Structured Review

InvivoGen mt1 d273
Analyses of 3D molecular envelope of soluble form of <t>MT1-MMP</t> variants A. Schematic representation of the Fc-fusion MT1-MMP variants <t>(sMT1-D273-Fc</t> and sMT1-N273-Fc). Whole ectodomain of MT1-MMP variants (Met 1 -G 543 , green) were fused with rabbit IgG Fc portion (red). B. Schematic representation of sMT1-Fc protein. IgG Fc region mediates homodimer formation through the disulfide bonds. Pro-peptide is removed upon secretion by proprotein convertases (PCs), and the enzyme is secreted as active form. C Scattering curve of sMT1-D 273 -Fc and sMT1-N 273 -Fc (scattering intensity vs. scattering angle, q = 4πsinθ/λ). The SEC-separated sample was exposed to X-rays in a 1.6 mm diameter, 10 μm thick quartz capillary flow cell, followed by data collection every 3 seconds. Frames which were captured around the elution peak were selected, then the background subtraction and averaging were done. Scattering curves form 2 variants are similar. D Pair-distance distribution function (P(r)) for sMT1-D 273 -Fc and sMT1-N 273 -Fc. P(r) calculations were done by using ScÅtter. The overall shape of P(r) functions are slightly different between 2 variants although D max values are almost same. E. Structural parameters which were derived from SAXS analysis F. The molecular mass calculated from the volume of correlation and amino acid composition for each Fc fusion MT1-MMP variant. G. The SEC-SAXS analyses of purified sMT1-D 273 -Fc and sMT1-N 273 -Fc. Top and lower panel shows the 90° rotated view of 3D molecular envelope models for sMT1-D 273 -Fc and sMT1-N 273 -Fc, respectively. The 3D envelope models were created by taking the mean of 40 individual models for each molecule. H. Analyses of gelatinolytic and collagenolytic activities of sMT1-D 273 -Fc and sMT1-N 273 -Fc. Purified sMT1-D 273 -Fc (17.5 ng) and sMT1-N 273 -Fc (17.5 ng) were reacted with gelatin for 4h at 37°C (left panel) or with type I collagen for 72h at 21°C (right panel). α1 and α2 chains for gelatin and collagen are indicated. Non-annotated arrows in the left panel indicate degraded fragments of gelatin. The ¾ and ¼ fragments of collagen α1 and α2 chains generated by collagenolysis are indicated for the right panel.
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1) Product Images from "An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren’s Disease"

Article Title: An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren’s Disease

Journal: bioRxiv

doi: 10.1101/2020.06.09.142513

Analyses of 3D molecular envelope of soluble form of MT1-MMP variants A. Schematic representation of the Fc-fusion MT1-MMP variants (sMT1-D273-Fc and sMT1-N273-Fc). Whole ectodomain of MT1-MMP variants (Met 1 -G 543 , green) were fused with rabbit IgG Fc portion (red). B. Schematic representation of sMT1-Fc protein. IgG Fc region mediates homodimer formation through the disulfide bonds. Pro-peptide is removed upon secretion by proprotein convertases (PCs), and the enzyme is secreted as active form. C Scattering curve of sMT1-D 273 -Fc and sMT1-N 273 -Fc (scattering intensity vs. scattering angle, q = 4πsinθ/λ). The SEC-separated sample was exposed to X-rays in a 1.6 mm diameter, 10 μm thick quartz capillary flow cell, followed by data collection every 3 seconds. Frames which were captured around the elution peak were selected, then the background subtraction and averaging were done. Scattering curves form 2 variants are similar. D Pair-distance distribution function (P(r)) for sMT1-D 273 -Fc and sMT1-N 273 -Fc. P(r) calculations were done by using ScÅtter. The overall shape of P(r) functions are slightly different between 2 variants although D max values are almost same. E. Structural parameters which were derived from SAXS analysis F. The molecular mass calculated from the volume of correlation and amino acid composition for each Fc fusion MT1-MMP variant. G. The SEC-SAXS analyses of purified sMT1-D 273 -Fc and sMT1-N 273 -Fc. Top and lower panel shows the 90° rotated view of 3D molecular envelope models for sMT1-D 273 -Fc and sMT1-N 273 -Fc, respectively. The 3D envelope models were created by taking the mean of 40 individual models for each molecule. H. Analyses of gelatinolytic and collagenolytic activities of sMT1-D 273 -Fc and sMT1-N 273 -Fc. Purified sMT1-D 273 -Fc (17.5 ng) and sMT1-N 273 -Fc (17.5 ng) were reacted with gelatin for 4h at 37°C (left panel) or with type I collagen for 72h at 21°C (right panel). α1 and α2 chains for gelatin and collagen are indicated. Non-annotated arrows in the left panel indicate degraded fragments of gelatin. The ¾ and ¼ fragments of collagen α1 and α2 chains generated by collagenolysis are indicated for the right panel.
Figure Legend Snippet: Analyses of 3D molecular envelope of soluble form of MT1-MMP variants A. Schematic representation of the Fc-fusion MT1-MMP variants (sMT1-D273-Fc and sMT1-N273-Fc). Whole ectodomain of MT1-MMP variants (Met 1 -G 543 , green) were fused with rabbit IgG Fc portion (red). B. Schematic representation of sMT1-Fc protein. IgG Fc region mediates homodimer formation through the disulfide bonds. Pro-peptide is removed upon secretion by proprotein convertases (PCs), and the enzyme is secreted as active form. C Scattering curve of sMT1-D 273 -Fc and sMT1-N 273 -Fc (scattering intensity vs. scattering angle, q = 4πsinθ/λ). The SEC-separated sample was exposed to X-rays in a 1.6 mm diameter, 10 μm thick quartz capillary flow cell, followed by data collection every 3 seconds. Frames which were captured around the elution peak were selected, then the background subtraction and averaging were done. Scattering curves form 2 variants are similar. D Pair-distance distribution function (P(r)) for sMT1-D 273 -Fc and sMT1-N 273 -Fc. P(r) calculations were done by using ScÅtter. The overall shape of P(r) functions are slightly different between 2 variants although D max values are almost same. E. Structural parameters which were derived from SAXS analysis F. The molecular mass calculated from the volume of correlation and amino acid composition for each Fc fusion MT1-MMP variant. G. The SEC-SAXS analyses of purified sMT1-D 273 -Fc and sMT1-N 273 -Fc. Top and lower panel shows the 90° rotated view of 3D molecular envelope models for sMT1-D 273 -Fc and sMT1-N 273 -Fc, respectively. The 3D envelope models were created by taking the mean of 40 individual models for each molecule. H. Analyses of gelatinolytic and collagenolytic activities of sMT1-D 273 -Fc and sMT1-N 273 -Fc. Purified sMT1-D 273 -Fc (17.5 ng) and sMT1-N 273 -Fc (17.5 ng) were reacted with gelatin for 4h at 37°C (left panel) or with type I collagen for 72h at 21°C (right panel). α1 and α2 chains for gelatin and collagen are indicated. Non-annotated arrows in the left panel indicate degraded fragments of gelatin. The ¾ and ¼ fragments of collagen α1 and α2 chains generated by collagenolysis are indicated for the right panel.

Techniques Used: Derivative Assay, Variant Assay, Purification, Generated

2) Product Images from "Sensitive and adaptable pharmacological control of CAR T cells through extracellular receptor dimerization"

Article Title: Sensitive and adaptable pharmacological control of CAR T cells through extracellular receptor dimerization

Journal: JCI Insight

doi: 10.1172/jci.insight.124430

DARIC T cells exhibit drug-mediated tumor control in vivo. ( A ) Outline of the in vivo experiment for testing CD19-DARIC T cells. The T cells were infused 11 days following tumor injection while drug dosing started 1 day prior to T cell injection. ( B ) Summary bioluminescence data for each drug control group. The UDT and the no-drug CAR/DARIC groups are the same for all figures, while the “+ drug” groups (light blue and red) represent the specific drug dose used for the group. Data points represent 5 mice. ( C ) Representative bioluminescence imaging at day 22 following tumor injection. ( D ) Bioluminescence tumor imaging of the AP2167 group followed for additional 20 days after drug dosing was stopped.
Figure Legend Snippet: DARIC T cells exhibit drug-mediated tumor control in vivo. ( A ) Outline of the in vivo experiment for testing CD19-DARIC T cells. The T cells were infused 11 days following tumor injection while drug dosing started 1 day prior to T cell injection. ( B ) Summary bioluminescence data for each drug control group. The UDT and the no-drug CAR/DARIC groups are the same for all figures, while the “+ drug” groups (light blue and red) represent the specific drug dose used for the group. Data points represent 5 mice. ( C ) Representative bioluminescence imaging at day 22 following tumor injection. ( D ) Bioluminescence tumor imaging of the AP2167 group followed for additional 20 days after drug dosing was stopped.

Techniques Used: In Vivo, Injection, Mouse Assay, Imaging

CD19-DARIC T cells are tumor reactive solely in the presence of a dimerization drug. ( A – C ) The CD19-CAR and CD19-DARIC T cells were cultured at a 1:1 ratio with fluorescent Nalm-6 target cells with or without different concentrations of either rapamycin or AP21967. Supernatant was collected 24 hours after culture initiation and cytokine levels were analyzed using IFN-γ–specific ELISA ( A and B ) or iQue QBead assay ( C ) ( n = 3). * P
Figure Legend Snippet: CD19-DARIC T cells are tumor reactive solely in the presence of a dimerization drug. ( A – C ) The CD19-CAR and CD19-DARIC T cells were cultured at a 1:1 ratio with fluorescent Nalm-6 target cells with or without different concentrations of either rapamycin or AP21967. Supernatant was collected 24 hours after culture initiation and cytokine levels were analyzed using IFN-γ–specific ELISA ( A and B ) or iQue QBead assay ( C ) ( n = 3). * P

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

CD19-DARIC T cells are potent even at low rapamycin concentrations and low antigen expression. ( A ) CD19-DARIC or CD19-CAR T cells were cultured with Nalm-6 cells for 24 hours in the presence of different concentrations of rapamycin. Cytokine production was analyzed using iQue QBeads. Data points represent 3 donors. ( B ) K562 cells were transfected with in vitro–transcribed mRNA encoding the CD19 antigen. The transfected cells were cultured for 24 hours and CD19 expression was analyzed by flow cytometry. The amount of CD19 mRNA for each transfection is shown on the left, and the CD19 MFI value is listed on the right. ( C ) CD19-transfected K562 cells were cultured with CD19-DARIC (red) or CD19-CAR (black) T cells in the presence of 20 nM AP21967. After a 24-hour incubation period, supernatant was collected for cytokine analysis and cells were cultured for an additional 72 hours (4 days total coculture). At the conclusion of the coculture period, the number of T cells in each well was counted. The dashed lines represent samples cultured without AP21967, while solid lines represent samples cultured in the presence of 20 nM AP21967. The gray line indicates untransduced control. ( D ) Cytokine production in 24-hour supernatants from C was analyzed using iQue QBeads. Data points represent 3 unique donors. * P
Figure Legend Snippet: CD19-DARIC T cells are potent even at low rapamycin concentrations and low antigen expression. ( A ) CD19-DARIC or CD19-CAR T cells were cultured with Nalm-6 cells for 24 hours in the presence of different concentrations of rapamycin. Cytokine production was analyzed using iQue QBeads. Data points represent 3 donors. ( B ) K562 cells were transfected with in vitro–transcribed mRNA encoding the CD19 antigen. The transfected cells were cultured for 24 hours and CD19 expression was analyzed by flow cytometry. The amount of CD19 mRNA for each transfection is shown on the left, and the CD19 MFI value is listed on the right. ( C ) CD19-transfected K562 cells were cultured with CD19-DARIC (red) or CD19-CAR (black) T cells in the presence of 20 nM AP21967. After a 24-hour incubation period, supernatant was collected for cytokine analysis and cells were cultured for an additional 72 hours (4 days total coculture). At the conclusion of the coculture period, the number of T cells in each well was counted. The dashed lines represent samples cultured without AP21967, while solid lines represent samples cultured in the presence of 20 nM AP21967. The gray line indicates untransduced control. ( D ) Cytokine production in 24-hour supernatants from C was analyzed using iQue QBeads. Data points represent 3 unique donors. * P

Techniques Used: Expressing, Cell Culture, Transfection, In Vitro, Flow Cytometry, Cytometry, Incubation

DARIC T cells recognize secondary antigens through the DARIC plug-in system. ( A ) Schematic of a DARIC signaling architecture in the presence or absence of a DARIC plug-in targeting a secondary antigen. ( B ) The recombinant CD19-DARIC plug-in was produced and purified from 293T cells using a rapamycin-based affinity column. The purified protein was analyzed using Coomassie blue staining. ( C ) Unmodified CD19-DARIC T cells were cocultured with K562-BCMA cells alone, in the presence of rapamycin, or in the presence of increasing concentration of rapamycin-preloaded BCMA plug-in. Cytokine production was analyzed by iQue QBeads. ( D ) The cytotoxicity and ( E ) cytokine production of BCMA-DARIC T cells cocultured with CD19 + Nalm-6 cells in the presence or absence of rapamycin-preloaded CD19 DARIC plug-in. ( F ) Schematic of adaptable CAR architecture, with an extracellular FRB* domain located next to the scFv able to bind recombinant DARIC plug-in scFv to target a secondary antigen. ( G ) The cytotoxicity and ( H ) IFN-γ cytokine production of BCMA-adaptable CAR following 24-hour coculture with K562-BCMA target cells was analyzed by FACS and QBeads, respectively. ( I ) The cytotoxicity and ( J ) cytokine production of control and BCMA-adaptable CAR in the presence of recombinant CD19-DARIC plug-in was analyzed after 24-hour coculture with Nalm-6 target cells. Data points represent 3 different donors.
Figure Legend Snippet: DARIC T cells recognize secondary antigens through the DARIC plug-in system. ( A ) Schematic of a DARIC signaling architecture in the presence or absence of a DARIC plug-in targeting a secondary antigen. ( B ) The recombinant CD19-DARIC plug-in was produced and purified from 293T cells using a rapamycin-based affinity column. The purified protein was analyzed using Coomassie blue staining. ( C ) Unmodified CD19-DARIC T cells were cocultured with K562-BCMA cells alone, in the presence of rapamycin, or in the presence of increasing concentration of rapamycin-preloaded BCMA plug-in. Cytokine production was analyzed by iQue QBeads. ( D ) The cytotoxicity and ( E ) cytokine production of BCMA-DARIC T cells cocultured with CD19 + Nalm-6 cells in the presence or absence of rapamycin-preloaded CD19 DARIC plug-in. ( F ) Schematic of adaptable CAR architecture, with an extracellular FRB* domain located next to the scFv able to bind recombinant DARIC plug-in scFv to target a secondary antigen. ( G ) The cytotoxicity and ( H ) IFN-γ cytokine production of BCMA-adaptable CAR following 24-hour coculture with K562-BCMA target cells was analyzed by FACS and QBeads, respectively. ( I ) The cytotoxicity and ( J ) cytokine production of control and BCMA-adaptable CAR in the presence of recombinant CD19-DARIC plug-in was analyzed after 24-hour coculture with Nalm-6 target cells. Data points represent 3 different donors.

Techniques Used: Recombinant, Produced, Purification, Affinity Column, Staining, Concentration Assay, FACS

CD19-DARIC T cells control tumor growth in vivo with nonimmunosuppressive rapamycin dosing. ( A ) Outline of the in vivo experiment for analyzing CD19-DARIC activity at low rapamycin dosing. ( B ) Summary bioluminescence reading for all the experimental groups. Black and blue dashed lines represent initial T cell injection and cessation of rapamycin dosing, respectively. Data presented as mean and standard deviation of 5 animals per group. ( C ) Trough rapamycin levels in whole blood were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Analysis was done at 24 hours (0.1 and 0.01) or 48 hours (0.1 q.a.d.) following the last rapamycin injection. ( D ) The bioluminescence traces for individual animals from each dose group shown in B are represented as black lines. The animals were tracked by regular imaging and rapamycin dosing was restarted when tumor regrowth was detected (red lines).
Figure Legend Snippet: CD19-DARIC T cells control tumor growth in vivo with nonimmunosuppressive rapamycin dosing. ( A ) Outline of the in vivo experiment for analyzing CD19-DARIC activity at low rapamycin dosing. ( B ) Summary bioluminescence reading for all the experimental groups. Black and blue dashed lines represent initial T cell injection and cessation of rapamycin dosing, respectively. Data presented as mean and standard deviation of 5 animals per group. ( C ) Trough rapamycin levels in whole blood were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Analysis was done at 24 hours (0.1 and 0.01) or 48 hours (0.1 q.a.d.) following the last rapamycin injection. ( D ) The bioluminescence traces for individual animals from each dose group shown in B are represented as black lines. The animals were tracked by regular imaging and rapamycin dosing was restarted when tumor regrowth was detected (red lines).

Techniques Used: In Vivo, Activity Assay, Injection, Standard Deviation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Imaging

CD19-DARIC transduction results in normal T cell development. ( A ) Schematic of the lentiviral vectors used in the study. SS, signal sequence; TM, transmembrane domain. ( B ) The CD19-DARIC T cells are inactive in the absence of drug (“OFF”) and addition of dimerizing agent brings the 2 domains together to turn the receptor “ON”. ( C ) T cells were transduced, expanded, and phenotyped after 10 days of expansion with CD62L and CD45RA. The T cell memory and naive compartments were identified on the basis of relative marker expression and the summary data are shown on the right. ( D ) The transduced cells were stained with fluorescently conjugated CD19-His antigen and analyzed by FACS. Summary of 3 donors is shown on the right, with the MFI values in red and the VCN values for each sample annotated above. ( E ) Western blot analysis of T cell lysates using CD3ζ- and 2A-specific antibodies. Relative intensity was determined using LICOR Western blot analysis software. ( F ) CD19-CAR or CD19-DARIC T cells were cultured for the indicated amount of time in the presence or absence of rapamycin. The expression of the CAR/DARIC construct was analyzed by staining with rabbit polyclonal anti–CD19 complex antibody. The summary of the staining data is shown below. Data are representative of at least 3 separate experiments, with 3 unique donors per experiment.
Figure Legend Snippet: CD19-DARIC transduction results in normal T cell development. ( A ) Schematic of the lentiviral vectors used in the study. SS, signal sequence; TM, transmembrane domain. ( B ) The CD19-DARIC T cells are inactive in the absence of drug (“OFF”) and addition of dimerizing agent brings the 2 domains together to turn the receptor “ON”. ( C ) T cells were transduced, expanded, and phenotyped after 10 days of expansion with CD62L and CD45RA. The T cell memory and naive compartments were identified on the basis of relative marker expression and the summary data are shown on the right. ( D ) The transduced cells were stained with fluorescently conjugated CD19-His antigen and analyzed by FACS. Summary of 3 donors is shown on the right, with the MFI values in red and the VCN values for each sample annotated above. ( E ) Western blot analysis of T cell lysates using CD3ζ- and 2A-specific antibodies. Relative intensity was determined using LICOR Western blot analysis software. ( F ) CD19-CAR or CD19-DARIC T cells were cultured for the indicated amount of time in the presence or absence of rapamycin. The expression of the CAR/DARIC construct was analyzed by staining with rabbit polyclonal anti–CD19 complex antibody. The summary of the staining data is shown below. Data are representative of at least 3 separate experiments, with 3 unique donors per experiment.

Techniques Used: Transduction, Sequencing, Marker, Expressing, Staining, FACS, Western Blot, Software, Cell Culture, Construct

3) Product Images from "An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren’s Disease"

Article Title: An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren’s Disease

Journal: bioRxiv

doi: 10.1101/2020.06.09.142513

Analyses of 3D molecular envelope of soluble form of MT1-MMP variants A. Schematic representation of the Fc-fusion MT1-MMP variants (sMT1-D273-Fc and sMT1-N273-Fc). Whole ectodomain of MT1-MMP variants (Met 1 -G 543 , green) were fused with rabbit IgG Fc portion (red). B. Schematic representation of sMT1-Fc protein. IgG Fc region mediates homodimer formation through the disulfide bonds. Pro-peptide is removed upon secretion by proprotein convertases (PCs), and the enzyme is secreted as active form. C Scattering curve of sMT1-D 273 -Fc and sMT1-N 273 -Fc (scattering intensity vs. scattering angle, q = 4πsinθ/λ). The SEC-separated sample was exposed to X-rays in a 1.6 mm diameter, 10 μm thick quartz capillary flow cell, followed by data collection every 3 seconds. Frames which were captured around the elution peak were selected, then the background subtraction and averaging were done. Scattering curves form 2 variants are similar. D Pair-distance distribution function (P(r)) for sMT1-D 273 -Fc and sMT1-N 273 -Fc. P(r) calculations were done by using ScÅtter. The overall shape of P(r) functions are slightly different between 2 variants although D max values are almost same. E. Structural parameters which were derived from SAXS analysis F. The molecular mass calculated from the volume of correlation and amino acid composition for each Fc fusion MT1-MMP variant. G. The SEC-SAXS analyses of purified sMT1-D 273 -Fc and sMT1-N 273 -Fc. Top and lower panel shows the 90° rotated view of 3D molecular envelope models for sMT1-D 273 -Fc and sMT1-N 273 -Fc, respectively. The 3D envelope models were created by taking the mean of 40 individual models for each molecule. H. Analyses of gelatinolytic and collagenolytic activities of sMT1-D 273 -Fc and sMT1-N 273 -Fc. Purified sMT1-D 273 -Fc (17.5 ng) and sMT1-N 273 -Fc (17.5 ng) were reacted with gelatin for 4h at 37°C (left panel) or with type I collagen for 72h at 21°C (right panel). α1 and α2 chains for gelatin and collagen are indicated. Non-annotated arrows in the left panel indicate degraded fragments of gelatin. The ¾ and ¼ fragments of collagen α1 and α2 chains generated by collagenolysis are indicated for the right panel.
Figure Legend Snippet: Analyses of 3D molecular envelope of soluble form of MT1-MMP variants A. Schematic representation of the Fc-fusion MT1-MMP variants (sMT1-D273-Fc and sMT1-N273-Fc). Whole ectodomain of MT1-MMP variants (Met 1 -G 543 , green) were fused with rabbit IgG Fc portion (red). B. Schematic representation of sMT1-Fc protein. IgG Fc region mediates homodimer formation through the disulfide bonds. Pro-peptide is removed upon secretion by proprotein convertases (PCs), and the enzyme is secreted as active form. C Scattering curve of sMT1-D 273 -Fc and sMT1-N 273 -Fc (scattering intensity vs. scattering angle, q = 4πsinθ/λ). The SEC-separated sample was exposed to X-rays in a 1.6 mm diameter, 10 μm thick quartz capillary flow cell, followed by data collection every 3 seconds. Frames which were captured around the elution peak were selected, then the background subtraction and averaging were done. Scattering curves form 2 variants are similar. D Pair-distance distribution function (P(r)) for sMT1-D 273 -Fc and sMT1-N 273 -Fc. P(r) calculations were done by using ScÅtter. The overall shape of P(r) functions are slightly different between 2 variants although D max values are almost same. E. Structural parameters which were derived from SAXS analysis F. The molecular mass calculated from the volume of correlation and amino acid composition for each Fc fusion MT1-MMP variant. G. The SEC-SAXS analyses of purified sMT1-D 273 -Fc and sMT1-N 273 -Fc. Top and lower panel shows the 90° rotated view of 3D molecular envelope models for sMT1-D 273 -Fc and sMT1-N 273 -Fc, respectively. The 3D envelope models were created by taking the mean of 40 individual models for each molecule. H. Analyses of gelatinolytic and collagenolytic activities of sMT1-D 273 -Fc and sMT1-N 273 -Fc. Purified sMT1-D 273 -Fc (17.5 ng) and sMT1-N 273 -Fc (17.5 ng) were reacted with gelatin for 4h at 37°C (left panel) or with type I collagen for 72h at 21°C (right panel). α1 and α2 chains for gelatin and collagen are indicated. Non-annotated arrows in the left panel indicate degraded fragments of gelatin. The ¾ and ¼ fragments of collagen α1 and α2 chains generated by collagenolysis are indicated for the right panel.

Techniques Used: Derivative Assay, Variant Assay, Purification, Generated

4) Product Images from "An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren’s Disease"

Article Title: An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren’s Disease

Journal: bioRxiv

doi: 10.1101/2020.06.09.142513

Analyses of 3D molecular envelope of soluble form of MT1-MMP variants A. Schematic representation of the Fc-fusion MT1-MMP variants (sMT1-D273-Fc and sMT1-N273-Fc). Whole ectodomain of MT1-MMP variants (Met 1 -G 543 , green) were fused with rabbit IgG Fc portion (red). B. Schematic representation of sMT1-Fc protein. IgG Fc region mediates homodimer formation through the disulfide bonds. Pro-peptide is removed upon secretion by proprotein convertases (PCs), and the enzyme is secreted as active form. C Scattering curve of sMT1-D 273 -Fc and sMT1-N 273 -Fc (scattering intensity vs. scattering angle, q = 4πsinθ/λ). The SEC-separated sample was exposed to X-rays in a 1.6 mm diameter, 10 μm thick quartz capillary flow cell, followed by data collection every 3 seconds. Frames which were captured around the elution peak were selected, then the background subtraction and averaging were done. Scattering curves form 2 variants are similar. D Pair-distance distribution function (P(r)) for sMT1-D 273 -Fc and sMT1-N 273 -Fc. P(r) calculations were done by using ScÅtter. The overall shape of P(r) functions are slightly different between 2 variants although D max values are almost same. E. Structural parameters which were derived from SAXS analysis F. The molecular mass calculated from the volume of correlation and amino acid composition for each Fc fusion MT1-MMP variant. G. The SEC-SAXS analyses of purified sMT1-D 273 -Fc and sMT1-N 273 -Fc. Top and lower panel shows the 90° rotated view of 3D molecular envelope models for sMT1-D 273 -Fc and sMT1-N 273 -Fc, respectively. The 3D envelope models were created by taking the mean of 40 individual models for each molecule. H. Analyses of gelatinolytic and collagenolytic activities of sMT1-D 273 -Fc and sMT1-N 273 -Fc. Purified sMT1-D 273 -Fc (17.5 ng) and sMT1-N 273 -Fc (17.5 ng) were reacted with gelatin for 4h at 37°C (left panel) or with type I collagen for 72h at 21°C (right panel). α1 and α2 chains for gelatin and collagen are indicated. Non-annotated arrows in the left panel indicate degraded fragments of gelatin. The ¾ and ¼ fragments of collagen α1 and α2 chains generated by collagenolysis are indicated for the right panel.
Figure Legend Snippet: Analyses of 3D molecular envelope of soluble form of MT1-MMP variants A. Schematic representation of the Fc-fusion MT1-MMP variants (sMT1-D273-Fc and sMT1-N273-Fc). Whole ectodomain of MT1-MMP variants (Met 1 -G 543 , green) were fused with rabbit IgG Fc portion (red). B. Schematic representation of sMT1-Fc protein. IgG Fc region mediates homodimer formation through the disulfide bonds. Pro-peptide is removed upon secretion by proprotein convertases (PCs), and the enzyme is secreted as active form. C Scattering curve of sMT1-D 273 -Fc and sMT1-N 273 -Fc (scattering intensity vs. scattering angle, q = 4πsinθ/λ). The SEC-separated sample was exposed to X-rays in a 1.6 mm diameter, 10 μm thick quartz capillary flow cell, followed by data collection every 3 seconds. Frames which were captured around the elution peak were selected, then the background subtraction and averaging were done. Scattering curves form 2 variants are similar. D Pair-distance distribution function (P(r)) for sMT1-D 273 -Fc and sMT1-N 273 -Fc. P(r) calculations were done by using ScÅtter. The overall shape of P(r) functions are slightly different between 2 variants although D max values are almost same. E. Structural parameters which were derived from SAXS analysis F. The molecular mass calculated from the volume of correlation and amino acid composition for each Fc fusion MT1-MMP variant. G. The SEC-SAXS analyses of purified sMT1-D 273 -Fc and sMT1-N 273 -Fc. Top and lower panel shows the 90° rotated view of 3D molecular envelope models for sMT1-D 273 -Fc and sMT1-N 273 -Fc, respectively. The 3D envelope models were created by taking the mean of 40 individual models for each molecule. H. Analyses of gelatinolytic and collagenolytic activities of sMT1-D 273 -Fc and sMT1-N 273 -Fc. Purified sMT1-D 273 -Fc (17.5 ng) and sMT1-N 273 -Fc (17.5 ng) were reacted with gelatin for 4h at 37°C (left panel) or with type I collagen for 72h at 21°C (right panel). α1 and α2 chains for gelatin and collagen are indicated. Non-annotated arrows in the left panel indicate degraded fragments of gelatin. The ¾ and ¼ fragments of collagen α1 and α2 chains generated by collagenolysis are indicated for the right panel.

Techniques Used: Derivative Assay, Variant Assay, Purification, Generated

5) Product Images from "Human GUCY2C-targeted chimeric antigen receptor (CAR)-expressing T cells eliminate colorectal cancer metastases"

Article Title: Human GUCY2C-targeted chimeric antigen receptor (CAR)-expressing T cells eliminate colorectal cancer metastases

Journal: Cancer immunology research

doi: 10.1158/2326-6066.CIR-16-0362

Generation of human GUCY2C-specific CAR-T cells (A) Recombinant 5F9 antibody was assessed by ELISA for specific binding to hGUCY2C ECD or BSA (negative control) plated at 1 μg/mL. Two-way ANOVA; **** p
Figure Legend Snippet: Generation of human GUCY2C-specific CAR-T cells (A) Recombinant 5F9 antibody was assessed by ELISA for specific binding to hGUCY2C ECD or BSA (negative control) plated at 1 μg/mL. Two-way ANOVA; **** p

Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control

6) Product Images from "Sensitive and adaptable pharmacological control of CAR T cells through extracellular receptor dimerization"

Article Title: Sensitive and adaptable pharmacological control of CAR T cells through extracellular receptor dimerization

Journal: JCI Insight

doi: 10.1172/jci.insight.124430

DARIC T cells recognize secondary antigens through the DARIC plug-in system. ( A ) Schematic of a DARIC signaling architecture in the presence or absence of a DARIC plug-in targeting a secondary antigen. ( B ) The recombinant CD19-DARIC plug-in was produced and purified from 293T cells using a rapamycin-based affinity column. The purified protein was analyzed using Coomassie blue staining. ( C ) Unmodified CD19-DARIC T cells were cocultured with K562-BCMA cells alone, in the presence of rapamycin, or in the presence of increasing concentration of rapamycin-preloaded BCMA plug-in. Cytokine production was analyzed by iQue QBeads. ( D ) The cytotoxicity and ( E ) cytokine production of BCMA-DARIC T cells cocultured with CD19 + Nalm-6 cells in the presence or absence of rapamycin-preloaded CD19 DARIC plug-in. ( F ) Schematic of adaptable CAR architecture, with an extracellular FRB* domain located next to the scFv able to bind recombinant DARIC plug-in scFv to target a secondary antigen. ( G ) The cytotoxicity and ( H ) IFN-γ cytokine production of BCMA-adaptable CAR following 24-hour coculture with K562-BCMA target cells was analyzed by FACS and QBeads, respectively. ( I ) The cytotoxicity and ( J ) cytokine production of control and BCMA-adaptable CAR in the presence of recombinant CD19-DARIC plug-in was analyzed after 24-hour coculture with Nalm-6 target cells. Data points represent 3 different donors.
Figure Legend Snippet: DARIC T cells recognize secondary antigens through the DARIC plug-in system. ( A ) Schematic of a DARIC signaling architecture in the presence or absence of a DARIC plug-in targeting a secondary antigen. ( B ) The recombinant CD19-DARIC plug-in was produced and purified from 293T cells using a rapamycin-based affinity column. The purified protein was analyzed using Coomassie blue staining. ( C ) Unmodified CD19-DARIC T cells were cocultured with K562-BCMA cells alone, in the presence of rapamycin, or in the presence of increasing concentration of rapamycin-preloaded BCMA plug-in. Cytokine production was analyzed by iQue QBeads. ( D ) The cytotoxicity and ( E ) cytokine production of BCMA-DARIC T cells cocultured with CD19 + Nalm-6 cells in the presence or absence of rapamycin-preloaded CD19 DARIC plug-in. ( F ) Schematic of adaptable CAR architecture, with an extracellular FRB* domain located next to the scFv able to bind recombinant DARIC plug-in scFv to target a secondary antigen. ( G ) The cytotoxicity and ( H ) IFN-γ cytokine production of BCMA-adaptable CAR following 24-hour coculture with K562-BCMA target cells was analyzed by FACS and QBeads, respectively. ( I ) The cytotoxicity and ( J ) cytokine production of control and BCMA-adaptable CAR in the presence of recombinant CD19-DARIC plug-in was analyzed after 24-hour coculture with Nalm-6 target cells. Data points represent 3 different donors.

Techniques Used: Recombinant, Produced, Purification, Affinity Column, Staining, Concentration Assay, FACS

7) Product Images from "Fibroblastic Reticular Cells Control Conduit Matrix Deposition during Lymph Node Expansion"

Article Title: Fibroblastic Reticular Cells Control Conduit Matrix Deposition during Lymph Node Expansion

Journal: Cell Reports

doi: 10.1016/j.celrep.2019.10.103

Regulation of Microtubule Organization by CLEC-2 via LL5β (A) Representative western blots showing LL5β expression in FRC cell lines. (B) Expression of LL5β mRNA relative to control FRCs by qPCR. Error bars represent means and SDs of 2 biological replicates. (C) Immunofluorescence of FRC cell lines in vitro . Maximum z stack projections. Scale bars, 10 μm. (D) Quantification of LL5β coverage in FRC cell lines as a percentage of the total perimeter. Each dot represents 1 cell. Error bars represent means and SDs. ∗∗∗∗ p
Figure Legend Snippet: Regulation of Microtubule Organization by CLEC-2 via LL5β (A) Representative western blots showing LL5β expression in FRC cell lines. (B) Expression of LL5β mRNA relative to control FRCs by qPCR. Error bars represent means and SDs of 2 biological replicates. (C) Immunofluorescence of FRC cell lines in vitro . Maximum z stack projections. Scale bars, 10 μm. (D) Quantification of LL5β coverage in FRC cell lines as a percentage of the total perimeter. Each dot represents 1 cell. Error bars represent means and SDs. ∗∗∗∗ p

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

Effects of CLEC-2 on ECM Production by FRCs (A) Gene Ontology analysis, genes regulated by CLEC-2-Fc ≥2-fold. RNA-seq data from 4 biological replicates per condition. (B) CLEC-2-Fc-regulated matrisomal gene cluster in a principal-component analysis (PCA) space. Arrows indicate time course. (C) Heatmaps of matrisomal genes regulated ≥2-fold by CLEC-2-Fc. Four biological replicates for each condition are shown. Color-coding represents Z score; row average is indicated (right). (D) Fibronectin (top) and collagen VI (bottom) representative immunofluorescence staining of in vitro FRC-derived matrices. Maximum z stack projections; scale bars, 20 μm. (E) Median gray intensity for ECM components. Each dot represents a region of interest, combined from 3 biological replicates. Error bars represent means and SDs. ∗ p
Figure Legend Snippet: Effects of CLEC-2 on ECM Production by FRCs (A) Gene Ontology analysis, genes regulated by CLEC-2-Fc ≥2-fold. RNA-seq data from 4 biological replicates per condition. (B) CLEC-2-Fc-regulated matrisomal gene cluster in a principal-component analysis (PCA) space. Arrows indicate time course. (C) Heatmaps of matrisomal genes regulated ≥2-fold by CLEC-2-Fc. Four biological replicates for each condition are shown. Color-coding represents Z score; row average is indicated (right). (D) Fibronectin (top) and collagen VI (bottom) representative immunofluorescence staining of in vitro FRC-derived matrices. Maximum z stack projections; scale bars, 20 μm. (E) Median gray intensity for ECM components. Each dot represents a region of interest, combined from 3 biological replicates. Error bars represent means and SDs. ∗ p

Techniques Used: RNA Sequencing Assay, Immunofluorescence, Staining, In Vitro, Derivative Assay

Phosphoproteomics of CLEC-2-Fc-Treated FRCs (A) Experimental setup, comparison of 5 control (untreated) and 5 CLEC-2-Fc-treated FRC cell lysates (biological replicates). (B) Waterfall plots showing proteome regulation by CLEC-2-Fc. (C) Control and CLEC-2-Fc-treated phosphoproteomes cluster in a PCA space. (D) Volcano plots showing statistical regulation of the CLEC-2-Fc-treated FRC’s phosphoproteome (n = 5, two-tailed t test, Gaussian regression). The number of regulated phosphosites is indicated. (E) Empirical parent kinase analysis. The bars represent the number of targets for kinases. Positive and negative values indicate higher or lower phosphorylation in CLEC-2-Fc-treated cells. (F) Gene Ontology analysis for biological processes. Each bar represents a biological process significantly enriched by binomial analysis.
Figure Legend Snippet: Phosphoproteomics of CLEC-2-Fc-Treated FRCs (A) Experimental setup, comparison of 5 control (untreated) and 5 CLEC-2-Fc-treated FRC cell lysates (biological replicates). (B) Waterfall plots showing proteome regulation by CLEC-2-Fc. (C) Control and CLEC-2-Fc-treated phosphoproteomes cluster in a PCA space. (D) Volcano plots showing statistical regulation of the CLEC-2-Fc-treated FRC’s phosphoproteome (n = 5, two-tailed t test, Gaussian regression). The number of regulated phosphosites is indicated. (E) Empirical parent kinase analysis. The bars represent the number of targets for kinases. Positive and negative values indicate higher or lower phosphorylation in CLEC-2-Fc-treated cells. (F) Gene Ontology analysis for biological processes. Each bar represents a biological process significantly enriched by binomial analysis.

Techniques Used: Two Tailed Test

8) Product Images from "Fibroblastic Reticular Cells Control Conduit Matrix Deposition during Lymph Node Expansion"

Article Title: Fibroblastic Reticular Cells Control Conduit Matrix Deposition during Lymph Node Expansion

Journal: Cell Reports

doi: 10.1016/j.celrep.2019.10.103

Regulation of Microtubule Organization by CLEC-2 via LL5β (A) Representative western blots showing LL5β expression in FRC cell lines. (B) Expression of LL5β mRNA relative to control FRCs by qPCR. Error bars represent means and SDs of 2 biological replicates. (C) Immunofluorescence of FRC cell lines in vitro . Maximum z stack projections. Scale bars, 10 μm. (D) Quantification of LL5β coverage in FRC cell lines as a percentage of the total perimeter. Each dot represents 1 cell. Error bars represent means and SDs. ∗∗∗∗ p
Figure Legend Snippet: Regulation of Microtubule Organization by CLEC-2 via LL5β (A) Representative western blots showing LL5β expression in FRC cell lines. (B) Expression of LL5β mRNA relative to control FRCs by qPCR. Error bars represent means and SDs of 2 biological replicates. (C) Immunofluorescence of FRC cell lines in vitro . Maximum z stack projections. Scale bars, 10 μm. (D) Quantification of LL5β coverage in FRC cell lines as a percentage of the total perimeter. Each dot represents 1 cell. Error bars represent means and SDs. ∗∗∗∗ p

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

Effects of CLEC-2 on ECM Production by FRCs (A) Gene Ontology analysis, genes regulated by CLEC-2-Fc ≥2-fold. RNA-seq data from 4 biological replicates per condition. (B) CLEC-2-Fc-regulated matrisomal gene cluster in a principal-component analysis (PCA) space. Arrows indicate time course. (C) Heatmaps of matrisomal genes regulated ≥2-fold by CLEC-2-Fc. Four biological replicates for each condition are shown. Color-coding represents Z score; row average is indicated (right). (D) Fibronectin (top) and collagen VI (bottom) representative immunofluorescence staining of in vitro FRC-derived matrices. Maximum z stack projections; scale bars, 20 μm. (E) Median gray intensity for ECM components. Each dot represents a region of interest, combined from 3 biological replicates. Error bars represent means and SDs. ∗ p
Figure Legend Snippet: Effects of CLEC-2 on ECM Production by FRCs (A) Gene Ontology analysis, genes regulated by CLEC-2-Fc ≥2-fold. RNA-seq data from 4 biological replicates per condition. (B) CLEC-2-Fc-regulated matrisomal gene cluster in a principal-component analysis (PCA) space. Arrows indicate time course. (C) Heatmaps of matrisomal genes regulated ≥2-fold by CLEC-2-Fc. Four biological replicates for each condition are shown. Color-coding represents Z score; row average is indicated (right). (D) Fibronectin (top) and collagen VI (bottom) representative immunofluorescence staining of in vitro FRC-derived matrices. Maximum z stack projections; scale bars, 20 μm. (E) Median gray intensity for ECM components. Each dot represents a region of interest, combined from 3 biological replicates. Error bars represent means and SDs. ∗ p

Techniques Used: RNA Sequencing Assay, Immunofluorescence, Staining, In Vitro, Derivative Assay

Phosphoproteomics of CLEC-2-Fc-Treated FRCs (A) Experimental setup, comparison of 5 control (untreated) and 5 CLEC-2-Fc-treated FRC cell lysates (biological replicates). (B) Waterfall plots showing proteome regulation by CLEC-2-Fc. (C) Control and CLEC-2-Fc-treated phosphoproteomes cluster in a PCA space. (D) Volcano plots showing statistical regulation of the CLEC-2-Fc-treated FRC’s phosphoproteome (n = 5, two-tailed t test, Gaussian regression). The number of regulated phosphosites is indicated. (E) Empirical parent kinase analysis. The bars represent the number of targets for kinases. Positive and negative values indicate higher or lower phosphorylation in CLEC-2-Fc-treated cells. (F) Gene Ontology analysis for biological processes. Each bar represents a biological process significantly enriched by binomial analysis.
Figure Legend Snippet: Phosphoproteomics of CLEC-2-Fc-Treated FRCs (A) Experimental setup, comparison of 5 control (untreated) and 5 CLEC-2-Fc-treated FRC cell lysates (biological replicates). (B) Waterfall plots showing proteome regulation by CLEC-2-Fc. (C) Control and CLEC-2-Fc-treated phosphoproteomes cluster in a PCA space. (D) Volcano plots showing statistical regulation of the CLEC-2-Fc-treated FRC’s phosphoproteome (n = 5, two-tailed t test, Gaussian regression). The number of regulated phosphosites is indicated. (E) Empirical parent kinase analysis. The bars represent the number of targets for kinases. Positive and negative values indicate higher or lower phosphorylation in CLEC-2-Fc-treated cells. (F) Gene Ontology analysis for biological processes. Each bar represents a biological process significantly enriched by binomial analysis.

Techniques Used: Two Tailed Test

9) Product Images from "An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren’s Disease"

Article Title: An SNP variant MT1-MMP with a defect in its collagenolytic activity confers the fibrotic phenotype of Dupuytren’s Disease

Journal: bioRxiv

doi: 10.1101/2020.06.09.142513

Effect of D 273 N substitution on biological activities of MT1-MMP A. COS7 cells were transfected with expression plasmids for FLAG-tagged MT1-D 273 , MT1-N 273 or empty plasmid, and subjected to fluorescently labeled gelatin film degradation assay. Cell surface MT1-MMPs were stained with anti-FLAG M2 antibody without permeabilizing cells. B. COS7 cells expressing MT1-D 273 (D 273 ) or MT1-N 273 (N 273 ) were cultured in proMMP-2-containing conditioned medium for 24h. ProMMP-2 in the media were analyzed by zymography (Zymo) and cell lysates by Western Blotting using anti-MT1-MMP Hpx domain antibody (MT1-MMP), and anti-actin as a loading control. C. COS7 cells expressing MT1-D 273 or MT1-N 273 were subjected to the collagen film degradation assay. Cells were transfected with MT1-D 273 (D), MT1-N 273 (N), both MT1-D 273 and MT1-N 273 in a 1:1 ratio (D/N), or empty plasmid (Mock). Images are representative of the collagen degradation activity of the cells, which is quantified in the bar chart. N=4 replicates for each transfection. Data represent mean ± SEM; ***p
Figure Legend Snippet: Effect of D 273 N substitution on biological activities of MT1-MMP A. COS7 cells were transfected with expression plasmids for FLAG-tagged MT1-D 273 , MT1-N 273 or empty plasmid, and subjected to fluorescently labeled gelatin film degradation assay. Cell surface MT1-MMPs were stained with anti-FLAG M2 antibody without permeabilizing cells. B. COS7 cells expressing MT1-D 273 (D 273 ) or MT1-N 273 (N 273 ) were cultured in proMMP-2-containing conditioned medium for 24h. ProMMP-2 in the media were analyzed by zymography (Zymo) and cell lysates by Western Blotting using anti-MT1-MMP Hpx domain antibody (MT1-MMP), and anti-actin as a loading control. C. COS7 cells expressing MT1-D 273 or MT1-N 273 were subjected to the collagen film degradation assay. Cells were transfected with MT1-D 273 (D), MT1-N 273 (N), both MT1-D 273 and MT1-N 273 in a 1:1 ratio (D/N), or empty plasmid (Mock). Images are representative of the collagen degradation activity of the cells, which is quantified in the bar chart. N=4 replicates for each transfection. Data represent mean ± SEM; ***p

Techniques Used: Transfection, Expressing, Plasmid Preparation, Labeling, Degradation Assay, Staining, Cell Culture, Zymography, Western Blot, Activity Assay

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Transformation Assay:

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Article Snippet: .. For other drug selection, transformed cells were recovered in YES media for 4 h in shaking incubator or overnight on the plate, and transferred to YES plate containing G418 (FORMEDIUM), Zeocin (Invivogen), Hygromycine B (FORMEDIUM and Roche) or ClonNat/Nourseothricin (WERNER BioAgents). ..

Selection:

Article Title: Sequential and counter-selectable cassettes for fission yeast
Article Snippet: .. For other drug selection, transformed cells were recovered in YES media for 4 h in shaking incubator or overnight on the plate, and transferred to YES plate containing G418 (FORMEDIUM), Zeocin (Invivogen), Hygromycine B (FORMEDIUM and Roche) or ClonNat/Nourseothricin (WERNER BioAgents). ..

Article Title: Kinetics of drug selection systems in mouse embryonic stem cells
Article Snippet: .. 2 × 107 ES cells were electroporated with 30 μg of linearized plasmid DNA at 800 V and 3 μF in a 0.4 cm cuvette using a Gene-Pulser (Bio-Rad) and then cultured in the presence of the drugs for selection, Puromycin (Nacalai tesque) Zeocin (Invivogen), Hygromycin B (HygroGold, Invivogen), L-Histidinol (Sigma), Blasticidin S (Invivogen) and G418 (Nacalai tesque), at indicated concentrations. .. Colonies were identified by Leishman (SIGMA) staining, and counted.

Plasmid Preparation:

Article Title: Kinetics of drug selection systems in mouse embryonic stem cells
Article Snippet: .. 2 × 107 ES cells were electroporated with 30 μg of linearized plasmid DNA at 800 V and 3 μF in a 0.4 cm cuvette using a Gene-Pulser (Bio-Rad) and then cultured in the presence of the drugs for selection, Puromycin (Nacalai tesque) Zeocin (Invivogen), Hygromycin B (HygroGold, Invivogen), L-Histidinol (Sigma), Blasticidin S (Invivogen) and G418 (Nacalai tesque), at indicated concentrations. .. Colonies were identified by Leishman (SIGMA) staining, and counted.

Article Title: ?-Globin Matrix Attachment Region Improves Stable Genomic Expression of the Sleeping Beauty Transposon
Article Snippet: .. The basic starting vectors were the kanamycin resistant pKT2 [ ]; HSB3 and HSB5 Sleeping Beauty transposase variants driven by mouse initiation factor promoter 4A1 which replaced the lacZ CDS in the parental pDRIVE01-eIF4AI(m) plasmid (InvivoGen, San Diego, CA) [ ]; SV40 enhancer was from pDRIVE03-SV40enh3-Alb(b) (InvivoGen); β-globin MAR, the bacterial zeocin resistance gene and RK6 origin of replication from pCpG-MCS (InvivoGen); and the zeocin resistance::green fluorescent protein fusion gene from psiRNA4-H1GFPzeo (InvivoGen). .. All DNA for transfection was prepared using Qiagen EndoFree plasmid kits (Valencia, CA) kits according to the recommended protocol.

Cell Culture:

Article Title: Kinetics of drug selection systems in mouse embryonic stem cells
Article Snippet: .. 2 × 107 ES cells were electroporated with 30 μg of linearized plasmid DNA at 800 V and 3 μF in a 0.4 cm cuvette using a Gene-Pulser (Bio-Rad) and then cultured in the presence of the drugs for selection, Puromycin (Nacalai tesque) Zeocin (Invivogen), Hygromycin B (HygroGold, Invivogen), L-Histidinol (Sigma), Blasticidin S (Invivogen) and G418 (Nacalai tesque), at indicated concentrations. .. Colonies were identified by Leishman (SIGMA) staining, and counted.

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