lipid mediated transfection  (Thermo Fisher)


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    Structured Review

    Thermo Fisher lipid mediated transfection
    Response to exogenous c-Myb expression of FeLV LTRs containing c-Myb binding site mutations. (A) . Diagram of the 21-bp triplication as contained in the FeLV-945 LTR, indicating the sequence of c-Myb binding sites across the repeat junctions of the triplication ( +/+ ). LTRs were constructed in which the first ( -/+ ) or second ( +/- ) binding site was mutated. (B) . Firefly luciferase reporter gene plasmids containing the FeLV LTR with wild type or mutant c-Myb binding sites (500 ng) were introduced by lipid-mediated <t>transfection</t> in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.
    Lipid Mediated Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    lipid mediated transfection - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Regulation of FeLV-945 by c-Myb binding and CBP recruitment to the LTR"

    Article Title: Regulation of FeLV-945 by c-Myb binding and CBP recruitment to the LTR

    Journal: Virology Journal

    doi: 10.1186/1743-422X-1-3

    Response to exogenous c-Myb expression of FeLV LTRs containing c-Myb binding site mutations. (A) . Diagram of the 21-bp triplication as contained in the FeLV-945 LTR, indicating the sequence of c-Myb binding sites across the repeat junctions of the triplication ( +/+ ). LTRs were constructed in which the first ( -/+ ) or second ( +/- ) binding site was mutated. (B) . Firefly luciferase reporter gene plasmids containing the FeLV LTR with wild type or mutant c-Myb binding sites (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.
    Figure Legend Snippet: Response to exogenous c-Myb expression of FeLV LTRs containing c-Myb binding site mutations. (A) . Diagram of the 21-bp triplication as contained in the FeLV-945 LTR, indicating the sequence of c-Myb binding sites across the repeat junctions of the triplication ( +/+ ). LTRs were constructed in which the first ( -/+ ) or second ( +/- ) binding site was mutated. (B) . Firefly luciferase reporter gene plasmids containing the FeLV LTR with wild type or mutant c-Myb binding sites (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.

    Techniques Used: Expressing, Binding Assay, Sequencing, Construct, Luciferase, Mutagenesis, Transfection, Plasmid Preparation, Activity Assay

    Regulation of FeLV replication in response to exogenous overexpression of CBP. K-562 cells were chronically infected with recombinant FeLV containing the LTR of FeLV-945 or FeLV-A/61E. A CBP expression plasmid was then introduced by lipid-mediated transfection. Culture supernatants were collected 3 days later and reverse transcription activity was quantified as a measure of virus production. Results are reported as cpm/ml of 3 H-TTP incorporated. The data shown were pooled from two independent experiments each performed in triplicate.
    Figure Legend Snippet: Regulation of FeLV replication in response to exogenous overexpression of CBP. K-562 cells were chronically infected with recombinant FeLV containing the LTR of FeLV-945 or FeLV-A/61E. A CBP expression plasmid was then introduced by lipid-mediated transfection. Culture supernatants were collected 3 days later and reverse transcription activity was quantified as a measure of virus production. Results are reported as cpm/ml of 3 H-TTP incorporated. The data shown were pooled from two independent experiments each performed in triplicate.

    Techniques Used: Over Expression, Infection, Recombinant, Expressing, Plasmid Preparation, Transfection, Activity Assay

    Response to exogenous c-Myb expression of FeLV LTRs containing variable numbers of the 21-bp element. Recombinant FeLV LTRs were constructed that contained 1, 2 or 3 copies of the 21-bp element and were cloned into a firefly luciferase reporter plasmid. LTR reporter plasmids or a 5X MRE positive control plasmid (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.
    Figure Legend Snippet: Response to exogenous c-Myb expression of FeLV LTRs containing variable numbers of the 21-bp element. Recombinant FeLV LTRs were constructed that contained 1, 2 or 3 copies of the 21-bp element and were cloned into a firefly luciferase reporter plasmid. LTR reporter plasmids or a 5X MRE positive control plasmid (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.

    Techniques Used: Expressing, Recombinant, Construct, Clone Assay, Luciferase, Plasmid Preparation, Positive Control, Transfection, Activity Assay

    2) Product Images from "The Vacuolar ATPase a2-subunit regulates Notch signaling in triple-negative breast cancer cells"

    Article Title: The Vacuolar ATPase a2-subunit regulates Notch signaling in triple-negative breast cancer cells

    Journal: Oncotarget

    doi:

    a2V inhibition increases Notch signaling in TNBC A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after transfection. Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.
    Figure Legend Snippet: a2V inhibition increases Notch signaling in TNBC A–D. TNBC cell line MDA-MB-231 was transfected with siRNA oligonucleotides against a2V or Notch1 along with scrambled control siRNA. Cells were harvested 48 hrs after transfection. Fold change in mRNA expression levels of (A) Notch receptors, (B) Notch Ligands and (C) Notch target genes is shown by qRT-PCR performed on the Notch signaling PCR array. Prior to fold-change calculation, the values were normalized to signal generated from endogenous control 18srRNA. (D) Protein level of Notch1 intracellular domain (N1ICD) following a2V gene silencing is shown by western blot analysis. β actin was used as loading control. E–I. MDA-MB-231 cells were treated with Vehicle Control (DMSO), Bafilomycin A1 (Baf A1 – 0.1 or 0.5 μM) or Gamma Secretase Inhibitor (GSI – 2 μM) for 24 hrs. (E) Protein level of Notch1 intracellular domain (N1ICD) following treatment with Baf A1 or GSI is shown by western blot. β actin was used as loading control. (F) Gene expression expression levels of Hes1 relative to endogenous control 18srRNA is shown. (G) Hes1 protein expression is shown by immunofluorescence (H and I). Independently, MDA-MB-231 and MDA-MB-468 were transfected with a RBP-j Notch reporter construct and then treated with Vehicle control, 0.5 μM BafA1 or 2 μM GSI for 24 hrs. Notch reporter levels in (H) MDA-MB-231 and (I) MDA-MB-468 as measured by luciferase assay. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 compared to control. RBPj: Recombinant Binding Protein Suppressor of Hairless.

    Techniques Used: Inhibition, Multiple Displacement Amplification, Transfection, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Generated, Western Blot, Immunofluorescence, Construct, Luciferase, Recombinant, Binding Assay

    a2V-ATPase inhibition enhances Wnt signaling in TNBC A. MDA-MB-231 cells were transfected with scrambled control or a2V siRNA and harvested after 48 hrs of transfection. Fold change in mRNA expression levels of Wnt signaling genes WNT4, β-catenin (CTNNB1), C-MYC and Cyclin D1 (CYCD1) was assessed by qRT PCR. Prior to fold--change calculation, the values were normalized to signal generated from endogenous control 18srRNA. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01 compared to control siRNA. (B and C) TNBC cells were grown on chamber slides and treated with B. vehicle control or C. 0.1 μM Baf A1 for 4 hours. Cells were fixed, permeabilized and processed for immunofluorescence microscopy. Localization of β-catenin (green) is shown. Nucleus was stained with DAPI (blue). Scale bars: 10 μm.
    Figure Legend Snippet: a2V-ATPase inhibition enhances Wnt signaling in TNBC A. MDA-MB-231 cells were transfected with scrambled control or a2V siRNA and harvested after 48 hrs of transfection. Fold change in mRNA expression levels of Wnt signaling genes WNT4, β-catenin (CTNNB1), C-MYC and Cyclin D1 (CYCD1) was assessed by qRT PCR. Prior to fold--change calculation, the values were normalized to signal generated from endogenous control 18srRNA. Data represent mean ± standard error, n = 4. * P ≤ 0.05, ** P ≤ 0.01 compared to control siRNA. (B and C) TNBC cells were grown on chamber slides and treated with B. vehicle control or C. 0.1 μM Baf A1 for 4 hours. Cells were fixed, permeabilized and processed for immunofluorescence microscopy. Localization of β-catenin (green) is shown. Nucleus was stained with DAPI (blue). Scale bars: 10 μm.

    Techniques Used: Inhibition, Multiple Displacement Amplification, Transfection, Expressing, Quantitative RT-PCR, Generated, Immunofluorescence, Microscopy, Staining

    3) Product Images from "Regulation of FeLV-945 by c-Myb binding and CBP recruitment to the LTR"

    Article Title: Regulation of FeLV-945 by c-Myb binding and CBP recruitment to the LTR

    Journal: Virology Journal

    doi: 10.1186/1743-422X-1-3

    Response to exogenous c-Myb expression of FeLV LTRs containing c-Myb binding site mutations. (A) . Diagram of the 21-bp triplication as contained in the FeLV-945 LTR, indicating the sequence of c-Myb binding sites across the repeat junctions of the triplication ( +/+ ). LTRs were constructed in which the first ( -/+ ) or second ( +/- ) binding site was mutated. (B) . Firefly luciferase reporter gene plasmids containing the FeLV LTR with wild type or mutant c-Myb binding sites (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.
    Figure Legend Snippet: Response to exogenous c-Myb expression of FeLV LTRs containing c-Myb binding site mutations. (A) . Diagram of the 21-bp triplication as contained in the FeLV-945 LTR, indicating the sequence of c-Myb binding sites across the repeat junctions of the triplication ( +/+ ). LTRs were constructed in which the first ( -/+ ) or second ( +/- ) binding site was mutated. (B) . Firefly luciferase reporter gene plasmids containing the FeLV LTR with wild type or mutant c-Myb binding sites (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.

    Techniques Used: Expressing, Binding Assay, Sequencing, Construct, Luciferase, Mutagenesis, Transfection, Plasmid Preparation, Activity Assay

    Regulation of FeLV replication in response to exogenous overexpression of CBP. K-562 cells were chronically infected with recombinant FeLV containing the LTR of FeLV-945 or FeLV-A/61E. A CBP expression plasmid was then introduced by lipid-mediated transfection. Culture supernatants were collected 3 days later and reverse transcription activity was quantified as a measure of virus production. Results are reported as cpm/ml of 3 H-TTP incorporated. The data shown were pooled from two independent experiments each performed in triplicate.
    Figure Legend Snippet: Regulation of FeLV replication in response to exogenous overexpression of CBP. K-562 cells were chronically infected with recombinant FeLV containing the LTR of FeLV-945 or FeLV-A/61E. A CBP expression plasmid was then introduced by lipid-mediated transfection. Culture supernatants were collected 3 days later and reverse transcription activity was quantified as a measure of virus production. Results are reported as cpm/ml of 3 H-TTP incorporated. The data shown were pooled from two independent experiments each performed in triplicate.

    Techniques Used: Over Expression, Infection, Recombinant, Expressing, Plasmid Preparation, Transfection, Activity Assay

    Response to exogenous c-Myb expression of FeLV LTRs containing variable numbers of the 21-bp element. Recombinant FeLV LTRs were constructed that contained 1, 2 or 3 copies of the 21-bp element and were cloned into a firefly luciferase reporter plasmid. LTR reporter plasmids or a 5X MRE positive control plasmid (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.
    Figure Legend Snippet: Response to exogenous c-Myb expression of FeLV LTRs containing variable numbers of the 21-bp element. Recombinant FeLV LTRs were constructed that contained 1, 2 or 3 copies of the 21-bp element and were cloned into a firefly luciferase reporter plasmid. LTR reporter plasmids or a 5X MRE positive control plasmid (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.

    Techniques Used: Expressing, Recombinant, Construct, Clone Assay, Luciferase, Plasmid Preparation, Positive Control, Transfection, Activity Assay

    4) Product Images from "Lithium promotes DNA stability and survival of ischemic retinal neurocytes by upregulating DNA ligase IV"

    Article Title: Lithium promotes DNA stability and survival of ischemic retinal neurocytes by upregulating DNA ligase IV

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.341

    Lithium treatment stimulates NHEJ repair in a rat model of retinal I/R injury. ( a ) Location of γ -H2AX foci coincides with CREB1 phosphorylation, Nrf-1 and ligase IV in retina after following I/R surgery. The γ -H2AX-positive cells were primarily located in the INL and GCL. Scale bars: 100 μ m. ( b ) γ -H2AX expression was quantified after I/R surgery by counting foci. These data are graphically represented (12 h, 37.0±3.74; 1d, 2.66±0.6; 3d, 0.8±0.5; 7d, 0.3±0.5) compared with controls (12 h, 70.33±6.55; 1d, 11.3±0.2; 3d, 4.5±0.5; 7d, 1.8±0.9). ( c ) DNA NHEJ assay in vivo . (The strategy of the NHEJ repair assay is shown in Figure 3d .) At 24 h after I/R surgery, intravitreal injection was performed with the linearized plasmid pEGFP-N1 following transfecting reagent following the manufacturer's instructions ( in vivo -jetPEI). At 48 h after transfection, the retina is fixed and sliced. GFP is detectable in the RGC layer. Scale bars: 100 μ m. ( d ) A comparison of GFP-positive cells in the rat retina. Lithium pretreatment promotes DNA NHEJ activity (Sham, 0.67±0.62; Sham+Li, 1.25±0.72; I/R surgery, 5.5±1.19; I/R surgery+Li, 13.25±1.83). n =8 for each group, * P
    Figure Legend Snippet: Lithium treatment stimulates NHEJ repair in a rat model of retinal I/R injury. ( a ) Location of γ -H2AX foci coincides with CREB1 phosphorylation, Nrf-1 and ligase IV in retina after following I/R surgery. The γ -H2AX-positive cells were primarily located in the INL and GCL. Scale bars: 100 μ m. ( b ) γ -H2AX expression was quantified after I/R surgery by counting foci. These data are graphically represented (12 h, 37.0±3.74; 1d, 2.66±0.6; 3d, 0.8±0.5; 7d, 0.3±0.5) compared with controls (12 h, 70.33±6.55; 1d, 11.3±0.2; 3d, 4.5±0.5; 7d, 1.8±0.9). ( c ) DNA NHEJ assay in vivo . (The strategy of the NHEJ repair assay is shown in Figure 3d .) At 24 h after I/R surgery, intravitreal injection was performed with the linearized plasmid pEGFP-N1 following transfecting reagent following the manufacturer's instructions ( in vivo -jetPEI). At 48 h after transfection, the retina is fixed and sliced. GFP is detectable in the RGC layer. Scale bars: 100 μ m. ( d ) A comparison of GFP-positive cells in the rat retina. Lithium pretreatment promotes DNA NHEJ activity (Sham, 0.67±0.62; Sham+Li, 1.25±0.72; I/R surgery, 5.5±1.19; I/R surgery+Li, 13.25±1.83). n =8 for each group, * P

    Techniques Used: Non-Homologous End Joining, Expressing, In Vivo, Injection, Plasmid Preparation, Transfection, Activity Assay

    5) Product Images from "Regulation of FeLV-945 by c-Myb binding and CBP recruitment to the LTR"

    Article Title: Regulation of FeLV-945 by c-Myb binding and CBP recruitment to the LTR

    Journal: Virology Journal

    doi: 10.1186/1743-422X-1-3

    Response to exogenous c-Myb expression of FeLV LTRs containing c-Myb binding site mutations. (A) . Diagram of the 21-bp triplication as contained in the FeLV-945 LTR, indicating the sequence of c-Myb binding sites across the repeat junctions of the triplication ( +/+ ). LTRs were constructed in which the first ( -/+ ) or second ( +/- ) binding site was mutated. (B) . Firefly luciferase reporter gene plasmids containing the FeLV LTR with wild type or mutant c-Myb binding sites (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.
    Figure Legend Snippet: Response to exogenous c-Myb expression of FeLV LTRs containing c-Myb binding site mutations. (A) . Diagram of the 21-bp triplication as contained in the FeLV-945 LTR, indicating the sequence of c-Myb binding sites across the repeat junctions of the triplication ( +/+ ). LTRs were constructed in which the first ( -/+ ) or second ( +/- ) binding site was mutated. (B) . Firefly luciferase reporter gene plasmids containing the FeLV LTR with wild type or mutant c-Myb binding sites (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.

    Techniques Used: Expressing, Binding Assay, Sequencing, Construct, Luciferase, Mutagenesis, Transfection, Plasmid Preparation, Activity Assay

    Regulation of FeLV replication in response to exogenous overexpression of CBP. K-562 cells were chronically infected with recombinant FeLV containing the LTR of FeLV-945 or FeLV-A/61E. A CBP expression plasmid was then introduced by lipid-mediated transfection. Culture supernatants were collected 3 days later and reverse transcription activity was quantified as a measure of virus production. Results are reported as cpm/ml of 3 H-TTP incorporated. The data shown were pooled from two independent experiments each performed in triplicate.
    Figure Legend Snippet: Regulation of FeLV replication in response to exogenous overexpression of CBP. K-562 cells were chronically infected with recombinant FeLV containing the LTR of FeLV-945 or FeLV-A/61E. A CBP expression plasmid was then introduced by lipid-mediated transfection. Culture supernatants were collected 3 days later and reverse transcription activity was quantified as a measure of virus production. Results are reported as cpm/ml of 3 H-TTP incorporated. The data shown were pooled from two independent experiments each performed in triplicate.

    Techniques Used: Over Expression, Infection, Recombinant, Expressing, Plasmid Preparation, Transfection, Activity Assay

    Response to exogenous c-Myb expression of FeLV LTRs containing variable numbers of the 21-bp element. Recombinant FeLV LTRs were constructed that contained 1, 2 or 3 copies of the 21-bp element and were cloned into a firefly luciferase reporter plasmid. LTR reporter plasmids or a 5X MRE positive control plasmid (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.
    Figure Legend Snippet: Response to exogenous c-Myb expression of FeLV LTRs containing variable numbers of the 21-bp element. Recombinant FeLV LTRs were constructed that contained 1, 2 or 3 copies of the 21-bp element and were cloned into a firefly luciferase reporter plasmid. LTR reporter plasmids or a 5X MRE positive control plasmid (500 ng) were introduced by lipid-mediated transfection in triplicate into feline embryonic fibroblasts (FEA) together with the Renilla luciferase reporter plasmid pRL-SV40 (5 ng) and a c-Myb expression plasmid in increasing concentrations (0 – 500 ng). Cell lysates were harvested 24 hours later and luciferase activity was quantified. Data are reported as a ratio of firefly to Renilla luciferase activity. Shown are data from a representative experiment repeated three times independently.

    Techniques Used: Expressing, Recombinant, Construct, Clone Assay, Luciferase, Plasmid Preparation, Positive Control, Transfection, Activity Assay

    6) Product Images from "Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells"

    Article Title: Identification of the endosomal sorting complex required for transport-I (ESCRT-I) as an important modulator of anti-miR uptake by cancer cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1367

    SKHEP1 exhibits productive uptake of anti-miR-21. ( A ) SKHEP1, A549 and HUCCT1 cells expressing miR-21 luciferase reporter were transfected with a dilution series of anti-miR-21 or MM control, and miR-21 luciferase reporter activity was measured at 24 h post-transfection. Mean ± SEM, n = 3. ( B ) Anti-miR-21 or MM control was added to the culture media of the SKHEP1, A549 and HUCCT1 miR-21 reporter cell lines. Luciferase activity was measured after 72 h of uptake. Mean ± SEM, n = 3. ( C ) SKHEP1 cells were treated with PBS or oligonucleotides (anti-miR-21 or MM control) at concentrations of either 1 or 10 μM. After 72 h of anti-miR treatment, total RNA was isolated and qPCR was performed to measure expression of the endogenous miR-21 targets ANKRD46 and DDAH1. Expression levels in PBS-treated samples are indicated by the dotted line. * P ≤ 0.05, Mean ± 1 SD, n = 2. ( D ) A dilution series of anti-miR-21 or two different MM controls was added to the culture media of SKHEP1 and relative cell number was quantified after 5 days treatment. Mean ± SEM, n = 3.
    Figure Legend Snippet: SKHEP1 exhibits productive uptake of anti-miR-21. ( A ) SKHEP1, A549 and HUCCT1 cells expressing miR-21 luciferase reporter were transfected with a dilution series of anti-miR-21 or MM control, and miR-21 luciferase reporter activity was measured at 24 h post-transfection. Mean ± SEM, n = 3. ( B ) Anti-miR-21 or MM control was added to the culture media of the SKHEP1, A549 and HUCCT1 miR-21 reporter cell lines. Luciferase activity was measured after 72 h of uptake. Mean ± SEM, n = 3. ( C ) SKHEP1 cells were treated with PBS or oligonucleotides (anti-miR-21 or MM control) at concentrations of either 1 or 10 μM. After 72 h of anti-miR treatment, total RNA was isolated and qPCR was performed to measure expression of the endogenous miR-21 targets ANKRD46 and DDAH1. Expression levels in PBS-treated samples are indicated by the dotted line. * P ≤ 0.05, Mean ± 1 SD, n = 2. ( D ) A dilution series of anti-miR-21 or two different MM controls was added to the culture media of SKHEP1 and relative cell number was quantified after 5 days treatment. Mean ± SEM, n = 3.

    Techniques Used: Expressing, Luciferase, Transfection, Activity Assay, Isolation, Real-time Polymerase Chain Reaction

    ESCRT-I restores anti-miR-21 uptake in cancer cell lines with inherently poor uptake. ( A ) A549 or ( B ) HUCCT1 cells expressing the miR-21 luciferase were transfected with siRNA targeting non-silencing (NS), TSG101 or VPS28. After 48 h siRNA knockdown, anti-miR-21 was added to culture media for an additional 48 h and then luciferase activity was measured. Mean ± SEM, n = 3. ( C ) A549 cell line was transfected with siRNA targeting NS, TSG101 or VPS28. At 24 h post-transfection the cells were treated by adding PBS, 1 μM anti-miR-21 or 1 μM MM control to the culture media. Following 72 h anti-miR treatment, RNA was isolated and quantitative PCR was performed to measure expression of ANKRD46, DDAH1. The dotted line represents expression of PBS-treated samples. Mean ± SD, n = 2. * P ≤ 0.05, ** P ≤ 0.01.
    Figure Legend Snippet: ESCRT-I restores anti-miR-21 uptake in cancer cell lines with inherently poor uptake. ( A ) A549 or ( B ) HUCCT1 cells expressing the miR-21 luciferase were transfected with siRNA targeting non-silencing (NS), TSG101 or VPS28. After 48 h siRNA knockdown, anti-miR-21 was added to culture media for an additional 48 h and then luciferase activity was measured. Mean ± SEM, n = 3. ( C ) A549 cell line was transfected with siRNA targeting NS, TSG101 or VPS28. At 24 h post-transfection the cells were treated by adding PBS, 1 μM anti-miR-21 or 1 μM MM control to the culture media. Following 72 h anti-miR treatment, RNA was isolated and quantitative PCR was performed to measure expression of ANKRD46, DDAH1. The dotted line represents expression of PBS-treated samples. Mean ± SD, n = 2. * P ≤ 0.05, ** P ≤ 0.01.

    Techniques Used: Expressing, Luciferase, Transfection, Activity Assay, Isolation, Real-time Polymerase Chain Reaction

    Knockdown of the ESCRT-I complex restores productive uptake in anti-miR-21 resistant clones. ( A ) SKHEP1 sensitive to anti-miR-21 were treated for 4 weeks with 2 μM anti-miR-21, subculturing the cells as needed. After 4 weeks, a polyclonal resistant population capable of growing in 2 μM anti-miR-21 was obtained. Limiting dilution cloning was performed to obtain resistant CLONE9 and CLONE15. ( B ) The SKHEP1, CLONE9 and CLONE15 cell lines were treated with a dilution series of anti-miR-21. The anti-miR-21 compound was added to the culture media and cells were grown for 5 days prior to quantification of cell number. ( C ) SKHEP1, CLONE9 and CLONE15 were transfected with a dilution series of anti-miR-21 and cell viability was quantified at 72 h post-transfection. ( D ) Anti-miR-21 or MM control was added to the culture media in the absence of cationic transfection lipid to SKHEP1, CLONE9 and CLONE15 expressing the miR-21 luciferase reporter. The cells were treated for 72 h with anti-miR and then luciferase activity was measured. ( E ) CLONE9 and CLONE15 were transfected with siRNA targeting non-silencing (NS), TSG101 or VPS28. After 48 h siRNA knockdown, anti-miR-21 was added to culture media and luciferase activity was measured after 48 h treatment. For all experiments data are plotted as Mean ± SEM, n = 3.
    Figure Legend Snippet: Knockdown of the ESCRT-I complex restores productive uptake in anti-miR-21 resistant clones. ( A ) SKHEP1 sensitive to anti-miR-21 were treated for 4 weeks with 2 μM anti-miR-21, subculturing the cells as needed. After 4 weeks, a polyclonal resistant population capable of growing in 2 μM anti-miR-21 was obtained. Limiting dilution cloning was performed to obtain resistant CLONE9 and CLONE15. ( B ) The SKHEP1, CLONE9 and CLONE15 cell lines were treated with a dilution series of anti-miR-21. The anti-miR-21 compound was added to the culture media and cells were grown for 5 days prior to quantification of cell number. ( C ) SKHEP1, CLONE9 and CLONE15 were transfected with a dilution series of anti-miR-21 and cell viability was quantified at 72 h post-transfection. ( D ) Anti-miR-21 or MM control was added to the culture media in the absence of cationic transfection lipid to SKHEP1, CLONE9 and CLONE15 expressing the miR-21 luciferase reporter. The cells were treated for 72 h with anti-miR and then luciferase activity was measured. ( E ) CLONE9 and CLONE15 were transfected with siRNA targeting non-silencing (NS), TSG101 or VPS28. After 48 h siRNA knockdown, anti-miR-21 was added to culture media and luciferase activity was measured after 48 h treatment. For all experiments data are plotted as Mean ± SEM, n = 3.

    Techniques Used: Clone Assay, Subculturing Assay, Transfection, Expressing, Luciferase, Activity Assay

    The ESCRT-I complex regulates uptake of anti-miR-21. ( A ) SKHEP1 cells were infected with an shRNA library and selected for stable transduction. At 3 days post-transduction, an aliquot of cells was collected and saved as a T0 time point. The remaining cells were split into two treatment groups and cultured for 13 days with either anti-miR-21 or MM control. At the end of 13 days, the cells were collected; genomic DNA was extracted and used as a template for PCR amplification of shRNA barcode which was sequenced on an Illuminia HiSeq2000. Two independent biological replicates of shRNA screen were performed. ( B ) Scatter plot of l og2 fold change (anti-miR-21 sequencing reads/MM control sequencing reads) from biological Replicate A and biological Replicate B. On average each gene was targeted by 5–6 shRNA. For each gene, the shRNA were ordered from high to low based on the log2 ratio (anti-miR-21/MM control). The value of the second lowest shRNA ratio for each gene is plotted with Replicate A on the ordinate and Replicate B on the abscissa. ( C ) SKHEP1 miR-21 luciferase cells were transfected with siRNA targeting non-silencing (NS), TSG101-siRNA#1, TSG101-siRNA#2 and TSG101-siRNA#3 or ( D ) siRNA targeting NS, VPS28-siRNA#1 and VPS28-siRNA#2. At 48 h post-transfection anti-miR-21 Chemistry A or Chemistry B was added to the culture media, and the cells were incubated for an additional 48 h, followed by measurement of miR-21 luciferase reporter activity. Luciferase expression was normalized to cell number and expressed as fold derepression compared to cells treated with media only. Mean ± SEM, n = 3. ( E ) SKHEP1 cells were transfected with siRNA targeting NS, TSG101 or VPS28. After 24 h knockdown with siRNA, the cells were treated by adding PBS, 1 μM anti-miR-21 or 1 μM MM control to the culture media. Following 72 h anti-miR treatment, RNA was isolated and expression of ANKRD46, DDAH1 was performed. Expression levels of PBS-treated samples are indicated by the dotted line. Data are plotted as Mean ± 1 SD, n = 2. * P ≤ 0.05, ** P ≤ 0.01.
    Figure Legend Snippet: The ESCRT-I complex regulates uptake of anti-miR-21. ( A ) SKHEP1 cells were infected with an shRNA library and selected for stable transduction. At 3 days post-transduction, an aliquot of cells was collected and saved as a T0 time point. The remaining cells were split into two treatment groups and cultured for 13 days with either anti-miR-21 or MM control. At the end of 13 days, the cells were collected; genomic DNA was extracted and used as a template for PCR amplification of shRNA barcode which was sequenced on an Illuminia HiSeq2000. Two independent biological replicates of shRNA screen were performed. ( B ) Scatter plot of l og2 fold change (anti-miR-21 sequencing reads/MM control sequencing reads) from biological Replicate A and biological Replicate B. On average each gene was targeted by 5–6 shRNA. For each gene, the shRNA were ordered from high to low based on the log2 ratio (anti-miR-21/MM control). The value of the second lowest shRNA ratio for each gene is plotted with Replicate A on the ordinate and Replicate B on the abscissa. ( C ) SKHEP1 miR-21 luciferase cells were transfected with siRNA targeting non-silencing (NS), TSG101-siRNA#1, TSG101-siRNA#2 and TSG101-siRNA#3 or ( D ) siRNA targeting NS, VPS28-siRNA#1 and VPS28-siRNA#2. At 48 h post-transfection anti-miR-21 Chemistry A or Chemistry B was added to the culture media, and the cells were incubated for an additional 48 h, followed by measurement of miR-21 luciferase reporter activity. Luciferase expression was normalized to cell number and expressed as fold derepression compared to cells treated with media only. Mean ± SEM, n = 3. ( E ) SKHEP1 cells were transfected with siRNA targeting NS, TSG101 or VPS28. After 24 h knockdown with siRNA, the cells were treated by adding PBS, 1 μM anti-miR-21 or 1 μM MM control to the culture media. Following 72 h anti-miR treatment, RNA was isolated and expression of ANKRD46, DDAH1 was performed. Expression levels of PBS-treated samples are indicated by the dotted line. Data are plotted as Mean ± 1 SD, n = 2. * P ≤ 0.05, ** P ≤ 0.01.

    Techniques Used: Infection, shRNA, Transduction, Cell Culture, Polymerase Chain Reaction, Amplification, Sequencing, Luciferase, Transfection, Incubation, Activity Assay, Expressing, Isolation

    7) Product Images from "The V-ATPase a2 isoform controls mammary gland development through Notch and TGF-β signaling"

    Article Title: The V-ATPase a2 isoform controls mammary gland development through Notch and TGF-β signaling

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.347

    Small interfering RNA (siRNa)-mediated knockdown of a2V in HMEpCs leads to the accumulation of cleaved Notch and secreted TGF- β 1. HMEpCs were grown in chamber slides and transfected with scrambled control or a2V siRNA and harvested after 48 h of transfection. Cells were fixed, permeabilized and processed for confocal immunofluorescence microscopy. ( a ) Double immunofluorescence staining of Notch 1 intracellular domain (green) with early endosome marker EEA1 (red). Nucleus was stained with DAPI (diamidino-2-phenylindole; blue). Scale bars: 10 μ m. ( b ) HMEpCs were seeded in 96-well plates and treated with scramble control or a2V siRNA. Cell supernatant was collected and secreted TGF- β was measured by enzyme-linked immunosorbent assay (ELISA) and expressed as pg/ml. Data represent mean±S.E., n =3. * P ≤0.05. ( c ) Following siRNA-mediated knockdown of a2V, HMEpCs were processed for confocal microscopy for nuclear localization of pSmad2 (green). Nucleus was stained with DAPI (blue). Scale bars: 10 μ m
    Figure Legend Snippet: Small interfering RNA (siRNa)-mediated knockdown of a2V in HMEpCs leads to the accumulation of cleaved Notch and secreted TGF- β 1. HMEpCs were grown in chamber slides and transfected with scrambled control or a2V siRNA and harvested after 48 h of transfection. Cells were fixed, permeabilized and processed for confocal immunofluorescence microscopy. ( a ) Double immunofluorescence staining of Notch 1 intracellular domain (green) with early endosome marker EEA1 (red). Nucleus was stained with DAPI (diamidino-2-phenylindole; blue). Scale bars: 10 μ m. ( b ) HMEpCs were seeded in 96-well plates and treated with scramble control or a2V siRNA. Cell supernatant was collected and secreted TGF- β was measured by enzyme-linked immunosorbent assay (ELISA) and expressed as pg/ml. Data represent mean±S.E., n =3. * P ≤0.05. ( c ) Following siRNA-mediated knockdown of a2V, HMEpCs were processed for confocal microscopy for nuclear localization of pSmad2 (green). Nucleus was stained with DAPI (blue). Scale bars: 10 μ m

    Techniques Used: Small Interfering RNA, Transfection, Immunofluorescence, Microscopy, Double Immunofluorescence Staining, Marker, Staining, Enzyme-linked Immunosorbent Assay, Confocal Microscopy

    8) Product Images from "The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma"

    Article Title: The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0128159

    siRNA screening and microarray analysis of MHCC97-H liver cancer cell line stably transfected with c-Met shRNA reveals EGFR pathway as a putative survival pathway in HCC. A) c-Met shRNA was stably transfected into the MHCC97-H cell line, which has constitutive c-Met activity. After puromycin selection, immunoblot determined c-Met knockdown in a c-Met + HCC cell line suppresses downstream signaling (c-Met, Akt, and Erk1/2 phosphorylation) compared to MHCC97-H cells stably expressing a scrambled shRNA. B) An XTT assay was performed to confirm the eight targets from the siRNA screen that had the greatest effect on cell viability in MHCC97-H c-Met KD cells. 10 nM siRNA and 0.2 ul RNAiMAX were used to transfect MHCC97-H c-Met KD cells and cell viability was determined at 48 hours post transfection. C) Ingenuity pathway analysis was conducted to compare microarray gene expression between MHCC97-H c-Met knockdown (KD) cells and MHCC97-H cells stably expressing a scrambled shRNA. The top seven enriched pathways are shown. D) A heatmap of the subset of the EGFR pathway gene set that is differentially expressed by microarray (Illumina human gene chip). A statistically significant (p
    Figure Legend Snippet: siRNA screening and microarray analysis of MHCC97-H liver cancer cell line stably transfected with c-Met shRNA reveals EGFR pathway as a putative survival pathway in HCC. A) c-Met shRNA was stably transfected into the MHCC97-H cell line, which has constitutive c-Met activity. After puromycin selection, immunoblot determined c-Met knockdown in a c-Met + HCC cell line suppresses downstream signaling (c-Met, Akt, and Erk1/2 phosphorylation) compared to MHCC97-H cells stably expressing a scrambled shRNA. B) An XTT assay was performed to confirm the eight targets from the siRNA screen that had the greatest effect on cell viability in MHCC97-H c-Met KD cells. 10 nM siRNA and 0.2 ul RNAiMAX were used to transfect MHCC97-H c-Met KD cells and cell viability was determined at 48 hours post transfection. C) Ingenuity pathway analysis was conducted to compare microarray gene expression between MHCC97-H c-Met knockdown (KD) cells and MHCC97-H cells stably expressing a scrambled shRNA. The top seven enriched pathways are shown. D) A heatmap of the subset of the EGFR pathway gene set that is differentially expressed by microarray (Illumina human gene chip). A statistically significant (p

    Techniques Used: Microarray, Stable Transfection, Transfection, shRNA, Activity Assay, Selection, Expressing, XTT Assay, Chromatin Immunoprecipitation

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    Protease Inhibitor:

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    Concentration Assay:

    Article Title: The arginine methyltransferase PRMT6 regulates cell proliferation and senescence through transcriptional repression of tumor suppressor genes
    Article Snippet: .. The following siRNA sequences were employed (sense strand indicated): siPRMT6_1 5′-GAGCAAGACACGGACGUUU-3′; siPRMT6_2 5′-GCAAGACACGGACGUUUCA-3′; siPRMT6_3 5′-GGAGGGAGAGUGACUUCAU-3′; siPRMT6_4 5′-GACGUUUCAGGAGAGAUCA-3′; siPRMT6_5 5′-CGGAACAGGUGGAUGCCAU-3′; sip21 (pool of three sequences) 5′-CGAUGGAACUUCGACUUUG-3′, 5′-GCGAUGGAACUUCGACUUU-3′ and 5′-GAUGGAACUUCGACUUUGU-3′; siLuci 5′-GAUUAUGUCCGGUUAUGUA-3′; siNon 5′-AUGAACGUGAAUUGCUCAA-3′; siScr 5′-CAUAAGCUGAGAUACUUCA-3′; siGFP 5′-GCAAGCUGACCCUGAAGUU-3′. siRNA transfections of U2OS (20 nM final concentration) were performed with the aid of Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. .. TIG3 BRAF-ER cells were transfected with siRNA (50 nM final concentration) using PEI or plasmid using FuGENE HD (Roche).

    Protein Extraction:

    Article Title: Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways
    Article Snippet: .. Dulbecco's Phosphate-Buffered Saline without Ca2+ and Mg2+ (DPBS, Lonza, Catalog# 17-512F) DMEM, no glucose (Life Technologies, catalog# 11966-025) Sodium pyruvate (Life Technologies, catalog# 11360-070) D-(+)-Galactose (Sigma-Aldrich, catalog# G5388-100G) Paraformaldehyde (Electron Microscopy Science, catalog #15714) Sytox red (Life Technologies, catalog #34859) Goat serum (Rockland, catalog #D204-00-0050) 96-well Acoustic uClear Flat-Bottom Black-Wall Plates (Greiner, catalog #655090) 10X RIPA Lysis Buffer (0.5M Tris-HCl, pH 7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) (Millipore, catalog #20-188) Sodium Dodecyl Sulfate (SDS, Sigma, catalog #L6026) Protease Inhibitor Cocktail (Sigma, catalog #P8340) Phosphatase Inhibitor Cocktail (Sigma, catalog #P0044) Halt Protease Inhibitor Cocktail, EDTA-free (Thermo Scientific, catalog #87785) M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific, catalog #78501) NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, catalog #28833) XDR Charge Separation Master Kit (ProteinSimple, catalog #CBS2001) pI Standard Ladder 3 (ProteinSimple, catalog #040-646) Premix G2, Pharmalyte pH 5-8 Separation Gradient (ProteinSimple, catalog #040-973) DMSO Inhibitor Mix (ProteinSimple, catalog #040-510) Wes™ 12-230 kDa Master Kit (ProteinSimple, catalog #PS-MK01 or PS-MK02) Bicine/CHAPS Lysis Buffer and Sample Diluent (ProteinSimple, catalog #040-764) Pierce BCA Protein Assay Kit (Thermo Scientific, catalog #23225) Lipofectamine RNAiMAX transfection reagent (Life Technologies, catalog #13778) Opti-MEM I Reduced Serum Medium (Life Technologies, catalog #31985-062) ON-TARGETplus SMARTpool Human ErbB2 siRNAs (GE Dharmacon, catalog #L-003126-00-0005) Non-targeting pool control siRNAs (GE Dharmacon, catalog #D-00180-10-05) Lapatinib (National Cancer Institute compound repository, NSC 727989) Neuregulin-1β (NRG) [Recombinant human neuregulin-1β (NRG1-β1/HRG1-β1 EGF domain), R & D Systems, catalog #396-HB-050] Trastuzumab (Genentech, Inc.) .. EGF/ErbB1 Receptor (D38B1) XP rabbit monoclonal antibody (Cell Signaling, catalog #4267S) ErbB2 Receptor (M45) rabbit polyclonal antibody (Cell Signaling, catalog #3250S) ErbB4 Receptor (111B2) rabbit monoclonal antibody (Cell Signaling, catalog #4795S) AKT (pan; C67E7) rabbit monoclonal antibody (Cell Signaling, catalog #4691S) Phospho-AKT (Ser473) rabbit polyclonal antibody (Cell Signaling, catalog # 9271) p42/44 MAPK (Erk1/2) rabbit polyclonal antibody (Cell Signaling catalog #9102) Phospho- p42/44 MAPK (Erk1/2) (Thr202/Tyr204) rabbit polyclonal (Cell Signaling, catalog #9101) Alpha-tubulin (11H10) rabbit monoclonal antibody (Cell Signaling, catalog #2125) Lamin B1(D4Q4Z) rabbit monoclonal antibody (Cell Signaling, catalog #12586) FOXO3a (D19A7) rabbit monoclonal antibody (Cell Signaling, catalog #12829) Phospho-FOXO3A (S253) rabbit polyclonal antibody (Abcam, catalog #ab47285) CREB (48H2) rabbit monoclonal antibody (Cell Signaling, catalog #9197) Phospho-CREB (Ser133) (87G3) rabbit monoclonal (Cell Signaling, catalog #9198) Amplified Rabbit or Mouse Secondary Antibody Detection Kit (ProteinSimple, catalog #041-126 or 041-127, respectively)

    BIA-KA:

    Article Title: Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways
    Article Snippet: .. Dulbecco's Phosphate-Buffered Saline without Ca2+ and Mg2+ (DPBS, Lonza, Catalog# 17-512F) DMEM, no glucose (Life Technologies, catalog# 11966-025) Sodium pyruvate (Life Technologies, catalog# 11360-070) D-(+)-Galactose (Sigma-Aldrich, catalog# G5388-100G) Paraformaldehyde (Electron Microscopy Science, catalog #15714) Sytox red (Life Technologies, catalog #34859) Goat serum (Rockland, catalog #D204-00-0050) 96-well Acoustic uClear Flat-Bottom Black-Wall Plates (Greiner, catalog #655090) 10X RIPA Lysis Buffer (0.5M Tris-HCl, pH 7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) (Millipore, catalog #20-188) Sodium Dodecyl Sulfate (SDS, Sigma, catalog #L6026) Protease Inhibitor Cocktail (Sigma, catalog #P8340) Phosphatase Inhibitor Cocktail (Sigma, catalog #P0044) Halt Protease Inhibitor Cocktail, EDTA-free (Thermo Scientific, catalog #87785) M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific, catalog #78501) NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, catalog #28833) XDR Charge Separation Master Kit (ProteinSimple, catalog #CBS2001) pI Standard Ladder 3 (ProteinSimple, catalog #040-646) Premix G2, Pharmalyte pH 5-8 Separation Gradient (ProteinSimple, catalog #040-973) DMSO Inhibitor Mix (ProteinSimple, catalog #040-510) Wes™ 12-230 kDa Master Kit (ProteinSimple, catalog #PS-MK01 or PS-MK02) Bicine/CHAPS Lysis Buffer and Sample Diluent (ProteinSimple, catalog #040-764) Pierce BCA Protein Assay Kit (Thermo Scientific, catalog #23225) Lipofectamine RNAiMAX transfection reagent (Life Technologies, catalog #13778) Opti-MEM I Reduced Serum Medium (Life Technologies, catalog #31985-062) ON-TARGETplus SMARTpool Human ErbB2 siRNAs (GE Dharmacon, catalog #L-003126-00-0005) Non-targeting pool control siRNAs (GE Dharmacon, catalog #D-00180-10-05) Lapatinib (National Cancer Institute compound repository, NSC 727989) Neuregulin-1β (NRG) [Recombinant human neuregulin-1β (NRG1-β1/HRG1-β1 EGF domain), R & D Systems, catalog #396-HB-050] Trastuzumab (Genentech, Inc.) .. EGF/ErbB1 Receptor (D38B1) XP rabbit monoclonal antibody (Cell Signaling, catalog #4267S) ErbB2 Receptor (M45) rabbit polyclonal antibody (Cell Signaling, catalog #3250S) ErbB4 Receptor (111B2) rabbit monoclonal antibody (Cell Signaling, catalog #4795S) AKT (pan; C67E7) rabbit monoclonal antibody (Cell Signaling, catalog #4691S) Phospho-AKT (Ser473) rabbit polyclonal antibody (Cell Signaling, catalog # 9271) p42/44 MAPK (Erk1/2) rabbit polyclonal antibody (Cell Signaling catalog #9102) Phospho- p42/44 MAPK (Erk1/2) (Thr202/Tyr204) rabbit polyclonal (Cell Signaling, catalog #9101) Alpha-tubulin (11H10) rabbit monoclonal antibody (Cell Signaling, catalog #2125) Lamin B1(D4Q4Z) rabbit monoclonal antibody (Cell Signaling, catalog #12586) FOXO3a (D19A7) rabbit monoclonal antibody (Cell Signaling, catalog #12829) Phospho-FOXO3A (S253) rabbit polyclonal antibody (Abcam, catalog #ab47285) CREB (48H2) rabbit monoclonal antibody (Cell Signaling, catalog #9197) Phospho-CREB (Ser133) (87G3) rabbit monoclonal (Cell Signaling, catalog #9198) Amplified Rabbit or Mouse Secondary Antibody Detection Kit (ProteinSimple, catalog #041-126 or 041-127, respectively)

    Lysis:

    Article Title: Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways
    Article Snippet: .. Dulbecco's Phosphate-Buffered Saline without Ca2+ and Mg2+ (DPBS, Lonza, Catalog# 17-512F) DMEM, no glucose (Life Technologies, catalog# 11966-025) Sodium pyruvate (Life Technologies, catalog# 11360-070) D-(+)-Galactose (Sigma-Aldrich, catalog# G5388-100G) Paraformaldehyde (Electron Microscopy Science, catalog #15714) Sytox red (Life Technologies, catalog #34859) Goat serum (Rockland, catalog #D204-00-0050) 96-well Acoustic uClear Flat-Bottom Black-Wall Plates (Greiner, catalog #655090) 10X RIPA Lysis Buffer (0.5M Tris-HCl, pH 7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) (Millipore, catalog #20-188) Sodium Dodecyl Sulfate (SDS, Sigma, catalog #L6026) Protease Inhibitor Cocktail (Sigma, catalog #P8340) Phosphatase Inhibitor Cocktail (Sigma, catalog #P0044) Halt Protease Inhibitor Cocktail, EDTA-free (Thermo Scientific, catalog #87785) M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific, catalog #78501) NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, catalog #28833) XDR Charge Separation Master Kit (ProteinSimple, catalog #CBS2001) pI Standard Ladder 3 (ProteinSimple, catalog #040-646) Premix G2, Pharmalyte pH 5-8 Separation Gradient (ProteinSimple, catalog #040-973) DMSO Inhibitor Mix (ProteinSimple, catalog #040-510) Wes™ 12-230 kDa Master Kit (ProteinSimple, catalog #PS-MK01 or PS-MK02) Bicine/CHAPS Lysis Buffer and Sample Diluent (ProteinSimple, catalog #040-764) Pierce BCA Protein Assay Kit (Thermo Scientific, catalog #23225) Lipofectamine RNAiMAX transfection reagent (Life Technologies, catalog #13778) Opti-MEM I Reduced Serum Medium (Life Technologies, catalog #31985-062) ON-TARGETplus SMARTpool Human ErbB2 siRNAs (GE Dharmacon, catalog #L-003126-00-0005) Non-targeting pool control siRNAs (GE Dharmacon, catalog #D-00180-10-05) Lapatinib (National Cancer Institute compound repository, NSC 727989) Neuregulin-1β (NRG) [Recombinant human neuregulin-1β (NRG1-β1/HRG1-β1 EGF domain), R & D Systems, catalog #396-HB-050] Trastuzumab (Genentech, Inc.) .. EGF/ErbB1 Receptor (D38B1) XP rabbit monoclonal antibody (Cell Signaling, catalog #4267S) ErbB2 Receptor (M45) rabbit polyclonal antibody (Cell Signaling, catalog #3250S) ErbB4 Receptor (111B2) rabbit monoclonal antibody (Cell Signaling, catalog #4795S) AKT (pan; C67E7) rabbit monoclonal antibody (Cell Signaling, catalog #4691S) Phospho-AKT (Ser473) rabbit polyclonal antibody (Cell Signaling, catalog # 9271) p42/44 MAPK (Erk1/2) rabbit polyclonal antibody (Cell Signaling catalog #9102) Phospho- p42/44 MAPK (Erk1/2) (Thr202/Tyr204) rabbit polyclonal (Cell Signaling, catalog #9101) Alpha-tubulin (11H10) rabbit monoclonal antibody (Cell Signaling, catalog #2125) Lamin B1(D4Q4Z) rabbit monoclonal antibody (Cell Signaling, catalog #12586) FOXO3a (D19A7) rabbit monoclonal antibody (Cell Signaling, catalog #12829) Phospho-FOXO3A (S253) rabbit polyclonal antibody (Abcam, catalog #ab47285) CREB (48H2) rabbit monoclonal antibody (Cell Signaling, catalog #9197) Phospho-CREB (Ser133) (87G3) rabbit monoclonal (Cell Signaling, catalog #9198) Amplified Rabbit or Mouse Secondary Antibody Detection Kit (ProteinSimple, catalog #041-126 or 041-127, respectively)

    Recombinant:

    Article Title: Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways
    Article Snippet: .. Dulbecco's Phosphate-Buffered Saline without Ca2+ and Mg2+ (DPBS, Lonza, Catalog# 17-512F) DMEM, no glucose (Life Technologies, catalog# 11966-025) Sodium pyruvate (Life Technologies, catalog# 11360-070) D-(+)-Galactose (Sigma-Aldrich, catalog# G5388-100G) Paraformaldehyde (Electron Microscopy Science, catalog #15714) Sytox red (Life Technologies, catalog #34859) Goat serum (Rockland, catalog #D204-00-0050) 96-well Acoustic uClear Flat-Bottom Black-Wall Plates (Greiner, catalog #655090) 10X RIPA Lysis Buffer (0.5M Tris-HCl, pH 7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA) (Millipore, catalog #20-188) Sodium Dodecyl Sulfate (SDS, Sigma, catalog #L6026) Protease Inhibitor Cocktail (Sigma, catalog #P8340) Phosphatase Inhibitor Cocktail (Sigma, catalog #P0044) Halt Protease Inhibitor Cocktail, EDTA-free (Thermo Scientific, catalog #87785) M-PER® Mammalian Protein Extraction Reagent (Thermo Scientific, catalog #78501) NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, catalog #28833) XDR Charge Separation Master Kit (ProteinSimple, catalog #CBS2001) pI Standard Ladder 3 (ProteinSimple, catalog #040-646) Premix G2, Pharmalyte pH 5-8 Separation Gradient (ProteinSimple, catalog #040-973) DMSO Inhibitor Mix (ProteinSimple, catalog #040-510) Wes™ 12-230 kDa Master Kit (ProteinSimple, catalog #PS-MK01 or PS-MK02) Bicine/CHAPS Lysis Buffer and Sample Diluent (ProteinSimple, catalog #040-764) Pierce BCA Protein Assay Kit (Thermo Scientific, catalog #23225) Lipofectamine RNAiMAX transfection reagent (Life Technologies, catalog #13778) Opti-MEM I Reduced Serum Medium (Life Technologies, catalog #31985-062) ON-TARGETplus SMARTpool Human ErbB2 siRNAs (GE Dharmacon, catalog #L-003126-00-0005) Non-targeting pool control siRNAs (GE Dharmacon, catalog #D-00180-10-05) Lapatinib (National Cancer Institute compound repository, NSC 727989) Neuregulin-1β (NRG) [Recombinant human neuregulin-1β (NRG1-β1/HRG1-β1 EGF domain), R & D Systems, catalog #396-HB-050] Trastuzumab (Genentech, Inc.) .. EGF/ErbB1 Receptor (D38B1) XP rabbit monoclonal antibody (Cell Signaling, catalog #4267S) ErbB2 Receptor (M45) rabbit polyclonal antibody (Cell Signaling, catalog #3250S) ErbB4 Receptor (111B2) rabbit monoclonal antibody (Cell Signaling, catalog #4795S) AKT (pan; C67E7) rabbit monoclonal antibody (Cell Signaling, catalog #4691S) Phospho-AKT (Ser473) rabbit polyclonal antibody (Cell Signaling, catalog # 9271) p42/44 MAPK (Erk1/2) rabbit polyclonal antibody (Cell Signaling catalog #9102) Phospho- p42/44 MAPK (Erk1/2) (Thr202/Tyr204) rabbit polyclonal (Cell Signaling, catalog #9101) Alpha-tubulin (11H10) rabbit monoclonal antibody (Cell Signaling, catalog #2125) Lamin B1(D4Q4Z) rabbit monoclonal antibody (Cell Signaling, catalog #12586) FOXO3a (D19A7) rabbit monoclonal antibody (Cell Signaling, catalog #12829) Phospho-FOXO3A (S253) rabbit polyclonal antibody (Abcam, catalog #ab47285) CREB (48H2) rabbit monoclonal antibody (Cell Signaling, catalog #9197) Phospho-CREB (Ser133) (87G3) rabbit monoclonal (Cell Signaling, catalog #9198) Amplified Rabbit or Mouse Secondary Antibody Detection Kit (ProteinSimple, catalog #041-126 or 041-127, respectively)

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    Thermo Fisher lipid mediated transfection
    Lipid Mediated Transfection, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipid mediated transfection/product/Thermo Fisher
    Average 91 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    lipid mediated transfection - by Bioz Stars, 2020-07
    91/100 stars
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