p2a peptide sequences  (Addgene inc)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Novel FRET-Based Biosensors for Real-Time Monitoring of Estrogen Receptor Dimerization and Translocation Dynamics in Living Cells.

doi: 10.1002/advs.202406907

Figure Lengend Snippet: Figure 1. Optimization and Application of the Estrogen Receptor 𝛼𝛼Full-Length (FL) FRET Biosensor. A) Schematic representation and B) mode of action of ER 𝛼𝛼FL FRET biosensors. C) Bar graphs depict changes in the normalized FRET/ECFP emission ratio of the ER 𝛼𝛼FL CyOFP1-EV biosensor (EV; n = 39) and the ER 𝛼𝛼FL CyOFP1-P2A biosensor (P2A; n = 21) after 30 min of treatment with 10 μM 4-hydroxytamoxifen (****p < 0.0001). Mean values are shown above each bar. D) Time course of mean normalized FRET/ECFP emission ratio changes and E) representative images illustrate the FRET ratios of the ER 𝛼𝛼FL CyOFP1-P2A biosensor (CyOFP1; n = 26) and the ER 𝛼𝛼FL FRET biosensor (mNeonGreen; n = 27) before and after treatment

Article Snippet: Next, the EV linker and P2A peptide sequences were amplified from pGEMT-TPE2A-Mef2c-Tdtomato-Gata4-Tbx5 (Addgene plasmid #111818) and pCAGGS-6011nes (Addgene plasmid #108652) before being digested with BspEI/MluI.

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Novel FRET-Based Biosensors for Real-Time Monitoring of Estrogen Receptor Dimerization and Translocation Dynamics in Living Cells.

doi: 10.1002/advs.202406907

Figure Lengend Snippet: Figure 2. Optimization and Application of the Estrogen Receptor 𝛽𝛼Full-Length (FL) FRET Biosensor. A) Schematic representation and B) mode of action of ER 𝛽𝛼FL FRET biosensors. C) Bar graphs depict changes in the normalized FRET/ECFP emission ratio of the ER 𝛽𝛼FL CyOFP1-EV biosensor (EV; n = 33) and the ER 𝛽𝛼FL CyOFP1-P2A biosensor (P2A; n = 33) after 30 min of treatment with 10 μM 4-hydroxytamoxifen (****p < 0.0001, ns: not significant). D) Time course of changes in the mean normalized FRET/ECFP emission ratio and E) representative images illustrating the FRET ratios of the ER 𝛽𝛼FL CyOFP1-P2A biosensor (CyOFP1; n = 49) and the ER 𝛽𝛼FL FRET biosensor (mNeonGreen; n = 14) before and after treatment with 10

Article Snippet: Next, the EV linker and P2A peptide sequences were amplified from pGEMT-TPE2A-Mef2c-Tdtomato-Gata4-Tbx5 (Addgene plasmid #111818) and pCAGGS-6011nes (Addgene plasmid #108652) before being digested with BspEI/MluI.

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Novel FRET-Based Biosensors for Real-Time Monitoring of Estrogen Receptor Dimerization and Translocation Dynamics in Living Cells.

doi: 10.1002/advs.202406907

Figure Lengend Snippet: Figure 3. Optimization and Application of the Estrogen Receptor 𝛽𝛽Full-Length (FL) FRET Biosensor. A) Schematic representation and B) mode of action of ER 𝛽𝛽FL FRET biosensors. C) Bar graphs depict changes in the normalized FRET/ECFP emission ratio between the ER 𝛽𝛽FL CyOFP1-EV (EV; n = 31) and the ER 𝛽𝛽FL CyOFP1-P2A biosensors (P2A; n = 21) after treatment with 10 μM 17𝛽-estradiol for 30 min (****p < 0.0001, *p < 0.05). D) Time course of changes in the mean normalized FRET/ECFP emission ratio and E) representative images illustrating the FRET ratios of the ER 𝛽𝛽 FL CyOFP1-EV (CyOFP1; n = 31) and ER 𝛽𝛽FL FRET biosensors (mNeonGreen; n = 17) before and after treatment with 10 μM 17𝛽-estradiol (****p

Article Snippet: Next, the EV linker and P2A peptide sequences were amplified from pGEMT-TPE2A-Mef2c-Tdtomato-Gata4-Tbx5 (Addgene plasmid #111818) and pCAGGS-6011nes (Addgene plasmid #108652) before being digested with BspEI/MluI.

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Novel FRET-Based Biosensors for Real-Time Monitoring of Estrogen Receptor Dimerization and Translocation Dynamics in Living Cells.

doi: 10.1002/advs.202406907

Figure Lengend Snippet: Figure 4. Optimization and Application of the Estrogen Receptor 𝛽𝛽LBD FRET Biosensor. A) Schematic diagram and B) mode of action of ER 𝛽𝛽LBD FRET biosensors. C) Bar graphs depict changes in the normalized FRET/mTurquoise2 emission ratio between the ER 𝛽𝛽LBD N-N term-EV (EV; n = 5) and the ER 𝛽𝛽LBD N-N term-P2A biosensors (P2A; n = 5) after treatment with 10 μM 17𝛽-estradiol for 30 min (****p < 0.0001, **p < 0.01). D) Time course of changes in the mean normalized FRET/mTurquoiuse2 emission ratio and E) representative images illustrating the FRET ratios of the ER 𝛽𝛽 LBD N-N term-EV (N-N; n = 5) and the ER 𝛽𝛽LBD FRET biosensors (N-C; n = 13) before and after treatment with 10 μM 17𝛽-estradiol (****p < 0.0001),

Article Snippet: Next, the EV linker and P2A peptide sequences were amplified from pGEMT-TPE2A-Mef2c-Tdtomato-Gata4-Tbx5 (Addgene plasmid #111818) and pCAGGS-6011nes (Addgene plasmid #108652) before being digested with BspEI/MluI.

Techniques:

Journal: Viruses

Article Title: Factor-Dependent Internal Ribosome Entry Site and -1 Programmed Frameshifting Signal in the Bemisia-Associated Dicistrovirus 2

doi: 10.3390/v16050695

Figure Lengend Snippet: BaDV-2 IGR IRES activity in infectious clone and in cells. ( A ) Bicistronic reporters were transfected into S2 cells followed by mock infection or CrPV infection (MOI = 20). Cells were collected 6 h post transfection. Bicistronic RNAs tested contain the wild-type CrPV, mutant CrPV (CC6214-5 to GG to disrupt PKI base-pairing), wild-type BaDV-2 full-length IGR IRES (WT BaDV-2), BaDV-2 3′ DEL 5, and BaDV “minimal” IRES (double deletion at both 3′ and 5′ end. Luciferase activities were measured and normalized to wild-type CrPV with mock infection. Red and black dots represent data points from three independent experiments. A paired t -test was used to determine the p value and thus the significance levels. “n.s.” denotes the difference is not significant between the control groups and the experimental groups ( p > 0.05). Shown are the averages from at least three independent experiments ± standard deviation. ( B ) Schematic of infectious clone CrPV (CrPV, CrPV_IGR_IRES) and chimeric clones with CrPV IGR IRES replaced with BaDV-2 IGR IRES (BaDV-2 IGR IRES) and with P2A-site added after BaDV-2 IGR IRES (BaDV-2 IGR IRES+ P2A). ( C ) Infectious clone RNAs were incubated in Sf-21 extracts containing [35S]-methionine/cysteine and then monitored by SDS-PAGE analysis. Mock = mock transfection; OS = ORF1Stop; CrPV = pCrPV; BaDV = BaDV-2 IGR IRES; P2A = BaDV-2 IGR IRES + P2A. Shown is a representative gel from at least three independent experiments. ( D ) In vitro transcribed infectious clone RNAs were transfected into S2 cells for 144 h. VP2 expression was detected by immunoblotting. Shown are a representative SDS PAGE gel and the averages from at least three independent experiments ± standard deviation.

Article Snippet: BaDV-2 IGR IRES with a downstream P2A peptide sequence (Twist Biosciences) was cloned into the CrPV infectious clone [ ], replacing the CrPV IGR IRES.

Techniques: Activity Assay, Transfection, Infection, Mutagenesis, Luciferase, Control, Standard Deviation, Clone Assay, Incubation, SDS Page, In Vitro, Expressing, Western Blot