immunoprecipitates from the lysates of anti-CD40 antibody (1 μg/ml and 6 μg/ml) treated macrophages for 10 mins. c The Correlation Dimensions (CD) were calculated to quantify the dependency in the spatial organization of surface atoms. CD magnitude varies within 2.00 and 3.00 for protein space. Results of this analysis too point unambiguously to similar organizational principles between dependencies (correlations) amongst surface atoms of H-Ras and K-Ras and their marked difference to that in N-Ras isoform. d Investigations were carried out on representative structures, viz., on pdb id.: 3K9L for H-Ras, pdb id.: 3GFT for K-Ras and pdb id.: 3CON for N-Ras. Extent of symmetry for all six aforementioned biophysical properties show that H-Ras and K-Ras molecules have a similar scheme of structural organization in interior and exterior of the molecule, which differs from that observed in N-Ras molecule. Such differences are significant because sequence identity between H-Ras, K-Ras and N-Ras molecules is found to be ≈ 89%.
Figure Legend Snippet: Ras isoforms differ in their effector specificity and Ras isoforms exhibit a difference in symmetry in functional site surface roughness. a H-Ras, K-Ras and N-Ras specific siRNAs modulated the anti-CD40 antibody (3 μg/ml)-induced downstream Ras effector proteins Raf-1 and PI-3 K in P388D1 cells, as assessed by Western blot. siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 10 min. b Analysis of association of H-Ras, K-Ras or N-Ras with PI3K or Raf in the co-immunoprecipitates from the lysates of anti-CD40 antibody (1 μg/ml and 6 μg/ml) treated macrophages for 10 mins. c The Correlation Dimensions (CD) were calculated to quantify the dependency in the spatial organization of surface atoms. CD magnitude varies within 2.00 and 3.00 for protein space. Results of this analysis too point unambiguously to similar organizational principles between dependencies (correlations) amongst surface atoms of H-Ras and K-Ras and their marked difference to that in N-Ras isoform. d Investigations were carried out on representative structures, viz., on pdb id.: 3K9L for H-Ras, pdb id.: 3GFT for K-Ras and pdb id.: 3CON for N-Ras. Extent of symmetry for all six aforementioned biophysical properties show that H-Ras and K-Ras molecules have a similar scheme of structural organization in interior and exterior of the molecule, which differs from that observed in N-Ras molecule. Such differences are significant because sequence identity between H-Ras, K-Ras and N-Ras molecules is found to be ≈ 89%.

Techniques Used: Functional Assay, Western Blot, Sequencing

Ras isoforms are differentially activated in CD40 signaling. a , b Immunoblot analysis of activated and total Ras in thioglycolate-elicited BALB/c-derived macrophages stimulated with various concentrations of anti-CD40 for 1, 5, 10, 15 and 20 mins ( a ). b . Immunoblot analysis of activated and total Ras in thioglycolate-elicited BALB/c-derived macrophages stimulated with various concentrations of CD40 ligand for 1, 5, 10, 15 and 20 mins ( b ). Densitometric quantifications of the blots are shown ( c , d ) Immunoblot analysis of activated H, K and N-Ras and total Ras in macrophages untreated or stimulated with 1, 3, 6 μg/ml of anti-CD40 ( c ) or 20, 50, 100 ng of CD40 ligand ( d ) for 7 min. Densitometric quantifications of the blots are shown.
Figure Legend Snippet: Ras isoforms are differentially activated in CD40 signaling. a , b Immunoblot analysis of activated and total Ras in thioglycolate-elicited BALB/c-derived macrophages stimulated with various concentrations of anti-CD40 for 1, 5, 10, 15 and 20 mins ( a ). b . Immunoblot analysis of activated and total Ras in thioglycolate-elicited BALB/c-derived macrophages stimulated with various concentrations of CD40 ligand for 1, 5, 10, 15 and 20 mins ( b ). Densitometric quantifications of the blots are shown ( c , d ) Immunoblot analysis of activated H, K and N-Ras and total Ras in macrophages untreated or stimulated with 1, 3, 6 μg/ml of anti-CD40 ( c ) or 20, 50, 100 ng of CD40 ligand ( d ) for 7 min. Densitometric quantifications of the blots are shown.

Techniques Used: Derivative Assay

Ras isoforms differentially modulate reciprocal CD40 signaling. a Immunoblot analysis for checking the specificity of siRNA used for silencing respective isoforms. Only the Isoform-specific siRNA but not the control siRNA silenced the Ras isoforms. b Control siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 15 min ; using Western blot CD40 (3 μg/ml)-induced p38MAPK or ERK-1/2 activation was assessed. c - e siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 15 min. siRNAs specific for H-Ras ( c ), K-Ras ( d ) and N-Ras ( e ) modulated the CD40 (3 μg/ml)-induced p38MAPK or ERK-1/2 activation, as assessed by Western blot. e - g siRNAs for H-Ras ( f ), K-Ras ( g ) and N-Ras ( h ) modulated the anti-CD40 antibody (3 μg/ml)-induced IL-12 or IL-10 productions as measured by using specific cytokine ELISA kits, in a macrophage-like mouse cell line P388D1. siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 48 h. Data represented as mean ± SEM; *, p ≤ 0.05; ** p ≤ 0.005 compared with anti-CD40 stimulated P388D1 cells.
Figure Legend Snippet: Ras isoforms differentially modulate reciprocal CD40 signaling. a Immunoblot analysis for checking the specificity of siRNA used for silencing respective isoforms. Only the Isoform-specific siRNA but not the control siRNA silenced the Ras isoforms. b Control siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 15 min ; using Western blot CD40 (3 μg/ml)-induced p38MAPK or ERK-1/2 activation was assessed. c - e siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 15 min. siRNAs specific for H-Ras ( c ), K-Ras ( d ) and N-Ras ( e ) modulated the CD40 (3 μg/ml)-induced p38MAPK or ERK-1/2 activation, as assessed by Western blot. e - g siRNAs for H-Ras ( f ), K-Ras ( g ) and N-Ras ( h ) modulated the anti-CD40 antibody (3 μg/ml)-induced IL-12 or IL-10 productions as measured by using specific cytokine ELISA kits, in a macrophage-like mouse cell line P388D1. siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 48 h. Data represented as mean ± SEM; *, p ≤ 0.05; ** p ≤ 0.005 compared with anti-CD40 stimulated P388D1 cells.

Techniques Used: Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay

Differential activation of GEFs modulates CD40-induced counteractive effector functions. a - c Immunoblot analyses of total and phosphorylated (p-) p38MAPK and ERK-1/2 in the anti-CD40 antibody (3 μg/ml) stimulated P388D1 cells: un-transfected or transfected with Sos-1/2, Vav and Ras-GRP specific siRNA. siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 15 min. Suppression of Sos-1/2 ( a ) reduced CD40 (3 μg/ml)-induced ERK-1/2 phosphorylation whereas silencing of Ras-GRP ( b ) and Vav ( c ) using respective siRNA reduced CD40 (3 μg/ml)-induced p38MAPK phosphorylation. d - f ELISA of IL-10 and IL-12p70 from the culture supernatants of the P388D1 cells transfected with the indicated GEF siRNA, followed by treatment with anti-CD40 antibody (3 μg/ml) for 48 h. respectively . p ≤ 0.05; ** p ≤ 0.005; *** p ≤ 0.0005 compared with control P388D1 cells.
Figure Legend Snippet: Differential activation of GEFs modulates CD40-induced counteractive effector functions. a - c Immunoblot analyses of total and phosphorylated (p-) p38MAPK and ERK-1/2 in the anti-CD40 antibody (3 μg/ml) stimulated P388D1 cells: un-transfected or transfected with Sos-1/2, Vav and Ras-GRP specific siRNA. siRNA treated P388D1 cells were stimulated with anti-CD40 antibody (3 μg/ml) for 15 min. Suppression of Sos-1/2 ( a ) reduced CD40 (3 μg/ml)-induced ERK-1/2 phosphorylation whereas silencing of Ras-GRP ( b ) and Vav ( c ) using respective siRNA reduced CD40 (3 μg/ml)-induced p38MAPK phosphorylation. d - f ELISA of IL-10 and IL-12p70 from the culture supernatants of the P388D1 cells transfected with the indicated GEF siRNA, followed by treatment with anti-CD40 antibody (3 μg/ml) for 48 h. respectively . p ≤ 0.05; ** p ≤ 0.005; *** p ≤ 0.0005 compared with control P388D1 cells.

Techniques Used: Activation Assay, Transfection, Enzyme-linked Immunosorbent Assay

Ras isoforms differentially associate with Ras-guanine nucleotide exchange factor (Ras-GEF) in CD40 signaling. a , b CD40 activates the GEFs in a dose-dependent manner. Higher dose activates Ras-GRP and Vav whereas lower dose activates Sos-1/2. Immunoblot analysis of Vav or phospho tyrosine, CD71, translocated (Tr) Sos-1/2 or RasGRP in the lysates of macrophages treated with the indicated concentrations of anti-CD40 antibody ( a ). Densitometric quantifications of the blots are shown in the bottom panel ( b ). c Analysis of the association of Sos-1/2, Ras-GRP and Vav after immunoprecipitation with H-Ras, K-Ras and N-Ras in the lysates of macrophages treated with the indicated doses of anti-CD40 antibody. H-Ras and K-Ras associate with Ras-GRP whereas N-Ras associates with Sos-1/2 in macrophages stimulated with the indicated doses of anti-CD40 antibody. Also shown is the association of Ras-GRP with Vav by co-immunoprecipitation in the above-mentioned macrophage lysates. Vav associated with Ras GRP. Cells were stimulated for 3 min. d The siRNA for GEFs (Sos-1/2, Vav, and Ras-GRP) silenced respective GEFs in siRNA treated-inset. e - g Immunoblot analysis of total Ras and activated Ras isoforms in the lysates of un-transfected or Sos-1/2-, Vav-, Ras-GRP-specific siRNA transfected P388D1 cells stimulated with anti-CD40 (3 μg/ml) for 7 min. Sos-1/2 siRNA reduced N-Ras activation ( e ) whereas Vav siRNA and Ras-GRP siRNA reduced H-Ras and K-Ras activation ( f , g ).
Figure Legend Snippet: Ras isoforms differentially associate with Ras-guanine nucleotide exchange factor (Ras-GEF) in CD40 signaling. a , b CD40 activates the GEFs in a dose-dependent manner. Higher dose activates Ras-GRP and Vav whereas lower dose activates Sos-1/2. Immunoblot analysis of Vav or phospho tyrosine, CD71, translocated (Tr) Sos-1/2 or RasGRP in the lysates of macrophages treated with the indicated concentrations of anti-CD40 antibody ( a ). Densitometric quantifications of the blots are shown in the bottom panel ( b ). c Analysis of the association of Sos-1/2, Ras-GRP and Vav after immunoprecipitation with H-Ras, K-Ras and N-Ras in the lysates of macrophages treated with the indicated doses of anti-CD40 antibody. H-Ras and K-Ras associate with Ras-GRP whereas N-Ras associates with Sos-1/2 in macrophages stimulated with the indicated doses of anti-CD40 antibody. Also shown is the association of Ras-GRP with Vav by co-immunoprecipitation in the above-mentioned macrophage lysates. Vav associated with Ras GRP. Cells were stimulated for 3 min. d The siRNA for GEFs (Sos-1/2, Vav, and Ras-GRP) silenced respective GEFs in siRNA treated-inset. e - g Immunoblot analysis of total Ras and activated Ras isoforms in the lysates of un-transfected or Sos-1/2-, Vav-, Ras-GRP-specific siRNA transfected P388D1 cells stimulated with anti-CD40 (3 μg/ml) for 7 min. Sos-1/2 siRNA reduced N-Ras activation ( e ) whereas Vav siRNA and Ras-GRP siRNA reduced H-Ras and K-Ras activation ( f , g ).

Techniques Used: Immunoprecipitation, Transfection, Activation Assay

24) Product Images from "A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic"

Article Title: A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004647

Localization of GFP-A-RAF, GFP-AR149 and endogenous A-RAF in mammalian cells. A. Top row: HeLa cells were transiently transfected with pEGFP bearing the indicated genes for 2 days. GFP fusion proteins were detected by fluorescence microscopy. GFP-A-RAF is present throughout the cytoplasm and accumulates around the nucleus. In contrast, GFP-AR149 labels punctate structures often aligned on strings. Strings were disassembled by treatment with Nocodazole. A C-RAF fragment orthologous to AR149 is GFP-C-RAF(C4). Representative images are shown. Scale bar = 10 µm. Middle row: HeLa cells were cotransfected with RFP-AR149 and GFP-ARF6 as described above. RFP and GFP fluorescences were recorded separately. Bottom row: HeLa cells were cotransfected with Myc-A-RAF and GFP-ARF6. After two days transfected cells were treated with digitonin to extract cytosol and processed for detection of A-RAF and ARF6 as described in Figure 3C . Boxed areas are shown at higher magnification. Arrows indicate vesicles with colocalization. Representative images are shown. Scale bar = 10 µm. B. Cells were fractionated using “ProteoExtract Subcellular Proteome Extraction Kit” (Calbiochem) and analyzed by immunoblotting. antibodies against following proteins were used as compartmental markers: vimentin as cytoskeletal marker, PARP as nuclear marker, 2MPK as cytosolic marker. Both A-RAF and AR149 are co-fractionating predominantly with cytoskeleton and cytosol. Small portion of AR149 was also found in plasma membrane fraction. C. HeLa cells were treated with digitonin to extract cytosol. After fixation and washing, immunofluorescence microscopy with antibodies against A-RAF and against β-tubulin was carried out. The boxed area is shown at higher magnification. Small A-RAF positive vesicles are at the periphery and line microtubules (arrows). Representative images are shown. Scale bar = 10 µm.
Figure Legend Snippet: Localization of GFP-A-RAF, GFP-AR149 and endogenous A-RAF in mammalian cells. A. Top row: HeLa cells were transiently transfected with pEGFP bearing the indicated genes for 2 days. GFP fusion proteins were detected by fluorescence microscopy. GFP-A-RAF is present throughout the cytoplasm and accumulates around the nucleus. In contrast, GFP-AR149 labels punctate structures often aligned on strings. Strings were disassembled by treatment with Nocodazole. A C-RAF fragment orthologous to AR149 is GFP-C-RAF(C4). Representative images are shown. Scale bar = 10 µm. Middle row: HeLa cells were cotransfected with RFP-AR149 and GFP-ARF6 as described above. RFP and GFP fluorescences were recorded separately. Bottom row: HeLa cells were cotransfected with Myc-A-RAF and GFP-ARF6. After two days transfected cells were treated with digitonin to extract cytosol and processed for detection of A-RAF and ARF6 as described in Figure 3C . Boxed areas are shown at higher magnification. Arrows indicate vesicles with colocalization. Representative images are shown. Scale bar = 10 µm. B. Cells were fractionated using “ProteoExtract Subcellular Proteome Extraction Kit” (Calbiochem) and analyzed by immunoblotting. antibodies against following proteins were used as compartmental markers: vimentin as cytoskeletal marker, PARP as nuclear marker, 2MPK as cytosolic marker. Both A-RAF and AR149 are co-fractionating predominantly with cytoskeleton and cytosol. Small portion of AR149 was also found in plasma membrane fraction. C. HeLa cells were treated with digitonin to extract cytosol. After fixation and washing, immunofluorescence microscopy with antibodies against A-RAF and against β-tubulin was carried out. The boxed area is shown at higher magnification. Small A-RAF positive vesicles are at the periphery and line microtubules (arrows). Representative images are shown. Scale bar = 10 µm.

Techniques Used: Transfection, Fluorescence, Microscopy, Marker, Immunofluorescence

25) Product Images from "TIMP-2 disrupts FGF-2-induced downstream signaling pathways"

Article Title: TIMP-2 disrupts FGF-2-induced downstream signaling pathways

Journal:

doi: 10.1016/j.mvr.2008.07.003

Effect of TIMP-2 on FGF-2/FGFR signaling pathways and cell proliferation in integrin α3 siRNA-transfected A549 cancer cells
Figure Legend Snippet: Effect of TIMP-2 on FGF-2/FGFR signaling pathways and cell proliferation in integrin α3 siRNA-transfected A549 cancer cells

Techniques Used: Transfection

Down-regulation of integrin α3 expression abrogates the suppressive effects of TIMP-2 on FGF-2-induced p42/44 MAPK and cell proliferation
Figure Legend Snippet: Down-regulation of integrin α3 expression abrogates the suppressive effects of TIMP-2 on FGF-2-induced p42/44 MAPK and cell proliferation

Techniques Used: Expressing

26) Product Images from "miR-518b Enhances Human Trophoblast Cell Proliferation Through Targeting Rap1b and Activating Ras-MAPK Signal"

Article Title: miR-518b Enhances Human Trophoblast Cell Proliferation Through Targeting Rap1b and Activating Ras-MAPK Signal

Journal: Frontiers in Endocrinology

doi: 10.3389/fendo.2018.00100

Localization of miR-518b and Rap1b in human placental villi. (A,B) The miR-518b expression was measured by in situ hybridization (blue) in human placental villi (A) and trophoblast cell column (B) at gestational weeks 7–9. (D,E) The localization of Rap1b was detected by immunohistochemistry in human placental villi (D) and trophoblast cell column (E) . (C,F) showed the negative control of miR-518b (scrambled miR) and Rap1b (rabbit igG). Scale bar represent 50 µm.
Figure Legend Snippet: Localization of miR-518b and Rap1b in human placental villi. (A,B) The miR-518b expression was measured by in situ hybridization (blue) in human placental villi (A) and trophoblast cell column (B) at gestational weeks 7–9. (D,E) The localization of Rap1b was detected by immunohistochemistry in human placental villi (D) and trophoblast cell column (E) . (C,F) showed the negative control of miR-518b (scrambled miR) and Rap1b (rabbit igG). Scale bar represent 50 µm.

Techniques Used: Expressing, In Situ Hybridization, Immunohistochemistry, Negative Control

RAS and MAPK are the downstream pathways of miR-518b and Rap1b. (A–C) Western blotting to measure the phosphorylation level of Raf-1 and ERK1/2 after transfected with miR-518b or scramble negative control (NC) and Rap1b-encoding plasmid (Rap1b) or control plasmid (Ctrl). (B,C) represent the statistical results of p-Raf-1 and p-ERK1/2 shown in (A) . (D) Change in miR-518b upon hypoxia condition (2% O 2 ). Each of the tests was replicated for three times. Data are presented as mean ± SEM. * P
Figure Legend Snippet: RAS and MAPK are the downstream pathways of miR-518b and Rap1b. (A–C) Western blotting to measure the phosphorylation level of Raf-1 and ERK1/2 after transfected with miR-518b or scramble negative control (NC) and Rap1b-encoding plasmid (Rap1b) or control plasmid (Ctrl). (B,C) represent the statistical results of p-Raf-1 and p-ERK1/2 shown in (A) . (D) Change in miR-518b upon hypoxia condition (2% O 2 ). Each of the tests was replicated for three times. Data are presented as mean ± SEM. * P

Techniques Used: Western Blot, Transfection, Negative Control, Plasmid Preparation

Expression pattern of Rap1b in preeclamptic placentas. (A,B) Real-time PCR to measure the mRNA expression of Rap1b in basal plate (A) and chorionic plate (B) of placentas from severe preeclamptic patients (sPE, n = 8) and gestational week-matched preterm labor (PTL, n = 8). (C–F) Western blotting to measure the protein level of Rap1b in basal plate (C) and chorionic plate (D) of the placentas from PTL and sPE patients, each of the lanes stood for one patient. (E) and (F) represent the statistical results of (C,D) , respectively. The correlation between the expression of miR-518b and Rap1b in placentas is shown as (G) . Data are presented as mean ± SEM. * P
Figure Legend Snippet: Expression pattern of Rap1b in preeclamptic placentas. (A,B) Real-time PCR to measure the mRNA expression of Rap1b in basal plate (A) and chorionic plate (B) of placentas from severe preeclamptic patients (sPE, n = 8) and gestational week-matched preterm labor (PTL, n = 8). (C–F) Western blotting to measure the protein level of Rap1b in basal plate (C) and chorionic plate (D) of the placentas from PTL and sPE patients, each of the lanes stood for one patient. (E) and (F) represent the statistical results of (C,D) , respectively. The correlation between the expression of miR-518b and Rap1b in placentas is shown as (G) . Data are presented as mean ± SEM. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Validation of Rap1b as a direct target of miR-518b in human trophoblast cells. (A–D) Change in Rap1b expression upon miR-518b overexpression in human trophoblast cells. The HTR8/SVneo cells were transfected with miR-518b or scramble negative control (NC). The efficiency of transfection was shown in (A) , and the expression of Rap1b was revealed by real-time PCR (B) and Western blotting (C,D) . (E) Schematic diagram of the plasmid construction for dual-luciferase report assay. (F) Dual-luciferase reporter assay in HTR8/SVneo cells transfected with wild-type (WT) or mutated (MUT) reporter plasmid, and with miR-518b or scramble NC. Each of the tests was replicated for three times. Data are presented as mean ± SEM. * P
Figure Legend Snippet: Validation of Rap1b as a direct target of miR-518b in human trophoblast cells. (A–D) Change in Rap1b expression upon miR-518b overexpression in human trophoblast cells. The HTR8/SVneo cells were transfected with miR-518b or scramble negative control (NC). The efficiency of transfection was shown in (A) , and the expression of Rap1b was revealed by real-time PCR (B) and Western blotting (C,D) . (E) Schematic diagram of the plasmid construction for dual-luciferase report assay. (F) Dual-luciferase reporter assay in HTR8/SVneo cells transfected with wild-type (WT) or mutated (MUT) reporter plasmid, and with miR-518b or scramble NC. Each of the tests was replicated for three times. Data are presented as mean ± SEM. * P

Techniques Used: Expressing, Over Expression, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Luciferase, Reporter Assay

miR-518b transfection accelerates the cell proliferation. (A) MTT assay to measure the cell viability in HTR8/SVneo cells. The cells were transfected with miR-518b together with Rap1b overexpression plasmid, and MTT assays were performed at 24, 48, and 72 h after transfection. (B) BrdU incorporation assay to measure the cell proliferation in HTR8/SVneo cells transfected with Rap1b overexpression plasmid. (C,D) Cell cycle assay for HTR8/SVneo cells transfected with Rap1b overexpression plasmid. (E,F) Costaining of propidium iodide (PI) and FITC-Annexin V for cell death/apoptosis assay for HTR8/SVneo cells transfected with Rap1b overexpression plasmid. MTT assay was replicated for five times, and other tests were replicated for three times. Data are presented as mean ± SEM. * P
Figure Legend Snippet: miR-518b transfection accelerates the cell proliferation. (A) MTT assay to measure the cell viability in HTR8/SVneo cells. The cells were transfected with miR-518b together with Rap1b overexpression plasmid, and MTT assays were performed at 24, 48, and 72 h after transfection. (B) BrdU incorporation assay to measure the cell proliferation in HTR8/SVneo cells transfected with Rap1b overexpression plasmid. (C,D) Cell cycle assay for HTR8/SVneo cells transfected with Rap1b overexpression plasmid. (E,F) Costaining of propidium iodide (PI) and FITC-Annexin V for cell death/apoptosis assay for HTR8/SVneo cells transfected with Rap1b overexpression plasmid. MTT assay was replicated for five times, and other tests were replicated for three times. Data are presented as mean ± SEM. * P

Techniques Used: Transfection, MTT Assay, Over Expression, Plasmid Preparation, BrdU Incorporation Assay, Cell Cycle Assay, Apoptosis Assay

27) Product Images from "Inhibitory effect of insulin-like growth factor-binding protein-7 (IGFBP7) on in vitro angiogenesis of vascular endothelial cells in the rat corpus luteum"

Article Title: Inhibitory effect of insulin-like growth factor-binding protein-7 (IGFBP7) on in vitro angiogenesis of vascular endothelial cells in the rat corpus luteum

Journal: The Journal of Reproduction and Development

doi: 10.1262/jrd.2014-069

Effects of IGFBP7 on VEGFA-induced phosphorylation of mitogen-activated protein kinases in LECs. LECs were treated with VEGFA (10 ng/ml) for 20 min following incubation with IGFBP7 (10 or 160 ng/ml) for 24 h. Cell lysate was subjected to Western blot analysis using the following primary antibodies: phospho-c-Raf (Ser338), phospho-mitogen-activated protein kinase kinase (MEK)1/2 (Ser217/221), phospho-ERK1/2 (Th202/Tyr204), and MEK1/2. Representative blots are shown. β-actin served as a loading control. BP7: IGFBP7.
Figure Legend Snippet: Effects of IGFBP7 on VEGFA-induced phosphorylation of mitogen-activated protein kinases in LECs. LECs were treated with VEGFA (10 ng/ml) for 20 min following incubation with IGFBP7 (10 or 160 ng/ml) for 24 h. Cell lysate was subjected to Western blot analysis using the following primary antibodies: phospho-c-Raf (Ser338), phospho-mitogen-activated protein kinase kinase (MEK)1/2 (Ser217/221), phospho-ERK1/2 (Th202/Tyr204), and MEK1/2. Representative blots are shown. β-actin served as a loading control. BP7: IGFBP7.

Techniques Used: Incubation, Western Blot

28) Product Images from "RACK1 Targets the Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Pathway To Link Integrin Engagement with Focal Adhesion Disassembly and Cell Motility †RACK1 Targets the Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Pathway To Link Integrin Engagement with Focal Adhesion Disassembly and Cell Motility † ▿"

Article Title: RACK1 Targets the Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Pathway To Link Integrin Engagement with Focal Adhesion Disassembly and Cell Motility †RACK1 Targets the Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Pathway To Link Integrin Engagement with Focal Adhesion Disassembly and Cell Motility † ▿

Journal:

doi: 10.1128/MCB.00598-07

RACK1 associates with components of the ERK pathway. (A) Coimmunoprecipitation of MP1 with RACK1 from CCL39 cells transiently transfected with either control vector or FLAG-RACK1 and MP1 constructs. RACK1 was precipitated and detected with FLAG M2 antibody.
Figure Legend Snippet: RACK1 associates with components of the ERK pathway. (A) Coimmunoprecipitation of MP1 with RACK1 from CCL39 cells transiently transfected with either control vector or FLAG-RACK1 and MP1 constructs. RACK1 was precipitated and detected with FLAG M2 antibody.

Techniques Used: Transfection, Plasmid Preparation, Construct

29) Product Images from "Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis"

Article Title: Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis

Journal: The Korean Journal of Parasitology

doi: 10.3347/kjp.2015.53.4.371

Effects of PI3K and MAPK inhibitors at TNF-α production in T. vaginalis-infected SiHa cells. (A) Viability of SiHa cells after treatment with inhibitors of PI3K and MAPK pathways. (B-E) SiHa cells were pretreated with 2, 20, and 200 nM of PI3K inhibitor wortmannin (B), 2, 10, and 50 μM of ERK1/2 inhibitor PD98059 (C), 1, 5, and 25 μM of p38 MAPK inhibitor SB203580 (D), and 2, 10, and 50 μM of JNK1/2 inhibitor SP600125 (E) for 1 hr, and treated with live T. vaginalis MOI 2 for 1, 2, and 4 hr. The levels of TNF-α (pg/ml) in the culture supernatants were analyzed by ELISA. Data are presented as the means±SD. * Statistically significant difference between 2 groups. ** P
Figure Legend Snippet: Effects of PI3K and MAPK inhibitors at TNF-α production in T. vaginalis-infected SiHa cells. (A) Viability of SiHa cells after treatment with inhibitors of PI3K and MAPK pathways. (B-E) SiHa cells were pretreated with 2, 20, and 200 nM of PI3K inhibitor wortmannin (B), 2, 10, and 50 μM of ERK1/2 inhibitor PD98059 (C), 1, 5, and 25 μM of p38 MAPK inhibitor SB203580 (D), and 2, 10, and 50 μM of JNK1/2 inhibitor SP600125 (E) for 1 hr, and treated with live T. vaginalis MOI 2 for 1, 2, and 4 hr. The levels of TNF-α (pg/ml) in the culture supernatants were analyzed by ELISA. Data are presented as the means±SD. * Statistically significant difference between 2 groups. ** P

Techniques Used: Infection, Enzyme-linked Immunosorbent Assay

30) Product Images from "Isoforms of Spectrin and Ankyrin Reflect the Functional Topography of the Mouse Kidney"

Article Title: Isoforms of Spectrin and Ankyrin Reflect the Functional Topography of the Mouse Kidney

Journal: PLoS ONE

doi: 10.1371/journal.pone.0142687

Spectrin and ankyrin abundance in kidney cortex and medulla. (A) Western blot analysis of cortical and medullary tissue. Three kidneys were analyzed; samples from two are shown as paired lanes. Aquaporin1 (AQP1) and aquaporin2 (AQP2) were used as regional markers, AQP1 being evenly distributed while AQP2 marked medullary tissue. (B) Immunofluorescent micrographs of renal cortex and medulla stained for the dominant spectrins. IM, inner medulla; OM, outer medulla. While αΙΙ spectrin staining is uniform, the distributions of βΙΙ vs. βΙΙΙ spectrin tend to be complementary, especially in the inner medullary region.
Figure Legend Snippet: Spectrin and ankyrin abundance in kidney cortex and medulla. (A) Western blot analysis of cortical and medullary tissue. Three kidneys were analyzed; samples from two are shown as paired lanes. Aquaporin1 (AQP1) and aquaporin2 (AQP2) were used as regional markers, AQP1 being evenly distributed while AQP2 marked medullary tissue. (B) Immunofluorescent micrographs of renal cortex and medulla stained for the dominant spectrins. IM, inner medulla; OM, outer medulla. While αΙΙ spectrin staining is uniform, the distributions of βΙΙ vs. βΙΙΙ spectrin tend to be complementary, especially in the inner medullary region.

Techniques Used: Western Blot, Staining

Spectrins associate with internal organelles. ImmunoEM micrographs highlight a pool of αΙΙ/βΙΙ spectrin in association with a variety of organelles including canaliculi (arrow heads) and coated vesicles (arrows) in the cytoplasm and along the lateral and apical membranes of proximal and distal tubule cells and the collecting duct. The boxed areas are enlarged in the right column. PC, principal cell; IC, intercalated cell.
Figure Legend Snippet: Spectrins associate with internal organelles. ImmunoEM micrographs highlight a pool of αΙΙ/βΙΙ spectrin in association with a variety of organelles including canaliculi (arrow heads) and coated vesicles (arrows) in the cytoplasm and along the lateral and apical membranes of proximal and distal tubule cells and the collecting duct. The boxed areas are enlarged in the right column. PC, principal cell; IC, intercalated cell.

Techniques Used:

Spectrin and ankyrin mRNA in mouse kidney. Spectrin and ankyrin mRNA expression were measured by RT-PCR and qPCR with primers summarized in Table 1 . (A) Amplimers were detected for spectrins βΙ, βΙΙ, βΙΙΙ, and αΙΙ and for ankyrins R (Ank1), G (Ank2), and B (Ank3). NC is no-RNA control. (B) The levels of the various transcripts varied widely, as measured by qPCR. Relative abundance is presented. Results shown for three separate determinations. Error bars ±1SD from mean. (Inset) RT-PCR detected two alternative transcripts of βΙΙ spectrin (βΙΙΣ1 βΙΙΣ2), but only one of two potential transcripts of βΙ spectrin.
Figure Legend Snippet: Spectrin and ankyrin mRNA in mouse kidney. Spectrin and ankyrin mRNA expression were measured by RT-PCR and qPCR with primers summarized in Table 1 . (A) Amplimers were detected for spectrins βΙ, βΙΙ, βΙΙΙ, and αΙΙ and for ankyrins R (Ank1), G (Ank2), and B (Ank3). NC is no-RNA control. (B) The levels of the various transcripts varied widely, as measured by qPCR. Relative abundance is presented. Results shown for three separate determinations. Error bars ±1SD from mean. (Inset) RT-PCR detected two alternative transcripts of βΙΙ spectrin (βΙΙΣ1 βΙΙΣ2), but only one of two potential transcripts of βΙ spectrin.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Distribution of alpha and beta spectrin. (A,B)The distribution of spectrins βΙΙ and βΙΙΙ relate to NKCC2 or calbindin1. NKCC2 marks the thick ascending loop of Henle (TAL); calbindin1 marks the distal convoluted tubule (DCT). Note that βΙΙΙ spectrin spares the TAL, but marks the DCT. (C) Co-stains of αΙΙ spectrin (red) with βΙΙ or βΙΙΙ spectrin (green) show that αII spectrin, present throughout the kidney, is complemented by either βΙΙ or βΙΙΙ spectrin.
Figure Legend Snippet: Distribution of alpha and beta spectrin. (A,B)The distribution of spectrins βΙΙ and βΙΙΙ relate to NKCC2 or calbindin1. NKCC2 marks the thick ascending loop of Henle (TAL); calbindin1 marks the distal convoluted tubule (DCT). Note that βΙΙΙ spectrin spares the TAL, but marks the DCT. (C) Co-stains of αΙΙ spectrin (red) with βΙΙ or βΙΙΙ spectrin (green) show that αII spectrin, present throughout the kidney, is complemented by either βΙΙ or βΙΙΙ spectrin.

Techniques Used:

31) Product Images from "The B Lymphocyte Adaptor Molecule of 32 kD (Bam32) Regulates B Cell Antigen Receptor Signaling and Cell Survival"

Article Title: The B Lymphocyte Adaptor Molecule of 32 kD (Bam32) Regulates B Cell Antigen Receptor Signaling and Cell Survival

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20011524

Impairment of BCR-induced NF-AT and NF-κB activation in the absence of Bam32. Cells transfected with NF-AT (top) or NF-κB (bottom) luciferase reporter construct were stimulated with graded dose of anti-μ (M4) for 6 h, lysed, and luciferase activity was assayed using a luminometer. The relative luciferase activity of medium and BCR stimulation was expressed in reference to that of PMA/ionomycin stimulation. The results were shown by average and SEM of triplicate cultures. One experiment representative of four independent experiments is shown.
Figure Legend Snippet: Impairment of BCR-induced NF-AT and NF-κB activation in the absence of Bam32. Cells transfected with NF-AT (top) or NF-κB (bottom) luciferase reporter construct were stimulated with graded dose of anti-μ (M4) for 6 h, lysed, and luciferase activity was assayed using a luminometer. The relative luciferase activity of medium and BCR stimulation was expressed in reference to that of PMA/ionomycin stimulation. The results were shown by average and SEM of triplicate cultures. One experiment representative of four independent experiments is shown.

Techniques Used: Activation Assay, Transfection, Luciferase, Construct, Activity Assay

32) Product Images from "Xentry, a new class of cell-penetrating peptide uniquely equipped for delivery of drugs"

Article Title: Xentry, a new class of cell-penetrating peptide uniquely equipped for delivery of drugs

Journal: Scientific Reports

doi: 10.1038/srep01661

Xentry-mediated delivery of antibody and siRNA cargoes into cells. (a) Uptake of Xentry-conjugated and unconjugated FITC-labelled rabbit IgG by HepG2 cells. Cell nuclei were stained blue with DAPI. (b) Representative cell showing staining of tubulin by a Cy3-labelled anti-β-tubulin antibody delivered to HepG2 cells by Xentry. HepG2 cells were incubated with the Xentry- anti-β-tubulin antibody conjugate for 1 h, washed, then incubated and photographed 24 h later. For comparison, a representative fixed and permeabilized cell is shown which has been stained by the free anti-β-tubulin antibody. The unconjugated anti-β-tubulin antibody could not penetrate and stain non-permeabilized HepG2 cells. (c) The abilities of a Xentry-conjugated and unconjugated anti-B-raf antibody to induce the apoptosis of WM-266-4 melanoma cells, as evidenced by staining of cells with annexin-V fluos (green). (d) A Xentry-KALA fusion peptide delivers an siRNA directed against B-raf transcripts into WM-266-4 melanoma cells, causing cell apoptosis as evidenced by staining of cells with annexin-V fluos (green). For comparison, the anti-B-raf siRNA was transfected into cells using the PolyMag agent. The siRNA was omitted from controls. Scale bar, 50 μm.
Figure Legend Snippet: Xentry-mediated delivery of antibody and siRNA cargoes into cells. (a) Uptake of Xentry-conjugated and unconjugated FITC-labelled rabbit IgG by HepG2 cells. Cell nuclei were stained blue with DAPI. (b) Representative cell showing staining of tubulin by a Cy3-labelled anti-β-tubulin antibody delivered to HepG2 cells by Xentry. HepG2 cells were incubated with the Xentry- anti-β-tubulin antibody conjugate for 1 h, washed, then incubated and photographed 24 h later. For comparison, a representative fixed and permeabilized cell is shown which has been stained by the free anti-β-tubulin antibody. The unconjugated anti-β-tubulin antibody could not penetrate and stain non-permeabilized HepG2 cells. (c) The abilities of a Xentry-conjugated and unconjugated anti-B-raf antibody to induce the apoptosis of WM-266-4 melanoma cells, as evidenced by staining of cells with annexin-V fluos (green). (d) A Xentry-KALA fusion peptide delivers an siRNA directed against B-raf transcripts into WM-266-4 melanoma cells, causing cell apoptosis as evidenced by staining of cells with annexin-V fluos (green). For comparison, the anti-B-raf siRNA was transfected into cells using the PolyMag agent. The siRNA was omitted from controls. Scale bar, 50 μm.

Techniques Used: Staining, Incubation, Transfection

33) Product Images from "Quercetin and Sorafenib as a Novel and Effective Couple in Programmed Cell Death Induction in Human Gliomas"

Article Title: Quercetin and Sorafenib as a Novel and Effective Couple in Programmed Cell Death Induction in Human Gliomas

Journal: Neurotoxicity Research

doi: 10.1007/s12640-013-9452-x

The level of Hsp27 and Hsp72 expression in T98G ( a , c ) and MOGGCCM ( b , d ) cells after transfection with specific anti-Hsp27 ( a , b ) or anti-Hsp72 ( c , d ) siRNA (si27 and si72, respectively) and subsequent quercetin (Q) and sorafenib (S) treatment, estimated by immunoblotting. T98G cells were incubated with 50 μM of quercetin and 0.75 μM sorafenib, while MOGGCCM cells with 30 μM (Q30) of quercetin and 0.75 μM of sorafenib. Protein level was normalised according to β-actin expression. C control, TR transfection reagent, TRsi transfection reagent with specific siRNA, SQ simultaneous quercetin and sorafenib treatment, * P
Figure Legend Snippet: The level of Hsp27 and Hsp72 expression in T98G ( a , c ) and MOGGCCM ( b , d ) cells after transfection with specific anti-Hsp27 ( a , b ) or anti-Hsp72 ( c , d ) siRNA (si27 and si72, respectively) and subsequent quercetin (Q) and sorafenib (S) treatment, estimated by immunoblotting. T98G cells were incubated with 50 μM of quercetin and 0.75 μM sorafenib, while MOGGCCM cells with 30 μM (Q30) of quercetin and 0.75 μM of sorafenib. Protein level was normalised according to β-actin expression. C control, TR transfection reagent, TRsi transfection reagent with specific siRNA, SQ simultaneous quercetin and sorafenib treatment, * P

Techniques Used: Expressing, Transfection, Incubation

The level of cytochrome c ( a cytoplasmic, b mitochondrial fraction), Ras ( c ), Raf ( d ), LC3 ( e ) and beclin 1 ( f ) expression with representative blots and the activity of caspase 3, 8, 9 ( g ) after sorafenib (S) and quercetin (Q) treatment for 48 h in T98G cells transfected with specific siRNA anti-Hsp27 (si27) and anti-Hsp72 (si72). The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, si27 or si72 specific siRNA blocking Hsp27 or Hsp72 expression, TR transfection reagent, TRsi transfection reagent with specific siRNA, * P
Figure Legend Snippet: The level of cytochrome c ( a cytoplasmic, b mitochondrial fraction), Ras ( c ), Raf ( d ), LC3 ( e ) and beclin 1 ( f ) expression with representative blots and the activity of caspase 3, 8, 9 ( g ) after sorafenib (S) and quercetin (Q) treatment for 48 h in T98G cells transfected with specific siRNA anti-Hsp27 (si27) and anti-Hsp72 (si72). The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, si27 or si72 specific siRNA blocking Hsp27 or Hsp72 expression, TR transfection reagent, TRsi transfection reagent with specific siRNA, * P

Techniques Used: Expressing, Activity Assay, Transfection, Blocking Assay

The effect of quercetin (Q) and sorafenib (S) on apoptosis, necrosis and autophagy induction in MOGGCCM ( a , c , e ) and T98G ( b , d , f ) cells transfected with specific siRNA anti-Hsp27 (si27) and anti-Hsp72 (si72). C control, TR transfection reagent, a , b cell death estimated by microscopic observation of cells stained with Hoechst 33342, propidium iodide, acridine orange, c , d apoptosis and necrosis induction estimated by flow cytometry with the Annexin V-FITC detection kit, e , f the mitochondrial membrane potential studied by flow cytometry after staining with DiOC 6 (3), * P
Figure Legend Snippet: The effect of quercetin (Q) and sorafenib (S) on apoptosis, necrosis and autophagy induction in MOGGCCM ( a , c , e ) and T98G ( b , d , f ) cells transfected with specific siRNA anti-Hsp27 (si27) and anti-Hsp72 (si72). C control, TR transfection reagent, a , b cell death estimated by microscopic observation of cells stained with Hoechst 33342, propidium iodide, acridine orange, c , d apoptosis and necrosis induction estimated by flow cytometry with the Annexin V-FITC detection kit, e , f the mitochondrial membrane potential studied by flow cytometry after staining with DiOC 6 (3), * P

Techniques Used: Transfection, Staining, Flow Cytometry, Cytometry

The level of cytochrome c ( a cytoplasmic, b mitochondrial fraction), Ras ( c ), Raf ( d ), LC3 ( e ) and beclin 1 ( f ) expression with representative blots and the activity of caspase 3, 8, 9 ( f ) after sorafenib (S) and quercetin (Q) treatment for 24 h in MOGGCCM cells transfected with specific siRNA anti-Hsp27 (si27) and anti-Hsp72 (si72). The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, si27 or si72 specific siRNA blocking Hsp27 or Hsp72 expression, TR transfection reagent, TRsi transfection reagent with specific siRNA * P
Figure Legend Snippet: The level of cytochrome c ( a cytoplasmic, b mitochondrial fraction), Ras ( c ), Raf ( d ), LC3 ( e ) and beclin 1 ( f ) expression with representative blots and the activity of caspase 3, 8, 9 ( f ) after sorafenib (S) and quercetin (Q) treatment for 24 h in MOGGCCM cells transfected with specific siRNA anti-Hsp27 (si27) and anti-Hsp72 (si72). The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, si27 or si72 specific siRNA blocking Hsp27 or Hsp72 expression, TR transfection reagent, TRsi transfection reagent with specific siRNA * P

Techniques Used: Expressing, Activity Assay, Transfection, Blocking Assay

The level of Hsp27 ( a ), Hsp72 ( b ), cytochrome c ( c cytoplasmic, d mitochondrial fraction), beclin 1 ( e ), LC3 ( f ), Ras ( g ) and Raf ( h ) expression with representative blots and the activity of caspase 3, 8, 9 ( i ) after sorafenib (S) and quercetin (Q) treatment for 48 h in T98G. The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, * P
Figure Legend Snippet: The level of Hsp27 ( a ), Hsp72 ( b ), cytochrome c ( c cytoplasmic, d mitochondrial fraction), beclin 1 ( e ), LC3 ( f ), Ras ( g ) and Raf ( h ) expression with representative blots and the activity of caspase 3, 8, 9 ( i ) after sorafenib (S) and quercetin (Q) treatment for 48 h in T98G. The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, * P

Techniques Used: Expressing, Activity Assay

The level of Hsp27 ( a ), Hsp72 ( b ), cytochrome c ( c cytoplasmic, d mitochondrial fraction), beclin 1 ( e ), LC3 ( f ), Ras ( g ) and Raf ( h ) expression with representative blots and the activity of caspase 3, 8, 9 (i) after sorafenib (S) and quercetin (Q) treatment for 24 h in MOGGCCM. The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, * P
Figure Legend Snippet: The level of Hsp27 ( a ), Hsp72 ( b ), cytochrome c ( c cytoplasmic, d mitochondrial fraction), beclin 1 ( e ), LC3 ( f ), Ras ( g ) and Raf ( h ) expression with representative blots and the activity of caspase 3, 8, 9 (i) after sorafenib (S) and quercetin (Q) treatment for 24 h in MOGGCCM. The data were normalised relative to β-actin (not shown). C control cells, SQ simultaneous drug treatment, * P

Techniques Used: Expressing, Activity Assay

34) Product Images from "Novel Histone Deacetylase Inhibitors with Enhanced Enzymatic Inhibition Effects and Potent in vitro and in vivo Anti-tumor Activities"

Article Title: Novel Histone Deacetylase Inhibitors with Enhanced Enzymatic Inhibition Effects and Potent in vitro and in vivo Anti-tumor Activities

Journal: ChemMedChem

doi: 10.1002/cmdc.201300297

Western blot analysis of c-Raf, p-Erk, Erk, p-Akt, Akt and histone H3 in U937 and MDA-MB-231 cell line after 24h of treatment with compounds ( D3 and D17 ) at 1 µM. Histone H3 was used as a loading control.
Figure Legend Snippet: Western blot analysis of c-Raf, p-Erk, Erk, p-Akt, Akt and histone H3 in U937 and MDA-MB-231 cell line after 24h of treatment with compounds ( D3 and D17 ) at 1 µM. Histone H3 was used as a loading control.

Techniques Used: Western Blot, Multiple Displacement Amplification

35) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Induces the Phosphatidylinositol 3-Kinase-PKC-?-MEK-ERK Signaling Pathway in Target Cells Early during Infection: Implications for Infectivity"

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Induces the Phosphatidylinositol 3-Kinase-PKC-?-MEK-ERK Signaling Pathway in Target Cells Early during Infection: Implications for Infectivity

Journal: Journal of Virology

doi: 10.1128/JVI.77.2.1524-1539.2003

HHV-8 induces MEK1/2 but not cRaf-1 in the target cells. (A) Kinetics of MEK1/2 induction. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 (lanes 2 to 6), or treated with 20% FBS for the indicated times. Cell lysates were resolved by SDS-10% PAGE and probed with anti-phospho-MEK1/2 antibodies. Membranes were stripped and reprobed with anti-MEK1/2 antibodies (middle panel) or with anti-β actin antibodies (bottom panel). (B) Inhibition of MEK1/2 inhibits the ERK1/2 activity induced by HHV-8. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 for 15 min (lane 2) or 30 min (lane 4), or incubated with 20% FBS for 15 min (lane 6). Cells were also preincubated with 10 μM MEK1/2 inhibitor U0126 for 1 h at 37°C and infected with virus in the presence of inhibitors for 15 and 30 min (lanes 3 and 5, respectively). Cell lysates were Western blotted and reacted with anti-phospho-ERK1/2 antibodies (top panel) or with anti-ERK2 antibodies (bottom panel). (C) HHV-8 does not induce cRaf-1 in the target cells. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 (lanes 2 to 6), or treated with LPS (1 μg/ml; lane 7) or TPA (10 nM; lane 8) for the indicated times. Lysates were either immunoprecipitated with total cRaf-1 antibody and subjected to a kinase assay with MEK as substrate (top panel) or used for Western analysis with either phospho-cRaf-1 or total cRaf-1 antibody (middle and lower panels, respectively).
Figure Legend Snippet: HHV-8 induces MEK1/2 but not cRaf-1 in the target cells. (A) Kinetics of MEK1/2 induction. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 (lanes 2 to 6), or treated with 20% FBS for the indicated times. Cell lysates were resolved by SDS-10% PAGE and probed with anti-phospho-MEK1/2 antibodies. Membranes were stripped and reprobed with anti-MEK1/2 antibodies (middle panel) or with anti-β actin antibodies (bottom panel). (B) Inhibition of MEK1/2 inhibits the ERK1/2 activity induced by HHV-8. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 for 15 min (lane 2) or 30 min (lane 4), or incubated with 20% FBS for 15 min (lane 6). Cells were also preincubated with 10 μM MEK1/2 inhibitor U0126 for 1 h at 37°C and infected with virus in the presence of inhibitors for 15 and 30 min (lanes 3 and 5, respectively). Cell lysates were Western blotted and reacted with anti-phospho-ERK1/2 antibodies (top panel) or with anti-ERK2 antibodies (bottom panel). (C) HHV-8 does not induce cRaf-1 in the target cells. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 (lanes 2 to 6), or treated with LPS (1 μg/ml; lane 7) or TPA (10 nM; lane 8) for the indicated times. Lysates were either immunoprecipitated with total cRaf-1 antibody and subjected to a kinase assay with MEK as substrate (top panel) or used for Western analysis with either phospho-cRaf-1 or total cRaf-1 antibody (middle and lower panels, respectively).

Techniques Used: Infection, Polyacrylamide Gel Electrophoresis, Inhibition, Activity Assay, Incubation, Western Blot, Immunoprecipitation, Kinase Assay

36) Product Images from "Pretreatment with DNA-damaging agents permits selective killing of checkpoint-deficient cells by microtubule-active drugs"

Article Title: Pretreatment with DNA-damaging agents permits selective killing of checkpoint-deficient cells by microtubule-active drugs

Journal: Journal of Clinical Investigation

doi:

Protection by p21 against PTX in p21 –/– . ( a ) Cells were infected with 10 moi Ad-p21, and after 24 hours, treated with 100 ng/mL PTX for 20 hours as indicated. Cells were lysed, and immunoblot for p53 and p21 was performed. ( b ) Cells were infected with 10 moi Ad-p21, and after 24 hours, cell-cycle analysis was performed. No doxorubicin or paclitaxel was added in this experiment. ( c ). Cells were infected with 10 moi Ad-p21, and after 24 hours, treated with 100 ng/mL PTX as indicated. MTT assays were performed after 2 days.
Figure Legend Snippet: Protection by p21 against PTX in p21 –/– . ( a ) Cells were infected with 10 moi Ad-p21, and after 24 hours, treated with 100 ng/mL PTX for 20 hours as indicated. Cells were lysed, and immunoblot for p53 and p21 was performed. ( b ) Cells were infected with 10 moi Ad-p21, and after 24 hours, cell-cycle analysis was performed. No doxorubicin or paclitaxel was added in this experiment. ( c ). Cells were infected with 10 moi Ad-p21, and after 24 hours, treated with 100 ng/mL PTX as indicated. MTT assays were performed after 2 days.

Techniques Used: Infection, Cell Cycle Assay, MTT Assay

Comparison of DOX- and p21-mediated cytoprotection. ( a ) HCT116 cells were treated with 100 ng/mL doxorubicin (DOX) or infected with 10 moi Ad-p21 (p21) for 36 hours, and then cell-cycle analysis was performed as described in Methods. The pie chart represents the percent of cells in each phase of the cell cycle: 1 = G1 phase; 2 = G2 phase; solid area = S phase. Control adenovirus, Ad-LacZ, did not change cell-cycle distribution. ( b ) HCT116 cells were pretreated with 100 ng/mL DOX for 16 hours or 10 moi Ad-p21 (p21) for 24 hours. A plus sign indicates that cells were treated with 100 ng/mL PTX for 20 hours; a minus sign indicates that they were left untreated. Cells were lysed after 20 hours, and p53, p21, Raf-1, and p120 were measured by immunoblot. ( c ) HCT116 cells were pretreated as already described here and then treated with 100 ng/mL PTX. After 48 hours the MTT assay was performed.
Figure Legend Snippet: Comparison of DOX- and p21-mediated cytoprotection. ( a ) HCT116 cells were treated with 100 ng/mL doxorubicin (DOX) or infected with 10 moi Ad-p21 (p21) for 36 hours, and then cell-cycle analysis was performed as described in Methods. The pie chart represents the percent of cells in each phase of the cell cycle: 1 = G1 phase; 2 = G2 phase; solid area = S phase. Control adenovirus, Ad-LacZ, did not change cell-cycle distribution. ( b ) HCT116 cells were pretreated with 100 ng/mL DOX for 16 hours or 10 moi Ad-p21 (p21) for 24 hours. A plus sign indicates that cells were treated with 100 ng/mL PTX for 20 hours; a minus sign indicates that they were left untreated. Cells were lysed after 20 hours, and p53, p21, Raf-1, and p120 were measured by immunoblot. ( c ) HCT116 cells were pretreated as already described here and then treated with 100 ng/mL PTX. After 48 hours the MTT assay was performed.

Techniques Used: Infection, Cell Cycle Assay, MTT Assay

Doxorubicin does not protect p21 –/– cells. ( a ) HCT116 and p21 –/– cells were incubated with 100 ng/mL doxorubicin for 24 hours and then assayed for mdm-2 and p21 proteins by immunoblot analysis. ( b ) HCT116 and p21 –/– cells were incubated with the indicated concentrations of doxorubicin for 24 hours, and then [ 3 H]thymidine incorporation was measured. ( c and d ) HCT116 ( c ) and p21 –/– cells ( d ) were pretreated with indicated dose of doxorubicin for 12 hours, and then cells were treated with 100 ng/mL PTX (gray bars) or left untreated (open bars). MTT test was performed after 2 days.
Figure Legend Snippet: Doxorubicin does not protect p21 –/– cells. ( a ) HCT116 and p21 –/– cells were incubated with 100 ng/mL doxorubicin for 24 hours and then assayed for mdm-2 and p21 proteins by immunoblot analysis. ( b ) HCT116 and p21 –/– cells were incubated with the indicated concentrations of doxorubicin for 24 hours, and then [ 3 H]thymidine incorporation was measured. ( c and d ) HCT116 ( c ) and p21 –/– cells ( d ) were pretreated with indicated dose of doxorubicin for 12 hours, and then cells were treated with 100 ng/mL PTX (gray bars) or left untreated (open bars). MTT test was performed after 2 days.

Techniques Used: Incubation, MTT Assay

Doxorubicin fails to protect p53 –/– cells. ( a ) Effects of doxorubicin on p53 and p21 proteins in HCT116, p21 –/– , and p53 –/– cells. Cells were incubated with (+) or without (–) 100 ng/mL doxorubicin for 16 hours and then assayed by immunoblot analysis. ( b ) Cells were pretreated with 100 ng/mL doxorubicin as indicated (DOX) and continued with or without 100 ng/mL paclitaxel (PTX). Cell survival was measured by MTT assay 2 days after PTX addition.
Figure Legend Snippet: Doxorubicin fails to protect p53 –/– cells. ( a ) Effects of doxorubicin on p53 and p21 proteins in HCT116, p21 –/– , and p53 –/– cells. Cells were incubated with (+) or without (–) 100 ng/mL doxorubicin for 16 hours and then assayed by immunoblot analysis. ( b ) Cells were pretreated with 100 ng/mL doxorubicin as indicated (DOX) and continued with or without 100 ng/mL paclitaxel (PTX). Cell survival was measured by MTT assay 2 days after PTX addition.

Techniques Used: Incubation, MTT Assay

Doxorubicin-induced G 2 arrest versus paclitaxel-induced mitotic arrest, Bcl-2 phosphorylation and cell death. HCT116 cells were incubated with 100 ng/mL doxorubicin (DOX) or left untreated. After 12–16 hours, 100 ng/mL paclitaxel (PTX) was added, if indicated. Pretreated cells continued in doxorubicin until harvest. ( a ) Cell-cycle distribution measured by FACS analysis was performed 24 hours after the addition of PTX. ( b ) Mitotic index was measured by DAPI staining after 24 hours after the addition of PTX. ( c ) Cells, treated as indicated, were photographed 16 hours after the addition of PTX. ( d ) Bcl-2 protein phosphorylation was demonstrated by immunoblot 12 hours after the addition of PTX. ( e ) Number of live cells, as measured by MTT assay, 2 days after the addition of PTX. ( f ) DNA replication, as measured by [ 3 H]thymidine incorporation, 16 hours after the addition of PTX. ( g ) The number of dead and live cells was counted with trypan blue staining 3 days after the addition of PTX. Open bars represent live cells (excluded trypan blue); filled bars represent dead cells (stained with trypan blue).
Figure Legend Snippet: Doxorubicin-induced G 2 arrest versus paclitaxel-induced mitotic arrest, Bcl-2 phosphorylation and cell death. HCT116 cells were incubated with 100 ng/mL doxorubicin (DOX) or left untreated. After 12–16 hours, 100 ng/mL paclitaxel (PTX) was added, if indicated. Pretreated cells continued in doxorubicin until harvest. ( a ) Cell-cycle distribution measured by FACS analysis was performed 24 hours after the addition of PTX. ( b ) Mitotic index was measured by DAPI staining after 24 hours after the addition of PTX. ( c ) Cells, treated as indicated, were photographed 16 hours after the addition of PTX. ( d ) Bcl-2 protein phosphorylation was demonstrated by immunoblot 12 hours after the addition of PTX. ( e ) Number of live cells, as measured by MTT assay, 2 days after the addition of PTX. ( f ) DNA replication, as measured by [ 3 H]thymidine incorporation, 16 hours after the addition of PTX. ( g ) The number of dead and live cells was counted with trypan blue staining 3 days after the addition of PTX. Open bars represent live cells (excluded trypan blue); filled bars represent dead cells (stained with trypan blue).

Techniques Used: Incubation, FACS, Staining, MTT Assay

37) Product Images from "Histone deacetylation of NIS promoter underlies BRAF V600E-promoted NIS silencing in thyroid cancer"

Article Title: Histone deacetylation of NIS promoter underlies BRAF V600E-promoted NIS silencing in thyroid cancer

Journal: Endocrine-Related Cancer

doi: 10.1530/ERC-13-0399

Global histone acetylation changes in PCCL3/BRAF and PCCL3 cells upon various treatments. 10 6 cells were planted in 12-well plates in each condition. PCCL3/BRAF cells were treated with 1 μg/ml DOX to induce BRAF V600E expression (left), PCCL3 cells were transiently expressed with BRAF V600E (middle), and PCCL3 cells were treated with DOX at 1 μg/ml or SAHA at 0.5 μM (right), followed by cell lysis and protein preparation for western-blotting 48 h later. BRAF V600E expression was successfully induced, accompanied by P-ERK activation and increase in global H3K9/14 acetylation. SAHA at 0.5 μM increased global histone H3K9/14 acetylation in both PCCL3/BRAF cells and WT PCCL3 cells. PCCL3 cells treated with DOX at 1 μg/ml did not show change in histone H3K9/14 acetylation, unlike treatment with SAHA. CON, control; DOX, doxycycline; p-ERK, phosphorylated ERK; H3K9/14ac, acetylated H3K9/14.
Figure Legend Snippet: Global histone acetylation changes in PCCL3/BRAF and PCCL3 cells upon various treatments. 10 6 cells were planted in 12-well plates in each condition. PCCL3/BRAF cells were treated with 1 μg/ml DOX to induce BRAF V600E expression (left), PCCL3 cells were transiently expressed with BRAF V600E (middle), and PCCL3 cells were treated with DOX at 1 μg/ml or SAHA at 0.5 μM (right), followed by cell lysis and protein preparation for western-blotting 48 h later. BRAF V600E expression was successfully induced, accompanied by P-ERK activation and increase in global H3K9/14 acetylation. SAHA at 0.5 μM increased global histone H3K9/14 acetylation in both PCCL3/BRAF cells and WT PCCL3 cells. PCCL3 cells treated with DOX at 1 μg/ml did not show change in histone H3K9/14 acetylation, unlike treatment with SAHA. CON, control; DOX, doxycycline; p-ERK, phosphorylated ERK; H3K9/14ac, acetylated H3K9/14.

Techniques Used: Expressing, Lysis, Western Blot, Activation Assay

The role of BRAF V600E/MAP kinase pathway in modulating histone acetylation at the human NIS promoter in thyroid cancer BCPAP cells. (A) Treatment of BCPAP cells with the MEK inhibitor AZD6244 (AZD) at 1 μM and the BRAF V600E inhibitor PLX4032 (PLX) at 1 μM for 48 h completely suppressed downstream P-ERK and had no significant effect on global H3K9/14 acetylation. The histone deacetylases inhibitor SAHA at 0.5 μM increased global H3K9/14 acetylation in BCPAP cells. (B) Effects of various inhibitors on H3K9/14 acetylation at the human NIS promoter. ChIP assay was used to analyze histone acetylation. AZD increased H3K9/14 acetylation at regions P1 and P2, PLX increased H3K9/14 acetylation at region P1, and SAHA increased H3K9/14 acetylation at regions P1 and P2 of the human NIS promoter. (C) Effects of various inhibitors on H4K16 acetylation at the human NIS promoter. Both AZD and PLX increased H4K16 acetylation at region P1 of the human NIS promoter and SAHA increased H4K16 acetylation in all the regions. The levels of histone acetylation were expressed as fraction of the input DNA. Each bar represents the mean value ± s.e.m. of at least three different experiments. The human NIS promoter regions are as presented in Table 1 . P-ERK, phosphorylated ERK; H3K9/14ac, acetylated H3K9/14; CON, control; AZD, AZD6244; PLX, PLX4032. * P
Figure Legend Snippet: The role of BRAF V600E/MAP kinase pathway in modulating histone acetylation at the human NIS promoter in thyroid cancer BCPAP cells. (A) Treatment of BCPAP cells with the MEK inhibitor AZD6244 (AZD) at 1 μM and the BRAF V600E inhibitor PLX4032 (PLX) at 1 μM for 48 h completely suppressed downstream P-ERK and had no significant effect on global H3K9/14 acetylation. The histone deacetylases inhibitor SAHA at 0.5 μM increased global H3K9/14 acetylation in BCPAP cells. (B) Effects of various inhibitors on H3K9/14 acetylation at the human NIS promoter. ChIP assay was used to analyze histone acetylation. AZD increased H3K9/14 acetylation at regions P1 and P2, PLX increased H3K9/14 acetylation at region P1, and SAHA increased H3K9/14 acetylation at regions P1 and P2 of the human NIS promoter. (C) Effects of various inhibitors on H4K16 acetylation at the human NIS promoter. Both AZD and PLX increased H4K16 acetylation at region P1 of the human NIS promoter and SAHA increased H4K16 acetylation in all the regions. The levels of histone acetylation were expressed as fraction of the input DNA. Each bar represents the mean value ± s.e.m. of at least three different experiments. The human NIS promoter regions are as presented in Table 1 . P-ERK, phosphorylated ERK; H3K9/14ac, acetylated H3K9/14; CON, control; AZD, AZD6244; PLX, PLX4032. * P

Techniques Used: Chromatin Immunoprecipitation

Effect of BRAF V600E on H3K9/14 acetylation in the rat NIS promoter in rat thyroid cells. (A) In PCCL3/BRAF cells, DOX at 1 μg/ml induced BRAF V600E expression after 48 h, accompanied by decrease in H3K9/14 acetylation in the regions P1, P2, and P3 of the NIS promoter, most dramatically in P1. SAHA at 0.5 μM increased H3K9/14 acetylation in regions P1, P3, P4, and NUE. (B) In PCCL3 cells, transient expression of BRAF V600E decreased H3K9/14 acetylation at exon 1 and P1 of the NIS promoter while transfection with WT-BRAF had no effect. Histone acetylation status was analyzed by ChIP. The levels of H3K9K14 acetylation are expressed as fraction of the input DNA (material before immunoprecipitation). Threshold cycles ( C t) were determined for ChIP samples and the input DNA, and the relative amount of immunoprecipitated DNA (% ChIP signal per input DNA) was calculated as 100 2Δ C t . Each bar represents the mean value ± s.e.m. of at least three different experiments. The promoter regions are as presented in Table 1 . EXON, exon 1; NUE, nuclear upstream enhancer; CON, control; DOX, doxycycline; rNIS, rat NIS. * P
Figure Legend Snippet: Effect of BRAF V600E on H3K9/14 acetylation in the rat NIS promoter in rat thyroid cells. (A) In PCCL3/BRAF cells, DOX at 1 μg/ml induced BRAF V600E expression after 48 h, accompanied by decrease in H3K9/14 acetylation in the regions P1, P2, and P3 of the NIS promoter, most dramatically in P1. SAHA at 0.5 μM increased H3K9/14 acetylation in regions P1, P3, P4, and NUE. (B) In PCCL3 cells, transient expression of BRAF V600E decreased H3K9/14 acetylation at exon 1 and P1 of the NIS promoter while transfection with WT-BRAF had no effect. Histone acetylation status was analyzed by ChIP. The levels of H3K9K14 acetylation are expressed as fraction of the input DNA (material before immunoprecipitation). Threshold cycles ( C t) were determined for ChIP samples and the input DNA, and the relative amount of immunoprecipitated DNA (% ChIP signal per input DNA) was calculated as 100 2Δ C t . Each bar represents the mean value ± s.e.m. of at least three different experiments. The promoter regions are as presented in Table 1 . EXON, exon 1; NUE, nuclear upstream enhancer; CON, control; DOX, doxycycline; rNIS, rat NIS. * P

Techniques Used: Expressing, Transfection, Chromatin Immunoprecipitation, Immunoprecipitation

38) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Induces the Phosphatidylinositol 3-Kinase-PKC-?-MEK-ERK Signaling Pathway in Target Cells Early during Infection: Implications for Infectivity"

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Induces the Phosphatidylinositol 3-Kinase-PKC-?-MEK-ERK Signaling Pathway in Target Cells Early during Infection: Implications for Infectivity

Journal: Journal of Virology

doi: 10.1128/JVI.77.2.1524-1539.2003

HHV-8 induces MEK1/2 but not cRaf-1 in the target cells. (A) Kinetics of MEK1/2 induction. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 (lanes 2 to 6), or treated with 20% FBS for the indicated times. Cell lysates were resolved by SDS-10% PAGE and probed with anti-phospho-MEK1/2 antibodies. Membranes were stripped and reprobed with anti-MEK1/2 antibodies (middle panel) or with anti-β actin antibodies (bottom panel). (B) Inhibition of MEK1/2 inhibits the ERK1/2 activity induced by HHV-8. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 for 15 min (lane 2) or 30 min (lane 4), or incubated with 20% FBS for 15 min (lane 6). Cells were also preincubated with 10 μM MEK1/2 inhibitor U0126 for 1 h at 37°C and infected with virus in the presence of inhibitors for 15 and 30 min (lanes 3 and 5, respectively). Cell lysates were Western blotted and reacted with anti-phospho-ERK1/2 antibodies (top panel) or with anti-ERK2 antibodies (bottom panel). (C) HHV-8 does not induce cRaf-1 in the target cells. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 (lanes 2 to 6), or treated with LPS (1 μg/ml; lane 7) or TPA (10 nM; lane 8) for the indicated times. Lysates were either immunoprecipitated with total cRaf-1 antibody and subjected to a kinase assay with MEK as substrate (top panel) or used for Western analysis with either phospho-cRaf-1 or total cRaf-1 antibody (middle and lower panels, respectively).
Figure Legend Snippet: HHV-8 induces MEK1/2 but not cRaf-1 in the target cells. (A) Kinetics of MEK1/2 induction. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 (lanes 2 to 6), or treated with 20% FBS for the indicated times. Cell lysates were resolved by SDS-10% PAGE and probed with anti-phospho-MEK1/2 antibodies. Membranes were stripped and reprobed with anti-MEK1/2 antibodies (middle panel) or with anti-β actin antibodies (bottom panel). (B) Inhibition of MEK1/2 inhibits the ERK1/2 activity induced by HHV-8. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 for 15 min (lane 2) or 30 min (lane 4), or incubated with 20% FBS for 15 min (lane 6). Cells were also preincubated with 10 μM MEK1/2 inhibitor U0126 for 1 h at 37°C and infected with virus in the presence of inhibitors for 15 and 30 min (lanes 3 and 5, respectively). Cell lysates were Western blotted and reacted with anti-phospho-ERK1/2 antibodies (top panel) or with anti-ERK2 antibodies (bottom panel). (C) HHV-8 does not induce cRaf-1 in the target cells. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 (lanes 2 to 6), or treated with LPS (1 μg/ml; lane 7) or TPA (10 nM; lane 8) for the indicated times. Lysates were either immunoprecipitated with total cRaf-1 antibody and subjected to a kinase assay with MEK as substrate (top panel) or used for Western analysis with either phospho-cRaf-1 or total cRaf-1 antibody (middle and lower panels, respectively).

Techniques Used: Infection, Polyacrylamide Gel Electrophoresis, Inhibition, Activity Assay, Incubation, Western Blot, Immunoprecipitation, Kinase Assay

39) Product Images from "Ginkgolide B inhibits renal cyst development in in vitro and in vivo cyst models"

Article Title: Ginkgolide B inhibits renal cyst development in in vitro and in vivo cyst models

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00356.2011

Ginkgolide B (GB) inhibits Madin-Darby canine kidney (MDCK) cell cyst formation. A : representative light micrographs of MDCK cells cultured in collagen gels. Light micrographs were taken on day 6 after cell seeding. MDCK cells were cultured without forskolin (FSK; top ) or with 2 μM GB and without FSK (the second from top ) or with 10 μM FSK (third from top ) or 10 μM forskolin plus 2 μM GB ( bottom ). Scale bar = 50 μm. B : MDCK cyst formation rate. Open bars show the total numbers of colonies (cysts colonies plus noncyst colonies) per well on day 6 after MDCK cells were incubated without (control) or with GB at indicated concentrations in the presence of 10 μM FSK. Filled bars show the numbers of cysts with a diameter > 50 μm (means ± SD; n = 3). * P
Figure Legend Snippet: Ginkgolide B (GB) inhibits Madin-Darby canine kidney (MDCK) cell cyst formation. A : representative light micrographs of MDCK cells cultured in collagen gels. Light micrographs were taken on day 6 after cell seeding. MDCK cells were cultured without forskolin (FSK; top ) or with 2 μM GB and without FSK (the second from top ) or with 10 μM FSK (third from top ) or 10 μM forskolin plus 2 μM GB ( bottom ). Scale bar = 50 μm. B : MDCK cyst formation rate. Open bars show the total numbers of colonies (cysts colonies plus noncyst colonies) per well on day 6 after MDCK cells were incubated without (control) or with GB at indicated concentrations in the presence of 10 μM FSK. Filled bars show the numbers of cysts with a diameter > 50 μm (means ± SD; n = 3). * P

Techniques Used: Cell Culture, Incubation

40) Product Images from "Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA"

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

Journal: British Journal of Pharmacology

doi: 10.1111/j.1476-5381.2010.00869.x

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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used:

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)
Figure Legend 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)

Techniques Used:

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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used:

(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
Figure Legend 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

Techniques Used: Transfection

Related Articles

Dominant Negative Mutation:

Article Title: PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer
Article Snippet: .. Reagents The materials were purchased and obtained as follows: Recombinant human EGF, HGF and IGF-1 were from R & D Systems (Abingdon, UK); pCMV6-PP4R1 (NM_005134), pCMV6-PP4R2 (NM_174907), pCMV6-PP4C (NM_002720), pCMV6-PATZ1 variant 3 (NM_032052), and pCMV6-PATZ1 variant 4 (NM_032051) plasmids were from ORIGENE (Rockville, MD); PATZ1 variant 4 and PP4R2 genes from pCMV6-PATZ1 and pCMV6-PP4R2 plasmids subcloned into pCMV6-AC-mCFP plasmids (ORIGENE) were applied for the establishment of PATZ1- and PP4R2-transduced stable cells. pCMV2-IKKβ WT (wild type; WT), pCMV2-IKKβ S177ES181E (dominant active; DA) and pCMV2-IKKβ K44M (dominant negative; DN) were obtained from Addgene (Cambridge, MA; plasmid numbers 11103 and 11104) and were originally created by Dr. Anjana Rao laboratory. control siRNAs, PP4R1-siRNA, PP4R2-siRNA, PP4C-siRNA and PATZ1-siRNA were all from Santa Cruz Biotechnology (Santa Cruz, CA); two shRNA HuSH 29mer shRNA (TF302311A and TF302311C) constructs against PP4R2 in pRFP-C-RS vector and the non-effective 29-mer scrambled shRNA (TR30015) were from ORIGENE; mouse anti-Vimentin (V9) antibodies and recombinant PGE2 were from Sigma-Aldrich (St Louis, MO); phosphatidylinositol (3,4,5)-trisphosphate diC16 (PI(3,4,5)P3 diC16) were from Echelon Biosciences (Salt Lake City, UT); Anti-PP4R1 (ab70624), anti-PP4R2 (ab70631), anti-PP4C (ab16475), anti-PP4R3α (ab70635), anti-PP4R3β (ab70622), anti-PP4R4 (ab111419), and anti-PATZ1 (ab154025) rabbit polyclonal antibodies were from Abcam (Cambridge, UK); mouse anti-PP1 (E-9), mouse anti-PP5 (H-7), rabbit anti-PHB (H-80), rabbit anti-IKKα/β (H-470), mouse anti-β-actin (C-4), goat anti-COX-2 (M-19), rabbit anti-E-cadherin (H-108), rabbit anti-ZEB1 (H-102), rabbit anti-Snail (H-130), rabbit anti-Twist (H-81), and rabbit anti-PATZ1 (H-300) antibodies as well as horseradish peroxidase-conjugated anti-mouse IgG, horseradish peroxidase-conjugated anti-goat IgG and horseradish peroxidase-conjugated anti-rabbit IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-PI3Kp85, rabbit anti-PI3Kp85Y458 , rabbit anti-Akt, rabbit anti-phospho-AktSer473 , rabbit anti-phospho-IKKα/βS176/S180 and rabbit anti-phospho-NF-κBp65S536 , horseradish peroxidase-conjugated mouse-anti-rabbit IgG (Conformation Specific; L27A9) antibodies were from Cell Signaling Technology (Beverly, MA); and rabbit anti-IκB, rabbit anti-NF-κBp65 and rabbit anti-Raf-1 antibodies were from Millipore (Temecula, CA). .. Cell culture A549 cell line (ATCC: CCL-185), H1299 (ATCC: CRL-5803), CL1-0 and CL1-5 cells [ , ] were maintained in DMEM or RPMI medium (GibcoBRL Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; GibcoBRL Life Technologies) and 1% penicillin-streptomycin-neomycin (GibcoBRL Life Technologies).

Immunohistochemistry:

Article Title: Angiogenesis in the Primate Ovulatory Follicle Is Stimulated by Luteinizing Hormone via Prostaglandin E2 1
Article Snippet: .. To assess cAMP levels, cells were grown to 80% confluence, then switched to basal media with 1% fetal bovine serum overnight before treatment with PGE2 or PTGER agonists (at concentrations listed above for immunohistochemistry) or isoproterenol (2 μM; Sigma) in basal media for 8 h. Media were harvested and stored at −20°C until assay for cAMP by enzyme immunoassay (EIA; Cayman) as previously described [ ]. .. Proliferation was measured using the MTT Cell Proliferation Assay (ATCC).

shRNA:

Article Title: PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer
Article Snippet: .. Reagents The materials were purchased and obtained as follows: Recombinant human EGF, HGF and IGF-1 were from R & D Systems (Abingdon, UK); pCMV6-PP4R1 (NM_005134), pCMV6-PP4R2 (NM_174907), pCMV6-PP4C (NM_002720), pCMV6-PATZ1 variant 3 (NM_032052), and pCMV6-PATZ1 variant 4 (NM_032051) plasmids were from ORIGENE (Rockville, MD); PATZ1 variant 4 and PP4R2 genes from pCMV6-PATZ1 and pCMV6-PP4R2 plasmids subcloned into pCMV6-AC-mCFP plasmids (ORIGENE) were applied for the establishment of PATZ1- and PP4R2-transduced stable cells. pCMV2-IKKβ WT (wild type; WT), pCMV2-IKKβ S177ES181E (dominant active; DA) and pCMV2-IKKβ K44M (dominant negative; DN) were obtained from Addgene (Cambridge, MA; plasmid numbers 11103 and 11104) and were originally created by Dr. Anjana Rao laboratory. control siRNAs, PP4R1-siRNA, PP4R2-siRNA, PP4C-siRNA and PATZ1-siRNA were all from Santa Cruz Biotechnology (Santa Cruz, CA); two shRNA HuSH 29mer shRNA (TF302311A and TF302311C) constructs against PP4R2 in pRFP-C-RS vector and the non-effective 29-mer scrambled shRNA (TR30015) were from ORIGENE; mouse anti-Vimentin (V9) antibodies and recombinant PGE2 were from Sigma-Aldrich (St Louis, MO); phosphatidylinositol (3,4,5)-trisphosphate diC16 (PI(3,4,5)P3 diC16) were from Echelon Biosciences (Salt Lake City, UT); Anti-PP4R1 (ab70624), anti-PP4R2 (ab70631), anti-PP4C (ab16475), anti-PP4R3α (ab70635), anti-PP4R3β (ab70622), anti-PP4R4 (ab111419), and anti-PATZ1 (ab154025) rabbit polyclonal antibodies were from Abcam (Cambridge, UK); mouse anti-PP1 (E-9), mouse anti-PP5 (H-7), rabbit anti-PHB (H-80), rabbit anti-IKKα/β (H-470), mouse anti-β-actin (C-4), goat anti-COX-2 (M-19), rabbit anti-E-cadherin (H-108), rabbit anti-ZEB1 (H-102), rabbit anti-Snail (H-130), rabbit anti-Twist (H-81), and rabbit anti-PATZ1 (H-300) antibodies as well as horseradish peroxidase-conjugated anti-mouse IgG, horseradish peroxidase-conjugated anti-goat IgG and horseradish peroxidase-conjugated anti-rabbit IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-PI3Kp85, rabbit anti-PI3Kp85Y458 , rabbit anti-Akt, rabbit anti-phospho-AktSer473 , rabbit anti-phospho-IKKα/βS176/S180 and rabbit anti-phospho-NF-κBp65S536 , horseradish peroxidase-conjugated mouse-anti-rabbit IgG (Conformation Specific; L27A9) antibodies were from Cell Signaling Technology (Beverly, MA); and rabbit anti-IκB, rabbit anti-NF-κBp65 and rabbit anti-Raf-1 antibodies were from Millipore (Temecula, CA). .. Cell culture A549 cell line (ATCC: CCL-185), H1299 (ATCC: CRL-5803), CL1-0 and CL1-5 cells [ , ] were maintained in DMEM or RPMI medium (GibcoBRL Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; GibcoBRL Life Technologies) and 1% penicillin-streptomycin-neomycin (GibcoBRL Life Technologies).

Positive Control:

Article Title: Finasteride Enhances the Generation of Human Myeloid-Derived Suppressor Cells by Up-Regulating the COX2/PGE2 Pathway
Article Snippet: .. For PGE2 induction assay, which served as a positive control, PGE2 (Sigma-Aldrich) was added on the 4th day. .. For neutralization assay, PGE2 neutralizing antibody (Cayman Chemical, Ann Arbor, MI) was added on the 1st and 4th day.

Plasmid Preparation:

Article Title: PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer
Article Snippet: .. Reagents The materials were purchased and obtained as follows: Recombinant human EGF, HGF and IGF-1 were from R & D Systems (Abingdon, UK); pCMV6-PP4R1 (NM_005134), pCMV6-PP4R2 (NM_174907), pCMV6-PP4C (NM_002720), pCMV6-PATZ1 variant 3 (NM_032052), and pCMV6-PATZ1 variant 4 (NM_032051) plasmids were from ORIGENE (Rockville, MD); PATZ1 variant 4 and PP4R2 genes from pCMV6-PATZ1 and pCMV6-PP4R2 plasmids subcloned into pCMV6-AC-mCFP plasmids (ORIGENE) were applied for the establishment of PATZ1- and PP4R2-transduced stable cells. pCMV2-IKKβ WT (wild type; WT), pCMV2-IKKβ S177ES181E (dominant active; DA) and pCMV2-IKKβ K44M (dominant negative; DN) were obtained from Addgene (Cambridge, MA; plasmid numbers 11103 and 11104) and were originally created by Dr. Anjana Rao laboratory. control siRNAs, PP4R1-siRNA, PP4R2-siRNA, PP4C-siRNA and PATZ1-siRNA were all from Santa Cruz Biotechnology (Santa Cruz, CA); two shRNA HuSH 29mer shRNA (TF302311A and TF302311C) constructs against PP4R2 in pRFP-C-RS vector and the non-effective 29-mer scrambled shRNA (TR30015) were from ORIGENE; mouse anti-Vimentin (V9) antibodies and recombinant PGE2 were from Sigma-Aldrich (St Louis, MO); phosphatidylinositol (3,4,5)-trisphosphate diC16 (PI(3,4,5)P3 diC16) were from Echelon Biosciences (Salt Lake City, UT); Anti-PP4R1 (ab70624), anti-PP4R2 (ab70631), anti-PP4C (ab16475), anti-PP4R3α (ab70635), anti-PP4R3β (ab70622), anti-PP4R4 (ab111419), and anti-PATZ1 (ab154025) rabbit polyclonal antibodies were from Abcam (Cambridge, UK); mouse anti-PP1 (E-9), mouse anti-PP5 (H-7), rabbit anti-PHB (H-80), rabbit anti-IKKα/β (H-470), mouse anti-β-actin (C-4), goat anti-COX-2 (M-19), rabbit anti-E-cadherin (H-108), rabbit anti-ZEB1 (H-102), rabbit anti-Snail (H-130), rabbit anti-Twist (H-81), and rabbit anti-PATZ1 (H-300) antibodies as well as horseradish peroxidase-conjugated anti-mouse IgG, horseradish peroxidase-conjugated anti-goat IgG and horseradish peroxidase-conjugated anti-rabbit IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-PI3Kp85, rabbit anti-PI3Kp85Y458 , rabbit anti-Akt, rabbit anti-phospho-AktSer473 , rabbit anti-phospho-IKKα/βS176/S180 and rabbit anti-phospho-NF-κBp65S536 , horseradish peroxidase-conjugated mouse-anti-rabbit IgG (Conformation Specific; L27A9) antibodies were from Cell Signaling Technology (Beverly, MA); and rabbit anti-IκB, rabbit anti-NF-κBp65 and rabbit anti-Raf-1 antibodies were from Millipore (Temecula, CA). .. Cell culture A549 cell line (ATCC: CCL-185), H1299 (ATCC: CRL-5803), CL1-0 and CL1-5 cells [ , ] were maintained in DMEM or RPMI medium (GibcoBRL Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; GibcoBRL Life Technologies) and 1% penicillin-streptomycin-neomycin (GibcoBRL Life Technologies).

Construct:

Article Title: PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer
Article Snippet: .. Reagents The materials were purchased and obtained as follows: Recombinant human EGF, HGF and IGF-1 were from R & D Systems (Abingdon, UK); pCMV6-PP4R1 (NM_005134), pCMV6-PP4R2 (NM_174907), pCMV6-PP4C (NM_002720), pCMV6-PATZ1 variant 3 (NM_032052), and pCMV6-PATZ1 variant 4 (NM_032051) plasmids were from ORIGENE (Rockville, MD); PATZ1 variant 4 and PP4R2 genes from pCMV6-PATZ1 and pCMV6-PP4R2 plasmids subcloned into pCMV6-AC-mCFP plasmids (ORIGENE) were applied for the establishment of PATZ1- and PP4R2-transduced stable cells. pCMV2-IKKβ WT (wild type; WT), pCMV2-IKKβ S177ES181E (dominant active; DA) and pCMV2-IKKβ K44M (dominant negative; DN) were obtained from Addgene (Cambridge, MA; plasmid numbers 11103 and 11104) and were originally created by Dr. Anjana Rao laboratory. control siRNAs, PP4R1-siRNA, PP4R2-siRNA, PP4C-siRNA and PATZ1-siRNA were all from Santa Cruz Biotechnology (Santa Cruz, CA); two shRNA HuSH 29mer shRNA (TF302311A and TF302311C) constructs against PP4R2 in pRFP-C-RS vector and the non-effective 29-mer scrambled shRNA (TR30015) were from ORIGENE; mouse anti-Vimentin (V9) antibodies and recombinant PGE2 were from Sigma-Aldrich (St Louis, MO); phosphatidylinositol (3,4,5)-trisphosphate diC16 (PI(3,4,5)P3 diC16) were from Echelon Biosciences (Salt Lake City, UT); Anti-PP4R1 (ab70624), anti-PP4R2 (ab70631), anti-PP4C (ab16475), anti-PP4R3α (ab70635), anti-PP4R3β (ab70622), anti-PP4R4 (ab111419), and anti-PATZ1 (ab154025) rabbit polyclonal antibodies were from Abcam (Cambridge, UK); mouse anti-PP1 (E-9), mouse anti-PP5 (H-7), rabbit anti-PHB (H-80), rabbit anti-IKKα/β (H-470), mouse anti-β-actin (C-4), goat anti-COX-2 (M-19), rabbit anti-E-cadherin (H-108), rabbit anti-ZEB1 (H-102), rabbit anti-Snail (H-130), rabbit anti-Twist (H-81), and rabbit anti-PATZ1 (H-300) antibodies as well as horseradish peroxidase-conjugated anti-mouse IgG, horseradish peroxidase-conjugated anti-goat IgG and horseradish peroxidase-conjugated anti-rabbit IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-PI3Kp85, rabbit anti-PI3Kp85Y458 , rabbit anti-Akt, rabbit anti-phospho-AktSer473 , rabbit anti-phospho-IKKα/βS176/S180 and rabbit anti-phospho-NF-κBp65S536 , horseradish peroxidase-conjugated mouse-anti-rabbit IgG (Conformation Specific; L27A9) antibodies were from Cell Signaling Technology (Beverly, MA); and rabbit anti-IκB, rabbit anti-NF-κBp65 and rabbit anti-Raf-1 antibodies were from Millipore (Temecula, CA). .. Cell culture A549 cell line (ATCC: CCL-185), H1299 (ATCC: CRL-5803), CL1-0 and CL1-5 cells [ , ] were maintained in DMEM or RPMI medium (GibcoBRL Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; GibcoBRL Life Technologies) and 1% penicillin-streptomycin-neomycin (GibcoBRL Life Technologies).

Enzyme-linked Immunosorbent Assay:

Article Title: Angiogenesis in the Primate Ovulatory Follicle Is Stimulated by Luteinizing Hormone via Prostaglandin E2 1
Article Snippet: .. To assess cAMP levels, cells were grown to 80% confluence, then switched to basal media with 1% fetal bovine serum overnight before treatment with PGE2 or PTGER agonists (at concentrations listed above for immunohistochemistry) or isoproterenol (2 μM; Sigma) in basal media for 8 h. Media were harvested and stored at −20°C until assay for cAMP by enzyme immunoassay (EIA; Cayman) as previously described [ ]. .. Proliferation was measured using the MTT Cell Proliferation Assay (ATCC).

other:

Article Title: Lactate promotes PGE2 synthesis and gluconeogenesis in monocytes to benefit the growth of inflammation-associated colorectal tumor
Article Snippet: Reagents Lactate solution, deferoxamine mesylate (DFX, HIF-1α inducer), YC-1 (3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole, HIF-1α inhibitor), dactinomycin D and PGE2 were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA).

Blocking Assay:

Article Title: Low magnitude high frequency vibration induces RANKL via cyclooxygenase pathway in human periodontal ligament cells in vitro
Article Snippet: .. To study the involvement of PGE2 in vibration-induced RANKL expression, 10 μM of the non-specific COX inhibitor indomethacin (Sigma-Aldrich, St Louis, MO, USA) was added to the medium 30 min before the experiment to permit this compound to penetrate the cells and block it respective pathways. ..

Expressing:

Article Title: Low magnitude high frequency vibration induces RANKL via cyclooxygenase pathway in human periodontal ligament cells in vitro
Article Snippet: .. To study the involvement of PGE2 in vibration-induced RANKL expression, 10 μM of the non-specific COX inhibitor indomethacin (Sigma-Aldrich, St Louis, MO, USA) was added to the medium 30 min before the experiment to permit this compound to penetrate the cells and block it respective pathways. ..

Recombinant:

Article Title: PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer
Article Snippet: .. Reagents The materials were purchased and obtained as follows: Recombinant human EGF, HGF and IGF-1 were from R & D Systems (Abingdon, UK); pCMV6-PP4R1 (NM_005134), pCMV6-PP4R2 (NM_174907), pCMV6-PP4C (NM_002720), pCMV6-PATZ1 variant 3 (NM_032052), and pCMV6-PATZ1 variant 4 (NM_032051) plasmids were from ORIGENE (Rockville, MD); PATZ1 variant 4 and PP4R2 genes from pCMV6-PATZ1 and pCMV6-PP4R2 plasmids subcloned into pCMV6-AC-mCFP plasmids (ORIGENE) were applied for the establishment of PATZ1- and PP4R2-transduced stable cells. pCMV2-IKKβ WT (wild type; WT), pCMV2-IKKβ S177ES181E (dominant active; DA) and pCMV2-IKKβ K44M (dominant negative; DN) were obtained from Addgene (Cambridge, MA; plasmid numbers 11103 and 11104) and were originally created by Dr. Anjana Rao laboratory. control siRNAs, PP4R1-siRNA, PP4R2-siRNA, PP4C-siRNA and PATZ1-siRNA were all from Santa Cruz Biotechnology (Santa Cruz, CA); two shRNA HuSH 29mer shRNA (TF302311A and TF302311C) constructs against PP4R2 in pRFP-C-RS vector and the non-effective 29-mer scrambled shRNA (TR30015) were from ORIGENE; mouse anti-Vimentin (V9) antibodies and recombinant PGE2 were from Sigma-Aldrich (St Louis, MO); phosphatidylinositol (3,4,5)-trisphosphate diC16 (PI(3,4,5)P3 diC16) were from Echelon Biosciences (Salt Lake City, UT); Anti-PP4R1 (ab70624), anti-PP4R2 (ab70631), anti-PP4C (ab16475), anti-PP4R3α (ab70635), anti-PP4R3β (ab70622), anti-PP4R4 (ab111419), and anti-PATZ1 (ab154025) rabbit polyclonal antibodies were from Abcam (Cambridge, UK); mouse anti-PP1 (E-9), mouse anti-PP5 (H-7), rabbit anti-PHB (H-80), rabbit anti-IKKα/β (H-470), mouse anti-β-actin (C-4), goat anti-COX-2 (M-19), rabbit anti-E-cadherin (H-108), rabbit anti-ZEB1 (H-102), rabbit anti-Snail (H-130), rabbit anti-Twist (H-81), and rabbit anti-PATZ1 (H-300) antibodies as well as horseradish peroxidase-conjugated anti-mouse IgG, horseradish peroxidase-conjugated anti-goat IgG and horseradish peroxidase-conjugated anti-rabbit IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-PI3Kp85, rabbit anti-PI3Kp85Y458 , rabbit anti-Akt, rabbit anti-phospho-AktSer473 , rabbit anti-phospho-IKKα/βS176/S180 and rabbit anti-phospho-NF-κBp65S536 , horseradish peroxidase-conjugated mouse-anti-rabbit IgG (Conformation Specific; L27A9) antibodies were from Cell Signaling Technology (Beverly, MA); and rabbit anti-IκB, rabbit anti-NF-κBp65 and rabbit anti-Raf-1 antibodies were from Millipore (Temecula, CA). .. Cell culture A549 cell line (ATCC: CCL-185), H1299 (ATCC: CRL-5803), CL1-0 and CL1-5 cells [ , ] were maintained in DMEM or RPMI medium (GibcoBRL Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; GibcoBRL Life Technologies) and 1% penicillin-streptomycin-neomycin (GibcoBRL Life Technologies).

Variant Assay:

Article Title: PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer
Article Snippet: .. Reagents The materials were purchased and obtained as follows: Recombinant human EGF, HGF and IGF-1 were from R & D Systems (Abingdon, UK); pCMV6-PP4R1 (NM_005134), pCMV6-PP4R2 (NM_174907), pCMV6-PP4C (NM_002720), pCMV6-PATZ1 variant 3 (NM_032052), and pCMV6-PATZ1 variant 4 (NM_032051) plasmids were from ORIGENE (Rockville, MD); PATZ1 variant 4 and PP4R2 genes from pCMV6-PATZ1 and pCMV6-PP4R2 plasmids subcloned into pCMV6-AC-mCFP plasmids (ORIGENE) were applied for the establishment of PATZ1- and PP4R2-transduced stable cells. pCMV2-IKKβ WT (wild type; WT), pCMV2-IKKβ S177ES181E (dominant active; DA) and pCMV2-IKKβ K44M (dominant negative; DN) were obtained from Addgene (Cambridge, MA; plasmid numbers 11103 and 11104) and were originally created by Dr. Anjana Rao laboratory. control siRNAs, PP4R1-siRNA, PP4R2-siRNA, PP4C-siRNA and PATZ1-siRNA were all from Santa Cruz Biotechnology (Santa Cruz, CA); two shRNA HuSH 29mer shRNA (TF302311A and TF302311C) constructs against PP4R2 in pRFP-C-RS vector and the non-effective 29-mer scrambled shRNA (TR30015) were from ORIGENE; mouse anti-Vimentin (V9) antibodies and recombinant PGE2 were from Sigma-Aldrich (St Louis, MO); phosphatidylinositol (3,4,5)-trisphosphate diC16 (PI(3,4,5)P3 diC16) were from Echelon Biosciences (Salt Lake City, UT); Anti-PP4R1 (ab70624), anti-PP4R2 (ab70631), anti-PP4C (ab16475), anti-PP4R3α (ab70635), anti-PP4R3β (ab70622), anti-PP4R4 (ab111419), and anti-PATZ1 (ab154025) rabbit polyclonal antibodies were from Abcam (Cambridge, UK); mouse anti-PP1 (E-9), mouse anti-PP5 (H-7), rabbit anti-PHB (H-80), rabbit anti-IKKα/β (H-470), mouse anti-β-actin (C-4), goat anti-COX-2 (M-19), rabbit anti-E-cadherin (H-108), rabbit anti-ZEB1 (H-102), rabbit anti-Snail (H-130), rabbit anti-Twist (H-81), and rabbit anti-PATZ1 (H-300) antibodies as well as horseradish peroxidase-conjugated anti-mouse IgG, horseradish peroxidase-conjugated anti-goat IgG and horseradish peroxidase-conjugated anti-rabbit IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-PI3Kp85, rabbit anti-PI3Kp85Y458 , rabbit anti-Akt, rabbit anti-phospho-AktSer473 , rabbit anti-phospho-IKKα/βS176/S180 and rabbit anti-phospho-NF-κBp65S536 , horseradish peroxidase-conjugated mouse-anti-rabbit IgG (Conformation Specific; L27A9) antibodies were from Cell Signaling Technology (Beverly, MA); and rabbit anti-IκB, rabbit anti-NF-κBp65 and rabbit anti-Raf-1 antibodies were from Millipore (Temecula, CA). .. Cell culture A549 cell line (ATCC: CCL-185), H1299 (ATCC: CRL-5803), CL1-0 and CL1-5 cells [ , ] were maintained in DMEM or RPMI medium (GibcoBRL Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; GibcoBRL Life Technologies) and 1% penicillin-streptomycin-neomycin (GibcoBRL Life Technologies).

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    Millipore rabbit raf 1 specific primary antibody
    Intrathecal <t>Raf-1-selective</t> 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
    Rabbit Raf 1 Specific Primary Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit raf 1 specific primary antibody/product/Millipore
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rabbit raf 1 specific primary antibody - by Bioz Stars, 2020-07
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    99
    Millipore rabbit polyclonal anti phospho raf
    Intrathecal <t>Raf-1-selective</t> 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
    Rabbit Polyclonal Anti Phospho Raf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti phospho raf/product/Millipore
    Average 99 stars, based on 1 article reviews
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    rabbit polyclonal anti phospho raf - by Bioz Stars, 2020-07
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    Image Search Results


    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: Raf-1 protein level was detected by incubating with a rabbit Raf-1-specific primary antibody (1:1000, Millipore, Billerica, MA, USA) and a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:100 000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

    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: Raf-1 protein level was detected by incubating with a rabbit Raf-1-specific primary antibody (1:1000, Millipore, Billerica, MA, USA) and a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:100 000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

    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: Raf-1 protein level was detected by incubating with a rabbit Raf-1-specific primary antibody (1:1000, Millipore, Billerica, MA, USA) and a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:100 000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

    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: Raf-1 protein level was detected by incubating with a rabbit Raf-1-specific primary antibody (1:1000, Millipore, Billerica, MA, USA) and a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:100 000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

    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: Raf-1 protein level was detected by incubating with a rabbit Raf-1-specific primary antibody (1:1000, Millipore, Billerica, MA, USA) and a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:100 000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

    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: Raf-1 protein level was detected by incubating with a rabbit Raf-1-specific primary antibody (1:1000, Millipore, Billerica, MA, USA) and a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:100 000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

    Techniques: Transfection