Structured Review

Stratagene calmodulin insensitive atcpsf30 mutant
Demarcation of the RNA-binding domain of <t>AtCPSF30.</t> A, Binding of RNA by wild-type AtCPSF30 as a function of protein concentration. A total of 3.5 pmol of uniformly labeled RNA containing the polyadenylation signal of the pea rbcS-E9 gene was incubated with varying quantities of MBP-AtCPSF4 and RNA binding assessed by electrophoresis on native gels and autoradiography. The positions of the RNA-protein complex (complexes) and free RNA are indicated on the left. No protein was added for the sample in lane 8. Lanes 1 to 7 contained 10.5, 7.5, 4.5, 1.5, 0.75, 0.375, and 0.188 μ g of purified protein, respectively. B, RNA binding by mutant forms of AtCPSF30. On the left is a depiction of the different variants of AtCPSF30 that were produced as MBP fusion proteins. The central zinc-finger domain is represented with three black lines within the respective rectangular boxes, and the small domain that is responsible for <t>calmodulin</t> binding shown as a gray box within the larger representation. The clear box in the 30M representation indicates that the calmodulin-binding motif has been changed by mutation to be nonfunctional. Fusion proteins that bound RNA are listed under “RNA-binding,” and the plots of binding as a function of protein concentration given on the right. Fusion proteins for which no detectable RNA binding was observed are listed under “unable to bind RNA”; the plots for these proteins are not given, as they would coincide with the x axis.
Calmodulin Insensitive Atcpsf30 Mutant, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/calmodulin insensitive atcpsf30 mutant/product/Stratagene
Average 85 stars, based on 60 article reviews
Price from $9.99 to $1999.99
calmodulin insensitive atcpsf30 mutant - by Bioz Stars, 2020-09
85/100 stars

Images

1) Product Images from "Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1 [OA]"

Article Title: Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1 [OA]

Journal: Plant Physiology

doi: 10.1104/pp.105.070672

Demarcation of the RNA-binding domain of AtCPSF30. A, Binding of RNA by wild-type AtCPSF30 as a function of protein concentration. A total of 3.5 pmol of uniformly labeled RNA containing the polyadenylation signal of the pea rbcS-E9 gene was incubated with varying quantities of MBP-AtCPSF4 and RNA binding assessed by electrophoresis on native gels and autoradiography. The positions of the RNA-protein complex (complexes) and free RNA are indicated on the left. No protein was added for the sample in lane 8. Lanes 1 to 7 contained 10.5, 7.5, 4.5, 1.5, 0.75, 0.375, and 0.188 μ g of purified protein, respectively. B, RNA binding by mutant forms of AtCPSF30. On the left is a depiction of the different variants of AtCPSF30 that were produced as MBP fusion proteins. The central zinc-finger domain is represented with three black lines within the respective rectangular boxes, and the small domain that is responsible for calmodulin binding shown as a gray box within the larger representation. The clear box in the 30M representation indicates that the calmodulin-binding motif has been changed by mutation to be nonfunctional. Fusion proteins that bound RNA are listed under “RNA-binding,” and the plots of binding as a function of protein concentration given on the right. Fusion proteins for which no detectable RNA binding was observed are listed under “unable to bind RNA”; the plots for these proteins are not given, as they would coincide with the x axis.
Figure Legend Snippet: Demarcation of the RNA-binding domain of AtCPSF30. A, Binding of RNA by wild-type AtCPSF30 as a function of protein concentration. A total of 3.5 pmol of uniformly labeled RNA containing the polyadenylation signal of the pea rbcS-E9 gene was incubated with varying quantities of MBP-AtCPSF4 and RNA binding assessed by electrophoresis on native gels and autoradiography. The positions of the RNA-protein complex (complexes) and free RNA are indicated on the left. No protein was added for the sample in lane 8. Lanes 1 to 7 contained 10.5, 7.5, 4.5, 1.5, 0.75, 0.375, and 0.188 μ g of purified protein, respectively. B, RNA binding by mutant forms of AtCPSF30. On the left is a depiction of the different variants of AtCPSF30 that were produced as MBP fusion proteins. The central zinc-finger domain is represented with three black lines within the respective rectangular boxes, and the small domain that is responsible for calmodulin binding shown as a gray box within the larger representation. The clear box in the 30M representation indicates that the calmodulin-binding motif has been changed by mutation to be nonfunctional. Fusion proteins that bound RNA are listed under “RNA-binding,” and the plots of binding as a function of protein concentration given on the right. Fusion proteins for which no detectable RNA binding was observed are listed under “unable to bind RNA”; the plots for these proteins are not given, as they would coincide with the x axis.

Techniques Used: RNA Binding Assay, Binding Assay, Protein Concentration, Labeling, Incubation, Electrophoresis, Autoradiography, Purification, Mutagenesis, Produced

2) Product Images from "Discovery and Characterization of Novel Subtype-Selective Allosteric Agonists for the Investigation of M1 Receptor Function in the Central Nervous System"

Article Title: Discovery and Characterization of Novel Subtype-Selective Allosteric Agonists for the Investigation of M1 Receptor Function in the Central Nervous System

Journal: ACS Chemical Neuroscience

doi: 10.1021/cn900003h

Extracellular loop 3 and the first helical turn of transmembrane seven are involved in mediating the binding of VU0184670 to the M 1 receptor. (A) Site-directed mutagenesis data showing targeted mutagenesis effort of amino acid residues in the third extracellular loop and first turn of the seventh transmembrane span of the M 1 receptor. When a dose−response curve was constructed for novel agonists tested in a functional calcium mobilization assay on transiently transfected K392D/E397V/E401H triple-, EE397/401DD double-, and related E401 single-point mutant CHO-K1 cells, functional potency was nearly completely lost for VU0184670. Not much potency appeared to be lost when increasing concentrations of agonist were applied on cells bearing K392D, E397V, or E397D single mutations, but the E401H single mutation led to substantial losses in potency for both VU0184670 (EC 50 = 1.21 μM; E max = 29.4%) when compared with WT. Additionally, E401A and E401D mutations resulted in similar losses in potency. (B) Automated flexible ligand docking of VU0184670 (green) into a homology model of the M 1 receptor (blue ribbon) based on the X-ray crystal structure of the β2 adrenergic receptor (PDB ID 2RH1). Gradient energy-minimized models illustrated physically plausible low-energy complexes with potential to form up to four hydrogen bonds (dashed yellow lines) with E401 (orange), E397 (yellow), Y381, and Q181. (C) Complexes of VU0184670 allosteric agonist with the M 1 receptor (shown) were among the top 10 scoring docking poses (of 5000 models generated), were consistent with pronounced and graded effects from mutagenesis of E401/E397, and generally agree with ligand SAR profiles by placing the western aryl moiety toward extracellular space and the eastern ethyl carbamate moiety into a buried, sterically occluded region of the receptor extracellular binding domain involving I180, F182, L183, Y381(located beneath VU0184670), and W400 (space-filling representations). Results are for three independent experiments with errors given as ±SEM.
Figure Legend Snippet: Extracellular loop 3 and the first helical turn of transmembrane seven are involved in mediating the binding of VU0184670 to the M 1 receptor. (A) Site-directed mutagenesis data showing targeted mutagenesis effort of amino acid residues in the third extracellular loop and first turn of the seventh transmembrane span of the M 1 receptor. When a dose−response curve was constructed for novel agonists tested in a functional calcium mobilization assay on transiently transfected K392D/E397V/E401H triple-, EE397/401DD double-, and related E401 single-point mutant CHO-K1 cells, functional potency was nearly completely lost for VU0184670. Not much potency appeared to be lost when increasing concentrations of agonist were applied on cells bearing K392D, E397V, or E397D single mutations, but the E401H single mutation led to substantial losses in potency for both VU0184670 (EC 50 = 1.21 μM; E max = 29.4%) when compared with WT. Additionally, E401A and E401D mutations resulted in similar losses in potency. (B) Automated flexible ligand docking of VU0184670 (green) into a homology model of the M 1 receptor (blue ribbon) based on the X-ray crystal structure of the β2 adrenergic receptor (PDB ID 2RH1). Gradient energy-minimized models illustrated physically plausible low-energy complexes with potential to form up to four hydrogen bonds (dashed yellow lines) with E401 (orange), E397 (yellow), Y381, and Q181. (C) Complexes of VU0184670 allosteric agonist with the M 1 receptor (shown) were among the top 10 scoring docking poses (of 5000 models generated), were consistent with pronounced and graded effects from mutagenesis of E401/E397, and generally agree with ligand SAR profiles by placing the western aryl moiety toward extracellular space and the eastern ethyl carbamate moiety into a buried, sterically occluded region of the receptor extracellular binding domain involving I180, F182, L183, Y381(located beneath VU0184670), and W400 (space-filling representations). Results are for three independent experiments with errors given as ±SEM.

Techniques Used: Binding Assay, Mutagenesis, Construct, Functional Assay, Calcium Mobilization Assay, Transfection, Generated, Western Blot

3) Product Images from "The neuronal RhoA GEF, Tech, interacts with the synaptic multi-PDZ-domain-containing protein, MUPP1"

Article Title: The neuronal RhoA GEF, Tech, interacts with the synaptic multi-PDZ-domain-containing protein, MUPP1

Journal:

doi: 10.1111/j.1471-4159.2008.05472.x

Role of MUPP1 PDZ domains 10 and 13 in Tech interaction
Figure Legend Snippet: Role of MUPP1 PDZ domains 10 and 13 in Tech interaction

Techniques Used:

4) Product Images from "A role for planar cell polarity signaling in angiogenesis"

Article Title: A role for planar cell polarity signaling in angiogenesis

Journal: Angiogenesis

doi: 10.1007/s10456-008-9116-2

Dvl2 mutants disrupt endothelial cell polarity. a Diagram of HA-Dvl2 constructs in pCMV5 (HA-tags on the N-terminal of each construct are not depicted). Each of the functional domains have been individually deleted. In addition, one construct was generated
Figure Legend Snippet: Dvl2 mutants disrupt endothelial cell polarity. a Diagram of HA-Dvl2 constructs in pCMV5 (HA-tags on the N-terminal of each construct are not depicted). Each of the functional domains have been individually deleted. In addition, one construct was generated

Techniques Used: Construct, Functional Assay, Generated

A PCP-deficient point-mutation form of Dvl2 (K446M-Dvl2) disrupts endothelial cell proliferation. a Western blot of Dvl2 constructs in MPE whole cell lysates. b Expression of WT or DIX-deleted forms of Dvl2 did not affect MPE cell proliferation. MPE cells
Figure Legend Snippet: A PCP-deficient point-mutation form of Dvl2 (K446M-Dvl2) disrupts endothelial cell proliferation. a Western blot of Dvl2 constructs in MPE whole cell lysates. b Expression of WT or DIX-deleted forms of Dvl2 did not affect MPE cell proliferation. MPE cells

Techniques Used: Mutagenesis, Western Blot, Construct, Expressing

PCP signaling inhibition disrupts endothelial cell migration in a p53-indpendant manner. a Migration of MPE cells transiently co-transfected with various Dvl2/GFP constructs (**, P
Figure Legend Snippet: PCP signaling inhibition disrupts endothelial cell migration in a p53-indpendant manner. a Migration of MPE cells transiently co-transfected with various Dvl2/GFP constructs (**, P

Techniques Used: Inhibition, Migration, Transfection, Construct

5) Product Images from "Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs ▿"

Article Title: Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs ▿

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.01739-08

Construction of molecular proviral clones containing deletions and the EGFP reporter gene. ΔX indicates a deletion of gag -PR, PR, RT, or IN. The deletions were generated in the 5′-hemigenomic molecular clone p83-2 by inverse PCR and self-ligation
Figure Legend Snippet: Construction of molecular proviral clones containing deletions and the EGFP reporter gene. ΔX indicates a deletion of gag -PR, PR, RT, or IN. The deletions were generated in the 5′-hemigenomic molecular clone p83-2 by inverse PCR and self-ligation

Techniques Used: Clone Assay, Generated, Inverse PCR, Ligation

6) Product Images from "The factor VIII C1 domain contributes to platelet binding"

Article Title: The factor VIII C1 domain contributes to platelet binding

Journal: Blood

doi: 10.1182/blood-2007-01-068957

Binding of biotinylated C1C2–2296C (C1C2 b ) and fluorescein-labeled C1C2-2296C (C1C2*) to platelets. (A) Binding of C1C2 b to platelets was detected by PE-streptavidin (FL2 on the y-axis is relative fluorescence using the channel for phycoerythrin,
Figure Legend Snippet: Binding of biotinylated C1C2–2296C (C1C2 b ) and fluorescein-labeled C1C2-2296C (C1C2*) to platelets. (A) Binding of C1C2 b to platelets was detected by PE-streptavidin (FL2 on the y-axis is relative fluorescence using the channel for phycoerythrin,

Techniques Used: Binding Assay, Labeling, Fluorescence

Estimated number of fluorescein-labeled C1C2 (C1C2*)–binding sites per platelet. The top histogram shows fluorescent signals from a mixture of 7.6 μm beads, where 1 is the peak for unlabeled beads and 2 through 5 are signals from the same
Figure Legend Snippet: Estimated number of fluorescein-labeled C1C2 (C1C2*)–binding sites per platelet. The top histogram shows fluorescent signals from a mixture of 7.6 μm beads, where 1 is the peak for unlabeled beads and 2 through 5 are signals from the same

Techniques Used: Labeling, Binding Assay

Binding of FVIII, FVIIIa, C1C2, and C2 proteins to platelets
Figure Legend Snippet: Binding of FVIII, FVIIIa, C1C2, and C2 proteins to platelets

Techniques Used: Binding Assay

Binding of fluorescein-labeled C1C2-2296C (C1C2*) to platelets before (center panel) and after (bottom panel) activation. The results are consistent with a smaller population of lower-affinity C1C2-binding sites on platelets prior to activation, whereas
Figure Legend Snippet: Binding of fluorescein-labeled C1C2-2296C (C1C2*) to platelets before (center panel) and after (bottom panel) activation. The results are consistent with a smaller population of lower-affinity C1C2-binding sites on platelets prior to activation, whereas

Techniques Used: Binding Assay, Labeling, Activation Assay

Competition assays. Fluorescein-labeled C1C2 (C1C2*), C2 (C2*), or FVIIIa (FVIIIa*) were added to activated platelets. The bound, labeled proteins were competed by adding increasing amounts of unlabeled C2, C1C2, FVIII, or FVIIIa; alternatively, platelet
Figure Legend Snippet: Competition assays. Fluorescein-labeled C1C2 (C1C2*), C2 (C2*), or FVIIIa (FVIIIa*) were added to activated platelets. The bound, labeled proteins were competed by adding increasing amounts of unlabeled C2, C1C2, FVIII, or FVIIIa; alternatively, platelet

Techniques Used: Labeling

Expression and purification of the FVIII C1C2 domain. (A) Lanes 1 and 2 show the insoluble and soluble fractions of an E coli cell lysate (15% SDS-PAGE, Coomassie Blue staining; marker proteins on far left). (B) Refolded C1C2 run on a 15% acrylamide gel,
Figure Legend Snippet: Expression and purification of the FVIII C1C2 domain. (A) Lanes 1 and 2 show the insoluble and soluble fractions of an E coli cell lysate (15% SDS-PAGE, Coomassie Blue staining; marker proteins on far left). (B) Refolded C1C2 run on a 15% acrylamide gel,

Techniques Used: Expressing, Purification, SDS Page, Staining, Marker, Acrylamide Gel Assay

7) Product Images from "A Novel Domain within the DEAD-Box Protein DP103 Is Essential for Transcriptional Repression and Helicase Activity"

Article Title: A Novel Domain within the DEAD-Box Protein DP103 Is Essential for Transcriptional Repression and Helicase Activity

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.1.414-423.2003

DP103 exhibits RNA helicase activity in vitro. Helicase assays (5′→3′) were performed under standard conditions as described in Materials and Methods, with 50 fmol of either 32 P-labeled RNA/RNA, RNA/DNA, or DNA/DNA duplex substrates, along with His-tagged DP103 in the presence of ATP (3 mM) at 37°C. (A) The structure of the artificial RNA or DNA duplex substrates. (B) RNA helicase activity of DP103 is concentration dependent. A concentration range of full-length His-tagged DP103 (0, 3, 10, 30, and 50 ng) was examined. (C) A time course for the RNA helicase activity of full-length DP103 at the time points indicated. (D) Helicase assay with 50 ng of proteins as indicated, along with RNA/RNA (upper panel), RNA/DNA (middle panel), or DNA/DNA (lower panel) substrates. ATP (3 mM) was present unless indicated as being absent or replaced with ATP-γ-S (3 mM). All the helicase assays were performed for 15 min. Boiled double-stranded (ds) substrates or substrate incubated without protein at 37°C was used as control, indicating the position of released single-stranded (ss) or ds probe, respectively. 32 P-labeled short ss probe was also included in the experiments depicted in panels B and C to highlight the position of released ssRNA or ssDNA.
Figure Legend Snippet: DP103 exhibits RNA helicase activity in vitro. Helicase assays (5′→3′) were performed under standard conditions as described in Materials and Methods, with 50 fmol of either 32 P-labeled RNA/RNA, RNA/DNA, or DNA/DNA duplex substrates, along with His-tagged DP103 in the presence of ATP (3 mM) at 37°C. (A) The structure of the artificial RNA or DNA duplex substrates. (B) RNA helicase activity of DP103 is concentration dependent. A concentration range of full-length His-tagged DP103 (0, 3, 10, 30, and 50 ng) was examined. (C) A time course for the RNA helicase activity of full-length DP103 at the time points indicated. (D) Helicase assay with 50 ng of proteins as indicated, along with RNA/RNA (upper panel), RNA/DNA (middle panel), or DNA/DNA (lower panel) substrates. ATP (3 mM) was present unless indicated as being absent or replaced with ATP-γ-S (3 mM). All the helicase assays were performed for 15 min. Boiled double-stranded (ds) substrates or substrate incubated without protein at 37°C was used as control, indicating the position of released single-stranded (ss) or ds probe, respectively. 32 P-labeled short ss probe was also included in the experiments depicted in panels B and C to highlight the position of released ssRNA or ssDNA.

Techniques Used: Activity Assay, In Vitro, Labeling, Concentration Assay, Helicase Assay, Incubation

DP103 721-825 diminishes the expression of SF-1 target genes in the adrenocortical line. Y1 cells were transfected with increasing amounts (0, 1.0, and 3.0 μg) of GAL4-DP103 1-825 , GAL4-DP103 721-825 , or GAL4-DP103 1-727 . Steroidogenesis was stimulated with 0.1 μM of ACTH or water vehicles 18 h after transfection. Total RNA was isolated and processed for quantitative RT-PCR as described in Materials and Methods. (A) Expression of P450scc mRNA. (B) Expression of P450c21 mRNA. (C) Expression of StAR mRNA. (D) GAL4-DP103 fusion proteins do not diminish the expression of SF-1. Results (means ± SD) are expressed as fold expression over control (no ACTH, no GAL4-DP103) and represent three independent experiments, each performed in duplicate. (E) Progesterone levels in media from cultured Y1 cells transfected with GAL4-DP103 plasmids (3 μg) and collected over 24 h prior to hormone determination. Results (means ± SD) are expressed as fold over control (GAL4) and represent three to five independent experiments, each performed in duplicate. An asterisk denotes a P value of
Figure Legend Snippet: DP103 721-825 diminishes the expression of SF-1 target genes in the adrenocortical line. Y1 cells were transfected with increasing amounts (0, 1.0, and 3.0 μg) of GAL4-DP103 1-825 , GAL4-DP103 721-825 , or GAL4-DP103 1-727 . Steroidogenesis was stimulated with 0.1 μM of ACTH or water vehicles 18 h after transfection. Total RNA was isolated and processed for quantitative RT-PCR as described in Materials and Methods. (A) Expression of P450scc mRNA. (B) Expression of P450c21 mRNA. (C) Expression of StAR mRNA. (D) GAL4-DP103 fusion proteins do not diminish the expression of SF-1. Results (means ± SD) are expressed as fold expression over control (no ACTH, no GAL4-DP103) and represent three independent experiments, each performed in duplicate. (E) Progesterone levels in media from cultured Y1 cells transfected with GAL4-DP103 plasmids (3 μg) and collected over 24 h prior to hormone determination. Results (means ± SD) are expressed as fold over control (GAL4) and represent three to five independent experiments, each performed in duplicate. An asterisk denotes a P value of

Techniques Used: Expressing, Transfection, Isolation, Quantitative RT-PCR, Cell Culture

DP103 721-825 physically interacts with SF-1's PRD (aa 193 to 201) in vitro. Wild-type SF-1 (W) and PRD mutant SF-1 (M) cloned in pBSK were expressed and labeled with [ 35 S]methionine in vitro by using a TNT transcription-translation system. Labeled SF-1 was incubated with His-tagged DP103 fragments bound to Ni 2+ -NTA agarose resin. Bound SF-1 was detected by using SDS-10% PAGE and autoradiography. Input included 20% SF-1 that was used for each assay. The empty Ni 2+ -NTA agarose resin, reacted with wild-type or mutant SF-1, served as negative control.
Figure Legend Snippet: DP103 721-825 physically interacts with SF-1's PRD (aa 193 to 201) in vitro. Wild-type SF-1 (W) and PRD mutant SF-1 (M) cloned in pBSK were expressed and labeled with [ 35 S]methionine in vitro by using a TNT transcription-translation system. Labeled SF-1 was incubated with His-tagged DP103 fragments bound to Ni 2+ -NTA agarose resin. Bound SF-1 was detected by using SDS-10% PAGE and autoradiography. Input included 20% SF-1 that was used for each assay. The empty Ni 2+ -NTA agarose resin, reacted with wild-type or mutant SF-1, served as negative control.

Techniques Used: In Vitro, Mutagenesis, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography, Negative Control

DP103 interacts with the PRD in SF-1 (aa 193 to 201) through aa 721 to 825. (A) Schematic diagram depicting the main transcriptional regulatory domains of SF-1. A mutated PRD, known to abrogate SF-1 repression, is shown. (B) A diagram of DP103, denoting eight highly conserved motifs within the N-terminal region of DEAD-box family proteins as well as the nonconserved C-terminal region. (C) DP103 721-825 interacts with wild type SF-1 but not with PRD mutant SF-1. Interaction was detected by using a mammalian two-hybrid assay, with SF-1 120-462 fused downstream from GAL4 and DP103 fragments (as shown) fused to the VP16 activation domain. Plasmids were transiently transfected into CV-1 cells along with the GAL4 reporter plasmid ΔGKI. Results represent three independent experiments performed in duplicate, expressed as fold activation over control in which the empty VP16 plasmid was used and normalized to β-galactosidase activity.
Figure Legend Snippet: DP103 interacts with the PRD in SF-1 (aa 193 to 201) through aa 721 to 825. (A) Schematic diagram depicting the main transcriptional regulatory domains of SF-1. A mutated PRD, known to abrogate SF-1 repression, is shown. (B) A diagram of DP103, denoting eight highly conserved motifs within the N-terminal region of DEAD-box family proteins as well as the nonconserved C-terminal region. (C) DP103 721-825 interacts with wild type SF-1 but not with PRD mutant SF-1. Interaction was detected by using a mammalian two-hybrid assay, with SF-1 120-462 fused downstream from GAL4 and DP103 fragments (as shown) fused to the VP16 activation domain. Plasmids were transiently transfected into CV-1 cells along with the GAL4 reporter plasmid ΔGKI. Results represent three independent experiments performed in duplicate, expressed as fold activation over control in which the empty VP16 plasmid was used and normalized to β-galactosidase activity.

Techniques Used: Mutagenesis, Two Hybrid Assay, Activation Assay, Transfection, Plasmid Preparation, Activity Assay

DP103 harbors a repression domain at aa 721 to 825. (A) CV-1 cells were cotransfected with increasing amounts (0, 0.03, 0.1, 0.3, 1.0, and 3.0 μg) of either full-length DP103 or DP103 fragments, fused to GAL4, along with the reporter plasmid GAL4 × 5-tkLuc. Results (means ± SD) are expressed as cRLU normalized to β-galactosidase activity and represent three independent experiments, each performed in duplicate. (B) Western immunoblotting of the GAL4-DP103 fusion proteins analyzed in panel A, demonstrating equal cellular expression of transfected plasmids. Lysates (30 μg) were prepared from CV-1 cells that were transfected with 3 μg of each DP103 plasmid, separated by SDS-PAGE, and immunodetected by using anti-GAL4 antibody as described in Materials and Methods. (C) The DEAD-box protein p68 (3 μg, expressed as chimeric protein GAL4-p68) does not repress the reporter plasmid GAL4 × 5-tkLuc in CV-1 cells. Results are means of three independent experiments, each performed in duplicate, and are expressed as fold over control in which the empty GAL4 plasmid was used.
Figure Legend Snippet: DP103 harbors a repression domain at aa 721 to 825. (A) CV-1 cells were cotransfected with increasing amounts (0, 0.03, 0.1, 0.3, 1.0, and 3.0 μg) of either full-length DP103 or DP103 fragments, fused to GAL4, along with the reporter plasmid GAL4 × 5-tkLuc. Results (means ± SD) are expressed as cRLU normalized to β-galactosidase activity and represent three independent experiments, each performed in duplicate. (B) Western immunoblotting of the GAL4-DP103 fusion proteins analyzed in panel A, demonstrating equal cellular expression of transfected plasmids. Lysates (30 μg) were prepared from CV-1 cells that were transfected with 3 μg of each DP103 plasmid, separated by SDS-PAGE, and immunodetected by using anti-GAL4 antibody as described in Materials and Methods. (C) The DEAD-box protein p68 (3 μg, expressed as chimeric protein GAL4-p68) does not repress the reporter plasmid GAL4 × 5-tkLuc in CV-1 cells. Results are means of three independent experiments, each performed in duplicate, and are expressed as fold over control in which the empty GAL4 plasmid was used.

Techniques Used: Plasmid Preparation, Activity Assay, Western Blot, Expressing, Transfection, SDS Page

DP103 721-825 represses the transcription of SF-1 target promoters. (A) The concentration-dependent influence of GAL4-fused full-length or truncated DP103 on the transcriptional activity of SF-1. JEG3 cells were cotransfected with 0.05 μg of CMV-SF-1 and either GAL4-DP103 1-825 , GAL4-DP103 721-825 , or GAL4-DP103 1-727 (0, 0.1, 0.3, 1.0, and 3.0 μg), along with 0.5 μg of the SF-1 luciferase reporter plasmid S25. (B) The repression effect of DP103 is observed only in the presence of SF-1. Transfection was performed as described above, with 3 μg of GAL4-DP103 721-825 or GAL4-DP103 1-727 . The empty expression vector CMV-neo or GAL4 was used as control for SF-1 and DP103, respectively. (C) Concentration-dependent influence of GAL4-fused full-length or truncated DP103 on the transcriptional activity of an SF-1-responsive rat P450scc reporter. Transfection was performed as described above, with 0.5 μg of the SF-1 luciferase reporter plasmid P450scc. (D) SF-1 is required for the repression effect of DP103. The repression was abrogated when the two SF-1 binding elements in the P450scc promoter were mutated. Transfection was performed as described above. Results (means ± SD) are expressed as cRLU normalized to β-galactosidase activity and represent three independent experiments, each performed in duplicate.
Figure Legend Snippet: DP103 721-825 represses the transcription of SF-1 target promoters. (A) The concentration-dependent influence of GAL4-fused full-length or truncated DP103 on the transcriptional activity of SF-1. JEG3 cells were cotransfected with 0.05 μg of CMV-SF-1 and either GAL4-DP103 1-825 , GAL4-DP103 721-825 , or GAL4-DP103 1-727 (0, 0.1, 0.3, 1.0, and 3.0 μg), along with 0.5 μg of the SF-1 luciferase reporter plasmid S25. (B) The repression effect of DP103 is observed only in the presence of SF-1. Transfection was performed as described above, with 3 μg of GAL4-DP103 721-825 or GAL4-DP103 1-727 . The empty expression vector CMV-neo or GAL4 was used as control for SF-1 and DP103, respectively. (C) Concentration-dependent influence of GAL4-fused full-length or truncated DP103 on the transcriptional activity of an SF-1-responsive rat P450scc reporter. Transfection was performed as described above, with 0.5 μg of the SF-1 luciferase reporter plasmid P450scc. (D) SF-1 is required for the repression effect of DP103. The repression was abrogated when the two SF-1 binding elements in the P450scc promoter were mutated. Transfection was performed as described above. Results (means ± SD) are expressed as cRLU normalized to β-galactosidase activity and represent three independent experiments, each performed in duplicate.

Techniques Used: Concentration Assay, Activity Assay, Luciferase, Plasmid Preparation, Transfection, Expressing, Binding Assay

8) Product Images from "Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia"

Article Title: Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2013101040

Intracellular accumulation of Na,K-ATPase in response to energy depletion does not occur in cells expressing the S588D mutant of AS160. (A) Immunofluorescence analysis of the distribution of endogenous Na,K-ATPase. MDCK cells untreated (−) (I and III) or treated with energy depletion (AA/DG) (II and IV) are stained with an antibody directed against the Na,K-ATPase α -subunit ( α 5) and with anti-FLAG to detect exogenous AS160. Na,K-ATPase accumulates in intracellular compartments in response to AA/DG in cells stably transfected with AS160 WT-FLAG (II). However, Na,K-ATPase is not localized in intracellular structures in the AS160 S588D cell line treated with AA/DG (IV). (B) Cell surface biotinylation. MDCK WT or AS160 S588D cells are biotinylated at the basolateral surface, then exposed or not to energy depletion (AA/DG). The biotin that remained at the cell surface after the energy depletion is stripped by treating the cells with MesNa. The biotinylated sodium pump is detected with the antibody α 5 directed against the Na,K-ATPase α -subunit. Lysates are blotted with anti-AS160, anti-FLAG, and with anti– β -actin antibodies to assess total protein loading. (C) Quantification of the biotinylated Na,K-ATPase band intensity normalized to the β -actin levels. The amount of sodium pump that was initially biotinylated at the cell surface and that accumulated within the cell after energy depletion is lower in the AS160 S588D cells compared with the MDCK WT cell line. * P
Figure Legend Snippet: Intracellular accumulation of Na,K-ATPase in response to energy depletion does not occur in cells expressing the S588D mutant of AS160. (A) Immunofluorescence analysis of the distribution of endogenous Na,K-ATPase. MDCK cells untreated (−) (I and III) or treated with energy depletion (AA/DG) (II and IV) are stained with an antibody directed against the Na,K-ATPase α -subunit ( α 5) and with anti-FLAG to detect exogenous AS160. Na,K-ATPase accumulates in intracellular compartments in response to AA/DG in cells stably transfected with AS160 WT-FLAG (II). However, Na,K-ATPase is not localized in intracellular structures in the AS160 S588D cell line treated with AA/DG (IV). (B) Cell surface biotinylation. MDCK WT or AS160 S588D cells are biotinylated at the basolateral surface, then exposed or not to energy depletion (AA/DG). The biotin that remained at the cell surface after the energy depletion is stripped by treating the cells with MesNa. The biotinylated sodium pump is detected with the antibody α 5 directed against the Na,K-ATPase α -subunit. Lysates are blotted with anti-AS160, anti-FLAG, and with anti– β -actin antibodies to assess total protein loading. (C) Quantification of the biotinylated Na,K-ATPase band intensity normalized to the β -actin levels. The amount of sodium pump that was initially biotinylated at the cell surface and that accumulated within the cell after energy depletion is lower in the AS160 S588D cells compared with the MDCK WT cell line. * P

Techniques Used: Expressing, Mutagenesis, Immunofluorescence, Staining, Stable Transfection, Transfection

9) Product Images from "Expression of functional alternative telomerase RNA component gene in mouse brain and in motor neurons cells protects from oxidative stress"

Article Title: Expression of functional alternative telomerase RNA component gene in mouse brain and in motor neurons cells protects from oxidative stress

Journal: Oncotarget

doi: 10.18632/oncotarget.13049

Stable overexpression of mTERC and alTERC increased telomerase activity in NSC-34 cells A. mTERC and alTERC were cloned into a retroviral vector and stable transduction of NSC-34 cells was performed. The expression of mTERC and alTERC in the transduced cells and in the control untransduced (UTr) or transduced with the empty vector (NV) cells were detected by PCR using the appropriate mTERC and alTERC primers. NTC- control without cDNA. B. TERT protein was detected by Western blot analysis with anti-TERT antibody and C. quantification of TERT protein relatively to the control β-actin protein was performed by densitometric analysis using the EZQuant software. The data are means ± SD of 3 independent experiments. D. Telomerase activity was measured by TRAP assay and, E. quantified by densitometric analysis using the EZQuant software. The results are % of the control NV and are means±SD, t Test, p
Figure Legend Snippet: Stable overexpression of mTERC and alTERC increased telomerase activity in NSC-34 cells A. mTERC and alTERC were cloned into a retroviral vector and stable transduction of NSC-34 cells was performed. The expression of mTERC and alTERC in the transduced cells and in the control untransduced (UTr) or transduced with the empty vector (NV) cells were detected by PCR using the appropriate mTERC and alTERC primers. NTC- control without cDNA. B. TERT protein was detected by Western blot analysis with anti-TERT antibody and C. quantification of TERT protein relatively to the control β-actin protein was performed by densitometric analysis using the EZQuant software. The data are means ± SD of 3 independent experiments. D. Telomerase activity was measured by TRAP assay and, E. quantified by densitometric analysis using the EZQuant software. The results are % of the control NV and are means±SD, t Test, p

Techniques Used: Over Expression, Activity Assay, Clone Assay, Plasmid Preparation, Transduction, Expressing, Polymerase Chain Reaction, Western Blot, Software, TRAP Assay

10) Product Images from "Complete Nucleotide Sequence of Polyomavirus SA12"

Article Title: Complete Nucleotide Sequence of Polyomavirus SA12

Journal:

doi: 10.1128/JVI.79.20.13094-13104.2005

Early mRNA splice junctions. (A) Sequence homology of early RNA splice sites. Regions surrounding the early RNA splice sites (5′ large T donor, 5′ small t donor, and 3′ acceptor) from SA12, SV40, JCV, and BKV were aligned using
Figure Legend Snippet: Early mRNA splice junctions. (A) Sequence homology of early RNA splice sites. Regions surrounding the early RNA splice sites (5′ large T donor, 5′ small t donor, and 3′ acceptor) from SA12, SV40, JCV, and BKV were aligned using

Techniques Used: Sequencing

Mutation of the SA12 core ori to a consensus sequence does not enhance viral DNA replication or yield. (A) The sequence of the SA12 core ori is shown. Putative T antigen binding sites are underlined. The position of a T10A mutant that changes the left-most
Figure Legend Snippet: Mutation of the SA12 core ori to a consensus sequence does not enhance viral DNA replication or yield. (A) The sequence of the SA12 core ori is shown. Putative T antigen binding sites are underlined. The position of a T10A mutant that changes the left-most

Techniques Used: Mutagenesis, Sequencing, Binding Assay

SA12 regulatory region. The 401-bp regulatory region of SA12 extends from nucleotides 5124 to 294. Important regulatory elements are highlighted in color. Light green indicates the consensus pentanucleotide T antigen binding sequence, dark green indicates
Figure Legend Snippet: SA12 regulatory region. The 401-bp regulatory region of SA12 extends from nucleotides 5124 to 294. Important regulatory elements are highlighted in color. Light green indicates the consensus pentanucleotide T antigen binding sequence, dark green indicates

Techniques Used: Binding Assay, Sequencing

Quantitative PCR assay that distinguishes SA12 from other primate polyomaviruses.
Figure Legend Snippet: Quantitative PCR assay that distinguishes SA12 from other primate polyomaviruses.

Techniques Used: Real-time Polymerase Chain Reaction

SA12 genome. The SA12 genome is represented by the circle, with the numbers indicating base pair positions. The base pairs are numbered in a clockwise direction with base pair 1 being the GC bp of the third GAGGC repeat within the origin of DNA replication.
Figure Legend Snippet: SA12 genome. The SA12 genome is represented by the circle, with the numbers indicating base pair positions. The base pairs are numbered in a clockwise direction with base pair 1 being the GC bp of the third GAGGC repeat within the origin of DNA replication.

Techniques Used:

SA12 protein coding regions. The cDNA sequence of (A) large T antigen, (B) small t antigen, (C) VP1, (D) VP2, or (E) VP3 is displayed as the bottom sequence, and the translated protein sequence is displayed on top. The lengths of the proteins in numbers
Figure Legend Snippet: SA12 protein coding regions. The cDNA sequence of (A) large T antigen, (B) small t antigen, (C) VP1, (D) VP2, or (E) VP3 is displayed as the bottom sequence, and the translated protein sequence is displayed on top. The lengths of the proteins in numbers

Techniques Used: Sequencing

Sequence alignments of the variable regions and host range domains. The host range domains from SA12, BKV, and JCV were aligned (A) with and (B) without the host range domain of SV40. The variable regions from BKV, SA12, and JCV were aligned (C) with
Figure Legend Snippet: Sequence alignments of the variable regions and host range domains. The host range domains from SA12, BKV, and JCV were aligned (A) with and (B) without the host range domain of SV40. The variable regions from BKV, SA12, and JCV were aligned (C) with

Techniques Used: Sequencing

Expression of SA12 miRNAs. (A) A secondary-structure prediction of the SA12 hairpin, starting at nucleotide 2785, which is conserved among several polyomaviruses containing the pre-miRNA, is shown. (B) Probe diagram showing the sequence of the hairpin
Figure Legend Snippet: Expression of SA12 miRNAs. (A) A secondary-structure prediction of the SA12 hairpin, starting at nucleotide 2785, which is conserved among several polyomaviruses containing the pre-miRNA, is shown. (B) Probe diagram showing the sequence of the hairpin

Techniques Used: Expressing, Sequencing

11) Product Images from "NaV1.7 Gain-of-Function Mutations as a Continuum: A1632E Displays Physiological Changes Associated with Erythromelalgia and Paroxysmal Extreme Pain Disorder Mutations and Produces Symptoms of Both Disorders"

Article Title: NaV1.7 Gain-of-Function Mutations as a Continuum: A1632E Displays Physiological Changes Associated with Erythromelalgia and Paroxysmal Extreme Pain Disorder Mutations and Produces Symptoms of Both Disorders

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.3443-08.2008

Sequence alignment of DIV/S4–S5 linker from human sodium channels. Schematic of the topology of the sodium channel polypeptide showing the location of the A1632E near the C-terminal end of the linker joining segments S4 and S5 in domain IV. The A1632E substitution is noted in the sequence from Na V 1.7. The equivalent residue is conserved in all sodium channels except for Na V 1.9, in which the analogous residue is a serine (S1496).
Figure Legend Snippet: Sequence alignment of DIV/S4–S5 linker from human sodium channels. Schematic of the topology of the sodium channel polypeptide showing the location of the A1632E near the C-terminal end of the linker joining segments S4 and S5 in domain IV. The A1632E substitution is noted in the sequence from Na V 1.7. The equivalent residue is conserved in all sodium channels except for Na V 1.9, in which the analogous residue is a serine (S1496).

Techniques Used: Sequencing

Fast inactivation is slowed and incomplete in A1632E-expressing cells. A , The decaying phase of the inward sodium current elicited by the activation protocol was fit to a single-exponential for the voltage range of −35 to +35 mV. The average time constant is plotted as a function of voltage for WT cells (open circles; n = 8) and for A1632E cells (filled circles; n = 18) Error bars are ±SEM. B , Example traces elicited by pulses to −20 mV are shown superimposed and normalized to peak inward current to illustrate the difference in inactivation rate between WT-expressing (gray line) and A1632E-expressing (black line, arrow) cells. The end of the 100 ms trace (which was cutoff in the main panel to illustrate kinetics) is shown as an inset with an expanded vertical axis to show that A1632E current (black line, arrow) persists for at least 100 ms. C , Example traces elicited by pulses to +15 mV are superimposed and normalized to peak inward current to illustrate the greater difference in inactivation rate at this depolarized potential. The inset shows that, even at this depolarized potential, persistent current can still be resolved from A1632E cells. D , Example traces elicited by pulses to −50 mV (nearer to RMP range and near threshold for voltage activation) are superimposed and normalized to peak inward current to illustrate that persistent current is resolvable in this voltage range (inset, black line, arrow). Note the expanded scale in D . For display purposes, the WT traces were digitally filtered to 2 kHz in the main panels and filtered to 1 kHz in the insets, whereas the A1632E traces were digitally filtered to 1 kHz in the main panels of B and C and filtered to 500 Hz in D and all insets.
Figure Legend Snippet: Fast inactivation is slowed and incomplete in A1632E-expressing cells. A , The decaying phase of the inward sodium current elicited by the activation protocol was fit to a single-exponential for the voltage range of −35 to +35 mV. The average time constant is plotted as a function of voltage for WT cells (open circles; n = 8) and for A1632E cells (filled circles; n = 18) Error bars are ±SEM. B , Example traces elicited by pulses to −20 mV are shown superimposed and normalized to peak inward current to illustrate the difference in inactivation rate between WT-expressing (gray line) and A1632E-expressing (black line, arrow) cells. The end of the 100 ms trace (which was cutoff in the main panel to illustrate kinetics) is shown as an inset with an expanded vertical axis to show that A1632E current (black line, arrow) persists for at least 100 ms. C , Example traces elicited by pulses to +15 mV are superimposed and normalized to peak inward current to illustrate the greater difference in inactivation rate at this depolarized potential. The inset shows that, even at this depolarized potential, persistent current can still be resolved from A1632E cells. D , Example traces elicited by pulses to −50 mV (nearer to RMP range and near threshold for voltage activation) are superimposed and normalized to peak inward current to illustrate that persistent current is resolvable in this voltage range (inset, black line, arrow). Note the expanded scale in D . For display purposes, the WT traces were digitally filtered to 2 kHz in the main panels and filtered to 1 kHz in the insets, whereas the A1632E traces were digitally filtered to 1 kHz in the main panels of B and C and filtered to 500 Hz in D and all insets.

Techniques Used: Expressing, Activation Assay, Mass Spectrometry

Current-clamp recordings in transfected DRG neurons. The A1632E mutation decreases action potential threshold in small, current-clamped DRG neurons. A , Responses of a current-clamped DRG neuron transfected with hNa V 1.7r–WT DNA to a series of subthreshold and suprathreshold depolarizing current stimuli stepped in 5 pA increments. Two traces are displayed that show the threshold value for eliciting an action potential. RMP for this cell was −56 mV and threshold was 165 pA. B , Two traces are displayed that show the threshold value for a DRG neuron transfected with hNa V 1.7r–A1632E DNA. RMP for this cell was −57 mV and threshold was 55 pA. Example action potential responses from DRG neurons transfected with hNa V 1.7r–WT and hNa V 1.7r–A1632E DNA. C–E illustrate the responses elicited at currents approximately two and three times threshold as well as the stimuli that elicited the maximal response for DRG neurons transfected with hNa V 1.7r–WT DNA. F–H show the responses for DGR neurons transfected with hNa V 1.7r–A1632E DNA. These cells are the same as shown in A and B to determine action potential threshold.
Figure Legend Snippet: Current-clamp recordings in transfected DRG neurons. The A1632E mutation decreases action potential threshold in small, current-clamped DRG neurons. A , Responses of a current-clamped DRG neuron transfected with hNa V 1.7r–WT DNA to a series of subthreshold and suprathreshold depolarizing current stimuli stepped in 5 pA increments. Two traces are displayed that show the threshold value for eliciting an action potential. RMP for this cell was −56 mV and threshold was 165 pA. B , Two traces are displayed that show the threshold value for a DRG neuron transfected with hNa V 1.7r–A1632E DNA. RMP for this cell was −57 mV and threshold was 55 pA. Example action potential responses from DRG neurons transfected with hNa V 1.7r–WT and hNa V 1.7r–A1632E DNA. C–E illustrate the responses elicited at currents approximately two and three times threshold as well as the stimuli that elicited the maximal response for DRG neurons transfected with hNa V 1.7r–WT DNA. F–H show the responses for DGR neurons transfected with hNa V 1.7r–A1632E DNA. These cells are the same as shown in A and B to determine action potential threshold.

Techniques Used: Transfection, Mutagenesis

A1632E mutation effects slow-inactivation, repriming, and slow ramp currents. A , The slow-inactivation protocol consists of a 30 s conditioning pulse, followed by a 100 ms pulse to −120 mV to restore the fast-inactivation state and then pulsed to 0 mV for 50 ms to activate the fraction of available channels. The A1632E data (filled circles; n = 5) show greater availability at all potentials compared with wild-type (open circles; n = 5) with the difference being greatest in the voltage range of −90 to −70 mV. In addition, there is a more prominent fraction of channels that do not become slow inactivated at the potentials more positive to −20 mV. The smooth lines are Boltzmann fits with the following parameters: WT: I max = 0.99, I min = 0.058, V 1/2 = −66.7 mV, and k = 17.4; A1632E: I max = 1.0, I min = 0.2, V 1/2 = −63.2 mV, and k = 9.5. B , The A1632E mutation alters recovery from fast inactivation. From a holding potential of −100 mV, fast inactivation was initiated by a step to 0 mV for 20 ms, followed by a hyperpolarizing step to a recovery potential that varied in time (2–400 ms) and amplitude (−100 to −60 mV). The recovery period was followed by a second depolarizing test pulse to 0 mV. For each recovery potential, the peak inward current in response to the test pulse was normalized by the amplitude of the response to the inactivation step and plotted as a function of recovery period duration. The averages for cells expressing WT channels (open symbols; n = 7) and A1632E channels (filled symbols; n = 8) are shown with error bars ±SEM. Single-exponential functions were fit to these averaged data and are shown by solid lines for A1632E and dashed lines for WT. C , This panel illustrates the responses to a ramp pulse protocol that spans the range of −100 to 20 mV over 600 ms (0.2 mV/ms). The response has been rescaled as the percentage of peak inward current recorded during the activation I–V protocol. The peak currents from wild-type hNa V 1.7 are much larger than from the A1632E-expressing cells, thus scaling the noise to be quieter. In addition, for display purposes, the WT trace has been post-acquisition filtered to 1 kHz, and the A1632E trace has been post-acquisition filtered to 500 Hz. The average peak ramp current from the A1632E cells was 4.2 ± 0.3% at −44.7 ± 0.7 mV ( n = 12), whereas the peak ramp current from the WT cells averaged 0.77 ± 0.05% at −39.8 ± 0.7 mV ( n = 9). Error bars are 1 SD.
Figure Legend Snippet: A1632E mutation effects slow-inactivation, repriming, and slow ramp currents. A , The slow-inactivation protocol consists of a 30 s conditioning pulse, followed by a 100 ms pulse to −120 mV to restore the fast-inactivation state and then pulsed to 0 mV for 50 ms to activate the fraction of available channels. The A1632E data (filled circles; n = 5) show greater availability at all potentials compared with wild-type (open circles; n = 5) with the difference being greatest in the voltage range of −90 to −70 mV. In addition, there is a more prominent fraction of channels that do not become slow inactivated at the potentials more positive to −20 mV. The smooth lines are Boltzmann fits with the following parameters: WT: I max = 0.99, I min = 0.058, V 1/2 = −66.7 mV, and k = 17.4; A1632E: I max = 1.0, I min = 0.2, V 1/2 = −63.2 mV, and k = 9.5. B , The A1632E mutation alters recovery from fast inactivation. From a holding potential of −100 mV, fast inactivation was initiated by a step to 0 mV for 20 ms, followed by a hyperpolarizing step to a recovery potential that varied in time (2–400 ms) and amplitude (−100 to −60 mV). The recovery period was followed by a second depolarizing test pulse to 0 mV. For each recovery potential, the peak inward current in response to the test pulse was normalized by the amplitude of the response to the inactivation step and plotted as a function of recovery period duration. The averages for cells expressing WT channels (open symbols; n = 7) and A1632E channels (filled symbols; n = 8) are shown with error bars ±SEM. Single-exponential functions were fit to these averaged data and are shown by solid lines for A1632E and dashed lines for WT. C , This panel illustrates the responses to a ramp pulse protocol that spans the range of −100 to 20 mV over 600 ms (0.2 mV/ms). The response has been rescaled as the percentage of peak inward current recorded during the activation I–V protocol. The peak currents from wild-type hNa V 1.7 are much larger than from the A1632E-expressing cells, thus scaling the noise to be quieter. In addition, for display purposes, the WT trace has been post-acquisition filtered to 1 kHz, and the A1632E trace has been post-acquisition filtered to 500 Hz. The average peak ramp current from the A1632E cells was 4.2 ± 0.3% at −44.7 ± 0.7 mV ( n = 12), whereas the peak ramp current from the WT cells averaged 0.77 ± 0.05% at −39.8 ± 0.7 mV ( n = 9). Error bars are 1 SD.

Techniques Used: Mutagenesis, Mass Spectrometry, Expressing, Activation Assay

A1632E shifts activation and fast inactivation. A , B , Typical data traces recorded from HEK cell lines expressing either the WT ( A ) or the hNa V 1.7r–A1632E mutant ( B ) sodium channel. The current densities from the A1632E-expressing cell line were lower, with total peak inward currents averaging just under 1 nA. In comparison, the hNa V 1.7r-WT clone total peak current averaged near 3 nA. For display purposes, the traces were digitally filtered to 2 kHz. C , The average time-to-peak for the traces recorded during the activation protocol are plotted as a function of test potential with the WT ( n = 9) responses shown by open circles and the A1632E (AE) ( n = 18) responses are shown by filled circles. Error bars are ±SEM. D , Deactivation kinetics are derived from single-exponential fits to tail currents recorded in response to brief activating pulses (0 mV for 0.5 ms), followed by a repolarization to the indicated potential. Example data traces comparing the deactivation during repolarization to −50 mV are shown in the inset. The time constants are averaged and plotted with the WT ( n = 9) shown by open circles and the A1632E ( n = 7) shown by filled circles. Error bars are ±SEM. E , The peak inward currents elicited using either the activation or the fast-inactivation protocol were transformed into normalized conductance as described in Materials and Methods, and the average response at each test voltage was plotted using open symbols for WT ( n = 9) and filled symbols for A1632E ( n = 18). The activation data are plotted using circles and the fast-inactivation data are plotted using squares. Error bars are ±SEM. The smooth lines are Boltzmann fits to the average values. The small offset of the activation curve is a result of the small total currents recorded from A1632E-expressing cells so that the peak inward current obtained from the baseline fluctuations represents a few percentage of I max . The baseline offset from the fit of the A1632E inactivation data, however, likely corresponds to an incomplete inactivation resulting in persistent current. F , The predicted window current is larger for A1632E (solid lines) compared with WT (dashed lines).
Figure Legend Snippet: A1632E shifts activation and fast inactivation. A , B , Typical data traces recorded from HEK cell lines expressing either the WT ( A ) or the hNa V 1.7r–A1632E mutant ( B ) sodium channel. The current densities from the A1632E-expressing cell line were lower, with total peak inward currents averaging just under 1 nA. In comparison, the hNa V 1.7r-WT clone total peak current averaged near 3 nA. For display purposes, the traces were digitally filtered to 2 kHz. C , The average time-to-peak for the traces recorded during the activation protocol are plotted as a function of test potential with the WT ( n = 9) responses shown by open circles and the A1632E (AE) ( n = 18) responses are shown by filled circles. Error bars are ±SEM. D , Deactivation kinetics are derived from single-exponential fits to tail currents recorded in response to brief activating pulses (0 mV for 0.5 ms), followed by a repolarization to the indicated potential. Example data traces comparing the deactivation during repolarization to −50 mV are shown in the inset. The time constants are averaged and plotted with the WT ( n = 9) shown by open circles and the A1632E ( n = 7) shown by filled circles. Error bars are ±SEM. E , The peak inward currents elicited using either the activation or the fast-inactivation protocol were transformed into normalized conductance as described in Materials and Methods, and the average response at each test voltage was plotted using open symbols for WT ( n = 9) and filled symbols for A1632E ( n = 18). The activation data are plotted using circles and the fast-inactivation data are plotted using squares. Error bars are ±SEM. The smooth lines are Boltzmann fits to the average values. The small offset of the activation curve is a result of the small total currents recorded from A1632E-expressing cells so that the peak inward current obtained from the baseline fluctuations represents a few percentage of I max . The baseline offset from the fit of the A1632E inactivation data, however, likely corresponds to an incomplete inactivation resulting in persistent current. F , The predicted window current is larger for A1632E (solid lines) compared with WT (dashed lines).

Techniques Used: Activation Assay, Expressing, Mutagenesis, Derivative Assay, Mass Spectrometry, Transformation Assay

Current-clamp recordings from transfected trigeminal ganglion neurons. The A1632E mutation decreases action potential threshold in small, current-clamped trigeminal ganglion neurons. A , Responses of a current-clamped trigeminal neuron transfected with hNa V 1.7r–WT DNA to a series of subthreshold and suprathreshold depolarizing current stimuli stepped in 5 pA increments. Two traces are displayed that show the threshold value for eliciting an action potential. RMP for this cell was −54 mV and threshold was 155 pA. B , Two traces are displayed that show the threshold for a trigeminal neuron transfected with hNa V 1.7r–A1632E DNA. RMP for this cell was −54 mV and threshold was 100 pA. C–E illustrate the responses elicited at currents approximately two and three times threshold as well as the response to a stimulus that elicited the maximal response for trigeminal neurons transfected with hNa V 1.7r–WT DNA. F–H show the responses for trigeminal neurons transfected with hNa V 1.7r–A1632E DNA. These cells are the same as shown in A and B to determine action potential threshold.
Figure Legend Snippet: Current-clamp recordings from transfected trigeminal ganglion neurons. The A1632E mutation decreases action potential threshold in small, current-clamped trigeminal ganglion neurons. A , Responses of a current-clamped trigeminal neuron transfected with hNa V 1.7r–WT DNA to a series of subthreshold and suprathreshold depolarizing current stimuli stepped in 5 pA increments. Two traces are displayed that show the threshold value for eliciting an action potential. RMP for this cell was −54 mV and threshold was 155 pA. B , Two traces are displayed that show the threshold for a trigeminal neuron transfected with hNa V 1.7r–A1632E DNA. RMP for this cell was −54 mV and threshold was 100 pA. C–E illustrate the responses elicited at currents approximately two and three times threshold as well as the response to a stimulus that elicited the maximal response for trigeminal neurons transfected with hNa V 1.7r–WT DNA. F–H show the responses for trigeminal neurons transfected with hNa V 1.7r–A1632E DNA. These cells are the same as shown in A and B to determine action potential threshold.

Techniques Used: Transfection, Mutagenesis

A1632E expression increases excitability in DRG and trigeminal ganglion neurons. The mean number of action potentials (defined as action potentials that overshoot 0 mV) is quantified in response to a series of 1-s-long current injections ranging from 50 to 500 pA in 50 pA increments. A , The mean response was significantly higher ( p
Figure Legend Snippet: A1632E expression increases excitability in DRG and trigeminal ganglion neurons. The mean number of action potentials (defined as action potentials that overshoot 0 mV) is quantified in response to a series of 1-s-long current injections ranging from 50 to 500 pA in 50 pA increments. A , The mean response was significantly higher ( p

Techniques Used: Expressing

12) Product Images from "Purkinje Cell Ataxin-1 Modulates Climbing Fiber Synaptic Input in Developing and Adult Mouse Cerebellum"

Article Title: Purkinje Cell Ataxin-1 Modulates Climbing Fiber Synaptic Input in Developing and Adult Mouse Cerebellum

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.6311-11.2013

Abnormal Parallel Fiber Distribution in ATXN1[30Q]-D776 Mice (A) 350× immunofluorescent images of calbindin labeled PCs somata from wt FVB, panel 1 and ATXN1[30Q]-D776 /+ mice, panels 2 and 3. Atypical spines can be seen protruding from the PC soma and apical dendrite in ATXN1[30Q]-D776 /+ (white arrows, panel 2). Co-localization between the presynaptic parallel fiber-terminal marker terminal marker, VGLUT1 (yellow, panel 3), and somatic PC spines. (B) Diagram depiction of the disruption of CF (red) and parallel fiber synapses (yellow) and their relationship to PC spines in wt FVB and ATXN1[30Q]-D776 /+ mice. (C and D) 150× immunofluorescent images of spine development in SCA1 mice. ATXN1[30Q]-D776 /+ mice (C) display somatic spines at 5 wk of age and remain out to 1 year of age. (D) No atypical spines were detected at 5 wk of age in ATXN1[82Q]-S776/+ mice. (E) Time-course of the percentage of PCs displaying somatic spines. ATXN1[30Q]-D776 /+: n = 50 PCs examined; ATXN1[82Q]-S776 /+: n = 63.
Figure Legend Snippet: Abnormal Parallel Fiber Distribution in ATXN1[30Q]-D776 Mice (A) 350× immunofluorescent images of calbindin labeled PCs somata from wt FVB, panel 1 and ATXN1[30Q]-D776 /+ mice, panels 2 and 3. Atypical spines can be seen protruding from the PC soma and apical dendrite in ATXN1[30Q]-D776 /+ (white arrows, panel 2). Co-localization between the presynaptic parallel fiber-terminal marker terminal marker, VGLUT1 (yellow, panel 3), and somatic PC spines. (B) Diagram depiction of the disruption of CF (red) and parallel fiber synapses (yellow) and their relationship to PC spines in wt FVB and ATXN1[30Q]-D776 /+ mice. (C and D) 150× immunofluorescent images of spine development in SCA1 mice. ATXN1[30Q]-D776 /+ mice (C) display somatic spines at 5 wk of age and remain out to 1 year of age. (D) No atypical spines were detected at 5 wk of age in ATXN1[82Q]-S776/+ mice. (E) Time-course of the percentage of PCs displaying somatic spines. ATXN1[30Q]-D776 /+: n = 50 PCs examined; ATXN1[82Q]-S776 /+: n = 63.

Techniques Used: Mouse Assay, Labeling, Marker

Mice Expressing Both ATXN1[30Q]-D776 and ATXN1[82Q]-S776 Display Increased Severity of Disease at 9 Months of Age (A) Graph shows number of PCs with greater than 10 VGLUT2 synapses (left axis, red bars) and average number of VGLUT2 synapses per PC soma (right axis, black bars) in 5 week of age mice. ATXN1[30Q]-D776 / ATXN1[82Q]-S776 PC somata retain VGLUT2 synapses to a similar extent see in ATXN1[30Q]-D776 /+ mice. Greater than three mice were used for each genotype; wt FVB: n = 74 PCs examined; ATXN1[30Q]-D776 /+: n = 52; ATXN1[82Q]-S776 /+: n = 73; ATXN1[30Q]-D776 / ATXN1[82Q]-S776 : n = 50. (B) 60× immunofluorescent images of calbindin labeled PCs (red) and VGLUT2 labeled CF synapses (green) (C) Graph shows the average molecular thickness. Wt FVB: N = 5 mice; ATXN1[30Q]-D776 /+: N = 7; ATXN1[82Q]-S776 /+: N = 8; ATXN1[30Q]-D776 / ATXN1[82Q]-S776 : N = 6 (D) Graph of pathological severity score. Wt FVB: N = 5 mice; ATXN1[30Q]-D776 /+: N = 7; ATXN1[82Q]-S776 /+: N = 8; ATXN1[30Q]-D776 / ATXN1[82Q]-S776 : N = 6. *p
Figure Legend Snippet: Mice Expressing Both ATXN1[30Q]-D776 and ATXN1[82Q]-S776 Display Increased Severity of Disease at 9 Months of Age (A) Graph shows number of PCs with greater than 10 VGLUT2 synapses (left axis, red bars) and average number of VGLUT2 synapses per PC soma (right axis, black bars) in 5 week of age mice. ATXN1[30Q]-D776 / ATXN1[82Q]-S776 PC somata retain VGLUT2 synapses to a similar extent see in ATXN1[30Q]-D776 /+ mice. Greater than three mice were used for each genotype; wt FVB: n = 74 PCs examined; ATXN1[30Q]-D776 /+: n = 52; ATXN1[82Q]-S776 /+: n = 73; ATXN1[30Q]-D776 / ATXN1[82Q]-S776 : n = 50. (B) 60× immunofluorescent images of calbindin labeled PCs (red) and VGLUT2 labeled CF synapses (green) (C) Graph shows the average molecular thickness. Wt FVB: N = 5 mice; ATXN1[30Q]-D776 /+: N = 7; ATXN1[82Q]-S776 /+: N = 8; ATXN1[30Q]-D776 / ATXN1[82Q]-S776 : N = 6 (D) Graph of pathological severity score. Wt FVB: N = 5 mice; ATXN1[30Q]-D776 /+: N = 7; ATXN1[82Q]-S776 /+: N = 8; ATXN1[30Q]-D776 / ATXN1[82Q]-S776 : N = 6. *p

Techniques Used: Mouse Assay, Expressing, Labeling

CF-PC Extension is Restored when ATXN1[30Q]-D776 is Not Expressed During Cerebellar Development (A) 60× immunofluorescent images from primary fissure showing restored PC morphology (green) and CF extension (red) in mice not expressing ATXN1[30Q]-D776 during the first 3 wk of age. (B) Graph of distal extension of CF terminals onto the PC dendrites in cATXN1[30Q]-D776 mice. cATXN1[30Q]-D776 Off N=4; cATXN1[30Q]-D776 gene off/on N= 3 animals; cATXN1[30Q]-D776 On N = 4. (C) Graph of ATXN1 expression during development in ATXN1[30Q]-D776 /+ and cATXN1[30Q]-D776, ATXN1[82Q]-S776 , and ATXN1[30Q]-D776-K772T /+mice. Data are normalized to ATXN1 expression of ATXN1[30Q]-D776 /+ mice at p7. *p
Figure Legend Snippet: CF-PC Extension is Restored when ATXN1[30Q]-D776 is Not Expressed During Cerebellar Development (A) 60× immunofluorescent images from primary fissure showing restored PC morphology (green) and CF extension (red) in mice not expressing ATXN1[30Q]-D776 during the first 3 wk of age. (B) Graph of distal extension of CF terminals onto the PC dendrites in cATXN1[30Q]-D776 mice. cATXN1[30Q]-D776 Off N=4; cATXN1[30Q]-D776 gene off/on N= 3 animals; cATXN1[30Q]-D776 On N = 4. (C) Graph of ATXN1 expression during development in ATXN1[30Q]-D776 /+ and cATXN1[30Q]-D776, ATXN1[82Q]-S776 , and ATXN1[30Q]-D776-K772T /+mice. Data are normalized to ATXN1 expression of ATXN1[30Q]-D776 /+ mice at p7. *p

Techniques Used: Mouse Assay, Expressing

Climbing Fiber Development and Regression in Affected SCA1 Mice Normal development of CF/PC synapses is depicted in the top row. At early stages (P7) each PC is innervated by multiple CFs from the inferior olive (IO). As development proceeds (P14) activity of one CF strengthens, its terminals translocate up the proximal dendritic shaft of the PC. At 5 wk of age, and into the adult, one CF maintains its synapses with the PC dendritic tree segregated from the synaptic territory of parallel fibers. In ATXN1[82Q]-S776 mice development is mostly normal except that translocation of the dominant CF terminals up the PC dendritic tree is delayed and some of its terminals remain on the PC soma (5wk). As disease progresses in ATXN1 mice CF terminals regress from the PC dendritic tree (12wk-adult). In ATXN1[30Q]-D776 mice development is more substantially altered. Although one CF seems to be become dominant and translocates its terminals up the PC dendritic tree (P14) subsequent terminal translocation is more severely compromised and pruning of terminals from the PC soma fails to occur (5wk). As in ATXN1[82Q]-S776 mice, CF terminals that translocated up the PC dendritic tree regress with age (12wk-adult).
Figure Legend Snippet: Climbing Fiber Development and Regression in Affected SCA1 Mice Normal development of CF/PC synapses is depicted in the top row. At early stages (P7) each PC is innervated by multiple CFs from the inferior olive (IO). As development proceeds (P14) activity of one CF strengthens, its terminals translocate up the proximal dendritic shaft of the PC. At 5 wk of age, and into the adult, one CF maintains its synapses with the PC dendritic tree segregated from the synaptic territory of parallel fibers. In ATXN1[82Q]-S776 mice development is mostly normal except that translocation of the dominant CF terminals up the PC dendritic tree is delayed and some of its terminals remain on the PC soma (5wk). As disease progresses in ATXN1 mice CF terminals regress from the PC dendritic tree (12wk-adult). In ATXN1[30Q]-D776 mice development is more substantially altered. Although one CF seems to be become dominant and translocates its terminals up the PC dendritic tree (P14) subsequent terminal translocation is more severely compromised and pruning of terminals from the PC soma fails to occur (5wk). As in ATXN1[82Q]-S776 mice, CF terminals that translocated up the PC dendritic tree regress with age (12wk-adult).

Techniques Used: Mouse Assay, Activity Assay, Translocation Assay

Irregular Morphology and Impaired Climbing Fiber Synapse Elimination in Adult ATXN1[30Q]-D776 Mice (A) Representative immunofluorescent confocal images taken at 100× of cerebellar sections with anterograde tracer labeled CFs (red) and calbindin labeled PCs (green) (upper panel). Digital representation of the PC-CF interaction from cerebellar sections with GFP-expressing PCs (grey), anterogradely labeled CF (red) and immunolabeled CF terminals (yellow) (lower panel). Note the co-localization between anterogradely labeled CF and VGLUT2 puncta in both control and mutant mice. (B) Graph shows number of PCs with greater than 10 VGLUT2 synapses (left axis, red bars) and average number of VGLUT2 synapses per PC soma (right axis, black bars) in 5 week of age mice. Wt FVB (n = 65 PCs) and ATXN1[30Q]-S776/S776 (n = 56) mice show no PCs with greater than 10 VGLUT2 somatic synapses. ATXN1[30Q]-D776 /+ (n =56) mice fail to eliminate the most somatic synapses. The data are represented as mean ± SEM. (C) Graph shows number of VGLUT2 synapses on the PC cell bodies in mice 21 days of age. Wt FVB (n = 54 PCs), ATXN1[30Q]-S776/S776 (n = 52), ATXN1[30Q]-D776 /+ (n = 54), and ATXN1[82Q]-S776 /+ (n = 51). (D) Representative 100× images of isolated PCs (red) and VGLUT2 (green) with area of interest defined (yellow box).
Figure Legend Snippet: Irregular Morphology and Impaired Climbing Fiber Synapse Elimination in Adult ATXN1[30Q]-D776 Mice (A) Representative immunofluorescent confocal images taken at 100× of cerebellar sections with anterograde tracer labeled CFs (red) and calbindin labeled PCs (green) (upper panel). Digital representation of the PC-CF interaction from cerebellar sections with GFP-expressing PCs (grey), anterogradely labeled CF (red) and immunolabeled CF terminals (yellow) (lower panel). Note the co-localization between anterogradely labeled CF and VGLUT2 puncta in both control and mutant mice. (B) Graph shows number of PCs with greater than 10 VGLUT2 synapses (left axis, red bars) and average number of VGLUT2 synapses per PC soma (right axis, black bars) in 5 week of age mice. Wt FVB (n = 65 PCs) and ATXN1[30Q]-S776/S776 (n = 56) mice show no PCs with greater than 10 VGLUT2 somatic synapses. ATXN1[30Q]-D776 /+ (n =56) mice fail to eliminate the most somatic synapses. The data are represented as mean ± SEM. (C) Graph shows number of VGLUT2 synapses on the PC cell bodies in mice 21 days of age. Wt FVB (n = 54 PCs), ATXN1[30Q]-S776/S776 (n = 52), ATXN1[30Q]-D776 /+ (n = 54), and ATXN1[82Q]-S776 /+ (n = 51). (D) Representative 100× images of isolated PCs (red) and VGLUT2 (green) with area of interest defined (yellow box).

Techniques Used: Mouse Assay, Labeling, Expressing, Immunolabeling, Mutagenesis, Isolation

Purkinje Cell Response Evoked from Contralateral Inferior Olive Stimulation in 12 Weeks of Age Mice (A) Background immunofluorescent images from exposed in-vivo cerebellum (upper row) and compilation images of autofluorescent flavoprotein responses evoked by contralateral inferior olive stimulation (lower row). (B) Representative field potentials evoked by contralateral inferior olive stimulation. (C) Quantification of flavoprotein autofluorescence responses evoked from CIO stimulation. Wt FVB: n = 11 animals, 60 observations for autofluorescent images; ATXN1[30Q]-S776/S776 : n = 6, n = 30 observations; ATXN1[30Q]-D776 /+: n = 6, n = 30 observations; ATXN1[82Q]-S776 /+: n = 4, n = 32 observations. (D) Quantification of CF-mediated negativity (CF-N 1 ) of the FP showing greatest reduced response to CF stimulation in ATX1[82Q]-S776 /+ mice. Wt FVB: n = 5, 14 observations; ATXN1[30Q]-S776/S776 : n = 6, 30 observations; ATXN1[30Q]-D776 /+: n = 6, 30 observations; ATX1[82Q]-S776 /+: n = 4, 30 observations. Wt FVB and ATXN1[82Q]-S776 . * Denotes genotypes are statistically different from wT/FVB and ATX1[82Q]-S776 /+, p
Figure Legend Snippet: Purkinje Cell Response Evoked from Contralateral Inferior Olive Stimulation in 12 Weeks of Age Mice (A) Background immunofluorescent images from exposed in-vivo cerebellum (upper row) and compilation images of autofluorescent flavoprotein responses evoked by contralateral inferior olive stimulation (lower row). (B) Representative field potentials evoked by contralateral inferior olive stimulation. (C) Quantification of flavoprotein autofluorescence responses evoked from CIO stimulation. Wt FVB: n = 11 animals, 60 observations for autofluorescent images; ATXN1[30Q]-S776/S776 : n = 6, n = 30 observations; ATXN1[30Q]-D776 /+: n = 6, n = 30 observations; ATXN1[82Q]-S776 /+: n = 4, n = 32 observations. (D) Quantification of CF-mediated negativity (CF-N 1 ) of the FP showing greatest reduced response to CF stimulation in ATX1[82Q]-S776 /+ mice. Wt FVB: n = 5, 14 observations; ATXN1[30Q]-S776/S776 : n = 6, 30 observations; ATXN1[30Q]-D776 /+: n = 6, 30 observations; ATX1[82Q]-S776 /+: n = 4, 30 observations. Wt FVB and ATXN1[82Q]-S776 . * Denotes genotypes are statistically different from wT/FVB and ATX1[82Q]-S776 /+, p

Techniques Used: Mouse Assay, In Vivo

12 week old ATXN1[30Q]-D776 Purkinje Cells are Multiply-Innervated Immunofluorescent images of PC somata (grey) were examined to detect PCs with lone VGLUT2-positive CF synapses(red – white arrows) that do not co-localize with the anterograde tracer labeled CFs (green). (A) A wt FVB PC with no VGLUT2-positive puncta. (B) A CF multiply-innervated mouse model, L7-PKCI /+ PC with VGLUT2-positive somatic synapses (white arrows). (C) An ATXN1[30Q]-D776 /+ PC with VGLUT2-positive somatic synapses (white arrows). (D) An ATXN1[82Q]-D776 /+ PC with no VGLUT2-positive puncta. (E) Graph showing the percentage of PCs per genotype that have lone red VGLUT2-positive synapses (p = 0.002, chi-squared test). Greater than three mice were used for each genotype; wt FVB: n = 20 PCs examined; L7-PKCI /+: n = 40; ATXN1[30Q]-D776 /+: n = 39; ATXN1[82Q]-S776 /+: n = 43.
Figure Legend Snippet: 12 week old ATXN1[30Q]-D776 Purkinje Cells are Multiply-Innervated Immunofluorescent images of PC somata (grey) were examined to detect PCs with lone VGLUT2-positive CF synapses(red – white arrows) that do not co-localize with the anterograde tracer labeled CFs (green). (A) A wt FVB PC with no VGLUT2-positive puncta. (B) A CF multiply-innervated mouse model, L7-PKCI /+ PC with VGLUT2-positive somatic synapses (white arrows). (C) An ATXN1[30Q]-D776 /+ PC with VGLUT2-positive somatic synapses (white arrows). (D) An ATXN1[82Q]-D776 /+ PC with no VGLUT2-positive puncta. (E) Graph showing the percentage of PCs per genotype that have lone red VGLUT2-positive synapses (p = 0.002, chi-squared test). Greater than three mice were used for each genotype; wt FVB: n = 20 PCs examined; L7-PKCI /+: n = 40; ATXN1[30Q]-D776 /+: n = 39; ATXN1[82Q]-S776 /+: n = 43.

Techniques Used: Labeling, Mouse Assay

Nuclear Localization of ATXN1[30Q]-D776 is Critical for Disruption of Climbing Fiber Synapse Elimination (A) Western blot of ATXN1 protein levels in cerebellar lysates. ATXN1[30Q]-D776 /+ and ATXN1[30Q]-D776-K772T /+. (B) 100× immunofluorescent images of ATXN1 localization at PC layer. (C) Graph of average number of VGLUT2 synapses per PC soma (right axis, black bars) and number of PCs with greater than 10 VGLUT2 synapses (left axis, red bars) in ATXN1[30Q]-D776-K772T and ATXN1[30Q]-D776 /+ mice.
Figure Legend Snippet: Nuclear Localization of ATXN1[30Q]-D776 is Critical for Disruption of Climbing Fiber Synapse Elimination (A) Western blot of ATXN1 protein levels in cerebellar lysates. ATXN1[30Q]-D776 /+ and ATXN1[30Q]-D776-K772T /+. (B) 100× immunofluorescent images of ATXN1 localization at PC layer. (C) Graph of average number of VGLUT2 synapses per PC soma (right axis, black bars) and number of PCs with greater than 10 VGLUT2 synapses (left axis, red bars) in ATXN1[30Q]-D776-K772T and ATXN1[30Q]-D776 /+ mice.

Techniques Used: Western Blot, Mouse Assay

Altered Climbing Fiber Development in SCA1 Affected Mice (A) Immunofluorescent confocal images taken at 60× from p14 (upper panel) and 5 weeks of age (lower panel) cerebellar sections immunostained with Calbindin to identify PCs (red) and VGLUT2 to identify CF terminals (green). (B) Time-course of distal extension profile of CF terminals onto the PC dendrites at different stages in development. Wild type FVB and ATXN1[30Q]-S776/S776 mice have a steady progression until 5 weeks of age. SCA1 mice show no progression ( ATXN1[30Q]-D776 /+) or retarded progression ( ATXN1[82Q]-S776 /+). Number of animals (n) assessed for each genotype are; wt FVB: p14-4, p17-4, p21-4, 5wk-3, 12wk-5; ATX1[30Q]-S776/S776 : p14-4, p17-4, p21-3, 5wk-3, 12wk-7; ATXN1[82Q]-S776 /+: p14-3, p17-3, p21-3, 5wk-3, 12wk-6; and ATXN1[30Q]-D776 /+: p14-3, p17-4, p21-4, 5wk-3, 12wk-4. At 7 wk cATXN1[30Q]-D776 gene on, n = 3; and cATXN1[30Q]-D776 .
Figure Legend Snippet: Altered Climbing Fiber Development in SCA1 Affected Mice (A) Immunofluorescent confocal images taken at 60× from p14 (upper panel) and 5 weeks of age (lower panel) cerebellar sections immunostained with Calbindin to identify PCs (red) and VGLUT2 to identify CF terminals (green). (B) Time-course of distal extension profile of CF terminals onto the PC dendrites at different stages in development. Wild type FVB and ATXN1[30Q]-S776/S776 mice have a steady progression until 5 weeks of age. SCA1 mice show no progression ( ATXN1[30Q]-D776 /+) or retarded progression ( ATXN1[82Q]-S776 /+). Number of animals (n) assessed for each genotype are; wt FVB: p14-4, p17-4, p21-4, 5wk-3, 12wk-5; ATX1[30Q]-S776/S776 : p14-4, p17-4, p21-3, 5wk-3, 12wk-7; ATXN1[82Q]-S776 /+: p14-3, p17-3, p21-3, 5wk-3, 12wk-6; and ATXN1[30Q]-D776 /+: p14-3, p17-4, p21-4, 5wk-3, 12wk-4. At 7 wk cATXN1[30Q]-D776 gene on, n = 3; and cATXN1[30Q]-D776 .

Techniques Used: Mouse Assay

13) Product Images from "Generation of Mutant Murine Cytomegalovirus Strains from Overlapping Cosmid and Plasmid Clones"

Article Title: Generation of Mutant Murine Cytomegalovirus Strains from Overlapping Cosmid and Plasmid Clones

Journal: Journal of Virology

doi:

Cosmid and plasmid vectors for making infectious MCMV clone sets. (A) The PmeCOS cosmid vector was constructed by adding a pair of Pme I sites surrounding the Bam ) so that any cloned MCMV sequences can be removed intact. (B) The PmeSUB vector was derived from pACYC177 by replacement of the Kan r gene with the MCS from pBluescript. The MCS was surrounded by Pme I sites to allow excision of MCMV sequences.
Figure Legend Snippet: Cosmid and plasmid vectors for making infectious MCMV clone sets. (A) The PmeCOS cosmid vector was constructed by adding a pair of Pme I sites surrounding the Bam ) so that any cloned MCMV sequences can be removed intact. (B) The PmeSUB vector was derived from pACYC177 by replacement of the Kan r gene with the MCS from pBluescript. The MCS was surrounded by Pme I sites to allow excision of MCMV sequences.

Techniques Used: Plasmid Preparation, Construct, Clone Assay, Derivative Assay

Restriction digests of the infectious cosmid set. The complete set of infectious cosmids for reconstituting MCMV was digested with either Eco RI or Pme I, fractionated by agarose gel electrophoresis and stained with ethidium bromide. The released MCMV sequences and cosmid vector, PmeCOS, are indicated on the right. The molecular size standards (std) are Bst EII-digested λ DNA.
Figure Legend Snippet: Restriction digests of the infectious cosmid set. The complete set of infectious cosmids for reconstituting MCMV was digested with either Eco RI or Pme I, fractionated by agarose gel electrophoresis and stained with ethidium bromide. The released MCMV sequences and cosmid vector, PmeCOS, are indicated on the right. The molecular size standards (std) are Bst EII-digested λ DNA.

Techniques Used: Agarose Gel Electrophoresis, Staining, Plasmid Preparation

14) Product Images from "Nav1.7 mutations associated with paroxysmal extreme pain disorder, but not erythromelalgia, enhance Nav?4 peptide-mediated resurgent sodium currents"

Article Title: Nav1.7 mutations associated with paroxysmal extreme pain disorder, but not erythromelalgia, enhance Nav?4 peptide-mediated resurgent sodium currents

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2010.200915

Navβ4 peptide produces resurgent currents in Nav1.7 channels expressed in HEK293 cells A and B , resurgent current traces recorded from representative HEK293 cells expressing either Nav1.7 WT or mutant channels in the absence ( A ) and presence ( B ) of Navβ4 in the recording pipette. Note that the current amplitudes are scaled to reflect the relative size of resurgent currents in relation to WT and to better compare the currents between each construct in the absence and presence of the peptide. C , resurgent current amplitude, as measured as a percentage of the average peak transient current elicited at +20 mV obtained immediately prior to and following the resurgent current protocol as shown in D . For cells recorded in the absence of Navβ4: WT, n = 12; M1627K, n = 9; T1464I, n = 8; V1299F, n = 9; I848T, n = 8; L858H, n = 8. The n values for cells recorded in the presence of Navβ4 are shown on the bars in C . * P
Figure Legend Snippet: Navβ4 peptide produces resurgent currents in Nav1.7 channels expressed in HEK293 cells A and B , resurgent current traces recorded from representative HEK293 cells expressing either Nav1.7 WT or mutant channels in the absence ( A ) and presence ( B ) of Navβ4 in the recording pipette. Note that the current amplitudes are scaled to reflect the relative size of resurgent currents in relation to WT and to better compare the currents between each construct in the absence and presence of the peptide. C , resurgent current amplitude, as measured as a percentage of the average peak transient current elicited at +20 mV obtained immediately prior to and following the resurgent current protocol as shown in D . For cells recorded in the absence of Navβ4: WT, n = 12; M1627K, n = 9; T1464I, n = 8; V1299F, n = 9; I848T, n = 8; L858H, n = 8. The n values for cells recorded in the presence of Navβ4 are shown on the bars in C . * P

Techniques Used: Expressing, Mutagenesis, Transferring, Construct

Mutations within Nav1.7 have differential effects on current properties A ). B , current traces recorded from representative HEK293 cells expressing either Nav1.7 WT or mutant channels. Cells were held at −80 mV, and currents were elicited with 50 ms test pulses to potentials ranging from −80 to +60 mV.
Figure Legend Snippet: Mutations within Nav1.7 have differential effects on current properties A ). B , current traces recorded from representative HEK293 cells expressing either Nav1.7 WT or mutant channels. Cells were held at −80 mV, and currents were elicited with 50 ms test pulses to potentials ranging from −80 to +60 mV.

Techniques Used: Expressing, Mutagenesis, Mass Spectrometry

Dependence of resurgent current amplitude on duration of depolarizing voltage step between Nav1.7 WT and PEPD mutant channels Cells expressing WT, T1464I and M1627K channels were assayed for their ability to generate resurgent current using a depolarizing conditioning pulse to +30 mV at different pulse durations (8–256 ms) and then repolarized to a potential near which the peak resurgent current amplitude was observed for each channel construct in order to maximize the signal-to-noise ratio (see inset). For each individual recording, resurgent current amplitudes were normalized to the current amplitude at 8 ms and presented as a percentage of that amplitude and averaged for all cells in each group ( n = 8–9, * P
Figure Legend Snippet: Dependence of resurgent current amplitude on duration of depolarizing voltage step between Nav1.7 WT and PEPD mutant channels Cells expressing WT, T1464I and M1627K channels were assayed for their ability to generate resurgent current using a depolarizing conditioning pulse to +30 mV at different pulse durations (8–256 ms) and then repolarized to a potential near which the peak resurgent current amplitude was observed for each channel construct in order to maximize the signal-to-noise ratio (see inset). For each individual recording, resurgent current amplitudes were normalized to the current amplitude at 8 ms and presented as a percentage of that amplitude and averaged for all cells in each group ( n = 8–9, * P

Techniques Used: Mutagenesis, Expressing, Mass Spectrometry, Construct

Effects of Nav1.7 mutations and the Navβ4 peptide on open channel fast inactivation A , normalized current traces elicited by a step depolarization to +20 mV from representative HEK293 cells expressing either Nav1.7 WT or mutant channels in the absence (top) and presence (bottom) of the Navβ4 peptide in the recording pipette. B , bar graph displaying the average decay time constants in the absence (dark shaded bars) and presence (light shaded bars) of Navβ4 in the recording pipette ( n = 8–14, * P
Figure Legend Snippet: Effects of Nav1.7 mutations and the Navβ4 peptide on open channel fast inactivation A , normalized current traces elicited by a step depolarization to +20 mV from representative HEK293 cells expressing either Nav1.7 WT or mutant channels in the absence (top) and presence (bottom) of the Navβ4 peptide in the recording pipette. B , bar graph displaying the average decay time constants in the absence (dark shaded bars) and presence (light shaded bars) of Navβ4 in the recording pipette ( n = 8–14, * P

Techniques Used: Expressing, Mutagenesis, Transferring

15) Product Images from "Interrelationship Between Cytoplasmic Retroviral Gag Concentration and Gag-Membrane Association"

Article Title: Interrelationship Between Cytoplasmic Retroviral Gag Concentration and Gag-Membrane Association

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2013.11.025

Analysis of the correlation between cytoplasmic Gag-EYFP concentration and Gag-membrane association. A. Concentration dependence of HIV-1 Gag-EYFP membrane association. Shown is a graph of HIV-1 Gag-EYFP cytoplasmic concentration versus the presence of
Figure Legend Snippet: Analysis of the correlation between cytoplasmic Gag-EYFP concentration and Gag-membrane association. A. Concentration dependence of HIV-1 Gag-EYFP membrane association. Shown is a graph of HIV-1 Gag-EYFP cytoplasmic concentration versus the presence of

Techniques Used: Concentration Assay

Correlation of HIV-1 and HTLV-1 MA-EYFP membrane-binding with cytoplasmic concentration. A. HIV-1 MA-EYP is a cytosolic protein. Shown is a graph of HIV-1 MA-EYFP cytoplasmic concentration versus the presence of membrane-associated MA as determined by
Figure Legend Snippet: Correlation of HIV-1 and HTLV-1 MA-EYFP membrane-binding with cytoplasmic concentration. A. HIV-1 MA-EYP is a cytosolic protein. Shown is a graph of HIV-1 MA-EYFP cytoplasmic concentration versus the presence of membrane-associated MA as determined by

Techniques Used: Binding Assay, Concentration Assay

Correlation of HIV-1 and HTLV-1 G2A-Gag-EYFP membrane-binding with cytoplasmic concentration. A. HIV-1 G2A-Gag-EYFP is a cytoplasmic protein. Graph of HIV-1 G2A-Gag-EYFP cytoplasmic concentration versus the presence of membrane-bound G2A-Gag is shown
Figure Legend Snippet: Correlation of HIV-1 and HTLV-1 G2A-Gag-EYFP membrane-binding with cytoplasmic concentration. A. HIV-1 G2A-Gag-EYFP is a cytoplasmic protein. Graph of HIV-1 G2A-Gag-EYFP cytoplasmic concentration versus the presence of membrane-bound G2A-Gag is shown

Techniques Used: Binding Assay, Concentration Assay

16) Product Images from "Attenuation of IgE Affinity for Fc?RI Radically Reduces the Allergic Response in Vitro and in Vivo *"

Article Title: Attenuation of IgE Affinity for Fc?RI Radically Reduces the Allergic Response in Vitro and in Vivo *

Journal:

doi: 10.1074/jbc.M804742200

SPR analysis of NP-IgE ( A ) and R334S NP-IgE ( B ) binding to sFcεRIα in the concentration range 12–200 n m . The top panel shows binding curves overlaid with biphasic fits that best describe the interaction; the bottom panel shows
Figure Legend Snippet: SPR analysis of NP-IgE ( A ) and R334S NP-IgE ( B ) binding to sFcεRIα in the concentration range 12–200 n m . The top panel shows binding curves overlaid with biphasic fits that best describe the interaction; the bottom panel shows

Techniques Used: SPR Assay, Binding Assay, Concentration Assay

In vitro analysis of R334S and wild type NP-IgE-mediated effector cell activation. A, titration of (•) wild type and (□) R334S IgE-induced degranulation from sensitized RBL-SX38 cells. Values are an average of three measurements corrected
Figure Legend Snippet: In vitro analysis of R334S and wild type NP-IgE-mediated effector cell activation. A, titration of (•) wild type and (□) R334S IgE-induced degranulation from sensitized RBL-SX38 cells. Values are an average of three measurements corrected

Techniques Used: In Vitro, Activation Assay, Titration

In vivo analysis of NP-IgE- and R334S NP-IgE-mediated effector cell activation. A, inset, titration of NP IgE in ears of hFcεRI Tg mice to determine IgE dose required for maximal PCA. Data are expressed as a mean of all measurements (±S.E.).
Figure Legend Snippet: In vivo analysis of NP-IgE- and R334S NP-IgE-mediated effector cell activation. A, inset, titration of NP IgE in ears of hFcεRI Tg mice to determine IgE dose required for maximal PCA. Data are expressed as a mean of all measurements (±S.E.).

Techniques Used: In Vivo, Activation Assay, Titration, Mouse Assay

Biochemical analysis of wild type and R334S NP-IgE. A, analytical S200 10/300 GL Superdex gel filtration traces of NP-IgE and R334S NP-IgE after purification; run at 0.75 ml/min in 0.5 m Tris, 0.25 m NaCl, pH 7.2. B, 4–20% Tris glycine SDS-PAGE
Figure Legend Snippet: Biochemical analysis of wild type and R334S NP-IgE. A, analytical S200 10/300 GL Superdex gel filtration traces of NP-IgE and R334S NP-IgE after purification; run at 0.75 ml/min in 0.5 m Tris, 0.25 m NaCl, pH 7.2. B, 4–20% Tris glycine SDS-PAGE

Techniques Used: Filtration, Purification, SDS Page

17) Product Images from "Attenuation of IgE Affinity for Fc?RI Radically Reduces the Allergic Response in Vitro and in Vivo *"

Article Title: Attenuation of IgE Affinity for Fc?RI Radically Reduces the Allergic Response in Vitro and in Vivo *

Journal:

doi: 10.1074/jbc.M804742200

SPR analysis of NP-IgE ( A ) and R334S NP-IgE ( B ) binding to sFcεRIα in the concentration range 12–200 n m . The top panel shows binding curves overlaid with biphasic fits that best describe the interaction; the bottom panel shows
Figure Legend Snippet: SPR analysis of NP-IgE ( A ) and R334S NP-IgE ( B ) binding to sFcεRIα in the concentration range 12–200 n m . The top panel shows binding curves overlaid with biphasic fits that best describe the interaction; the bottom panel shows

Techniques Used: SPR Assay, Binding Assay, Concentration Assay

In vitro analysis of R334S and wild type NP-IgE-mediated effector cell activation. A, titration of (•) wild type and (□) R334S IgE-induced degranulation from sensitized RBL-SX38 cells. Values are an average of three measurements corrected
Figure Legend Snippet: In vitro analysis of R334S and wild type NP-IgE-mediated effector cell activation. A, titration of (•) wild type and (□) R334S IgE-induced degranulation from sensitized RBL-SX38 cells. Values are an average of three measurements corrected

Techniques Used: In Vitro, Activation Assay, Titration

In vivo analysis of NP-IgE- and R334S NP-IgE-mediated effector cell activation. A, inset, titration of NP IgE in ears of hFcεRI Tg mice to determine IgE dose required for maximal PCA. Data are expressed as a mean of all measurements (±S.E.).
Figure Legend Snippet: In vivo analysis of NP-IgE- and R334S NP-IgE-mediated effector cell activation. A, inset, titration of NP IgE in ears of hFcεRI Tg mice to determine IgE dose required for maximal PCA. Data are expressed as a mean of all measurements (±S.E.).

Techniques Used: In Vivo, Activation Assay, Titration, Mouse Assay

Biochemical analysis of wild type and R334S NP-IgE. A, analytical S200 10/300 GL Superdex gel filtration traces of NP-IgE and R334S NP-IgE after purification; run at 0.75 ml/min in 0.5 m Tris, 0.25 m NaCl, pH 7.2. B, 4–20% Tris glycine SDS-PAGE
Figure Legend Snippet: Biochemical analysis of wild type and R334S NP-IgE. A, analytical S200 10/300 GL Superdex gel filtration traces of NP-IgE and R334S NP-IgE after purification; run at 0.75 ml/min in 0.5 m Tris, 0.25 m NaCl, pH 7.2. B, 4–20% Tris glycine SDS-PAGE

Techniques Used: Filtration, Purification, SDS Page

18) Product Images from "Effects of individually silenced N-glycosylation sites and non-synonymous single-nucleotide polymorphisms on the fusogenic function of human syncytin-2"

Article Title: Effects of individually silenced N-glycosylation sites and non-synonymous single-nucleotide polymorphisms on the fusogenic function of human syncytin-2

Journal: Cell Adhesion & Migration

doi: 10.1080/19336918.2015.1093720

Schematic of the syncytin-2 gene structure indicating the location of candidate N-glycosylation sites and non-synonymous single-nucleotide polymorphic (SNP) sites and the establishment of the fusion assay for functional evaluation of syncytin-2 and its mutants. (A) Upper panel: structure of a human endogenous retrovirus (HERV)-FRD gene locus and its encoded envelope protein syncytin-2. 4,9 HERV is typically composed of 5′ and 3′ LTRs (long terminal repeats), gag (group-specific antigen gene), pol (polymerase), and env (envelope). Syncytin-2 encoded by the env gene is composed of SU and TM subunits. Lower panel: schematic diagram of syncytin-2 indicating the positions of 10 potential N-glycosylation sites (numbered 1–10) and 10 naturally occurring SNPs from SNP databases (http://www.ncbi.nlm.nih.gov/snp) in predicted functional domains (numbered 11–21). The positions of amino acids of different domains/motifs are indicated. SU, surface protein; TM, transmembrane protein; SP, signal peptide; FP, fusion peptide; HR, heptad repeat region; ISD, immunosuppressive domain; TMD, transmembrane domain. CTM, cytoplasmic domain. (B) Establishment of 293T and HeLa cell lines that stably express EmGFP. (C) Cell-cell fusion mediated by exogenous syncytin-2. The 293T-EmGFP cells were transfected with phCMV empty vector or phCMV-syncytin-2 for 36 h. HeLa-EmGFP cells were co-transfected with phCMV-syncytin-2 and pcDNA3.1-MFSD2A-FLAG for 36 h. Single-plasmid transfection with either phCMV-syncytin-2 or pcDNA3.1-MFSD2A-FLAG did not cause cell-cell fusion. All experiments were repeated 3 times. Bars = 100 µm.
Figure Legend Snippet: Schematic of the syncytin-2 gene structure indicating the location of candidate N-glycosylation sites and non-synonymous single-nucleotide polymorphic (SNP) sites and the establishment of the fusion assay for functional evaluation of syncytin-2 and its mutants. (A) Upper panel: structure of a human endogenous retrovirus (HERV)-FRD gene locus and its encoded envelope protein syncytin-2. 4,9 HERV is typically composed of 5′ and 3′ LTRs (long terminal repeats), gag (group-specific antigen gene), pol (polymerase), and env (envelope). Syncytin-2 encoded by the env gene is composed of SU and TM subunits. Lower panel: schematic diagram of syncytin-2 indicating the positions of 10 potential N-glycosylation sites (numbered 1–10) and 10 naturally occurring SNPs from SNP databases (http://www.ncbi.nlm.nih.gov/snp) in predicted functional domains (numbered 11–21). The positions of amino acids of different domains/motifs are indicated. SU, surface protein; TM, transmembrane protein; SP, signal peptide; FP, fusion peptide; HR, heptad repeat region; ISD, immunosuppressive domain; TMD, transmembrane domain. CTM, cytoplasmic domain. (B) Establishment of 293T and HeLa cell lines that stably express EmGFP. (C) Cell-cell fusion mediated by exogenous syncytin-2. The 293T-EmGFP cells were transfected with phCMV empty vector or phCMV-syncytin-2 for 36 h. HeLa-EmGFP cells were co-transfected with phCMV-syncytin-2 and pcDNA3.1-MFSD2A-FLAG for 36 h. Single-plasmid transfection with either phCMV-syncytin-2 or pcDNA3.1-MFSD2A-FLAG did not cause cell-cell fusion. All experiments were repeated 3 times. Bars = 100 µm.

Techniques Used: Single Vesicle Fusion Assay, Functional Assay, Stable Transfection, Transfection, Plasmid Preparation

19) Product Images from "MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia"

Article Title: MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia

Journal: PLoS Medicine

doi: 10.1371/journal.pmed.0030270

Flow Cytometry Analysis of BM and Spleen in Mice Transduced with MPLW515L and MPLWT (A) Flow-cytometry analysis of bone marrow cells shows a 4-fold increase in Mac1+/Gr1+ cells and a shift to a more immature erythroid population. There is a 15-fold increase in CD41+ cells in bone marrow expressing MPLW515L compared with MPLWT. (B) Flow-cytometry analysis of spleen cells shows a 10-fold increase in Mac1+/Gr1+ cells in MPLW515L. There is also a shift to a more immature erythroid population in MPLW515L with a greater percentage of CD71+/Ter119- cells. CD41+ cells are increased 30-fold in MPLW515L spleen cells compared with MPLWT spleen cells.
Figure Legend Snippet: Flow Cytometry Analysis of BM and Spleen in Mice Transduced with MPLW515L and MPLWT (A) Flow-cytometry analysis of bone marrow cells shows a 4-fold increase in Mac1+/Gr1+ cells and a shift to a more immature erythroid population. There is a 15-fold increase in CD41+ cells in bone marrow expressing MPLW515L compared with MPLWT. (B) Flow-cytometry analysis of spleen cells shows a 10-fold increase in Mac1+/Gr1+ cells in MPLW515L. There is also a shift to a more immature erythroid population in MPLW515L with a greater percentage of CD71+/Ter119- cells. CD41+ cells are increased 30-fold in MPLW515L spleen cells compared with MPLWT spleen cells.

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Transduction, Expressing

MPLW515L-Expressing Cells Show Hyper-Responsiveness to TPO (A) 32D MPLWT cells and 32D MPLW515L cells were cultured in triplicate at an initial concentration of 1 × 10 5 cells/mL, in RPMI/10% FBS containing TPO 2 ng/mL, 1 ng/mL, 0.1 ng/mL, 0.01 ng/mL, 0.001 ng/mL, or in the absence of TPO for four days, and then cell numbers were assessed. Error bars denote the standard deviation for each sample measured in triplicate. (B) UT7 MPLWT cells and UT7 MPLW515L cells were cultured in triplicate, at an initial concentration of 1 × 10 5 cells/mL, in IMDM/10% FBS containing TPO 5 ng/mL, 1 ng/mL, 0.1 ng/mL, 0.01 ng/mL, 0.001ng/mL, or in the absence of TPO for six days, and then cell numbers were assessed. Error bars denote the standard deviation for each sample measured in triplicate. (C) 32D MPLWT or 32D MPLW515L cells were deprived of cytokine overnight and then stimulated with TPO 5 ng/mL, 1 ng/mL, 0.1 ng/mL, 0.01 ng/mL, or without TPO for seven minutes and then analyzed by Western blot for phosphorylation of JAK2 and STAT3, demonstrating increased JAK2/STAT3 phosphorylation in response to TPO in 32D MPLW515L cells as compared with 32D-MPLWT cells. (D) Phosphorylation of JAK2, TYK2, STAT3, STAT5, AKT, and ERK in response to seven-minute stimulation with TPO (50ng/mL). 32D MPLWT or 32 MPLW515L cells were deprived of cytokine overnight and then stimulated with TPO 50 ng/mL for seven minutes and then analyzed by Western blot for phosphorylation of JAK2, TYK2, STAT3, STAT5, AKT, and ERK, demonstrating increased phosphorylation of these proteins in response to TPO in 32D MPLW515L cells as compared with 32D MPLWT cells.
Figure Legend Snippet: MPLW515L-Expressing Cells Show Hyper-Responsiveness to TPO (A) 32D MPLWT cells and 32D MPLW515L cells were cultured in triplicate at an initial concentration of 1 × 10 5 cells/mL, in RPMI/10% FBS containing TPO 2 ng/mL, 1 ng/mL, 0.1 ng/mL, 0.01 ng/mL, 0.001 ng/mL, or in the absence of TPO for four days, and then cell numbers were assessed. Error bars denote the standard deviation for each sample measured in triplicate. (B) UT7 MPLWT cells and UT7 MPLW515L cells were cultured in triplicate, at an initial concentration of 1 × 10 5 cells/mL, in IMDM/10% FBS containing TPO 5 ng/mL, 1 ng/mL, 0.1 ng/mL, 0.01 ng/mL, 0.001ng/mL, or in the absence of TPO for six days, and then cell numbers were assessed. Error bars denote the standard deviation for each sample measured in triplicate. (C) 32D MPLWT or 32D MPLW515L cells were deprived of cytokine overnight and then stimulated with TPO 5 ng/mL, 1 ng/mL, 0.1 ng/mL, 0.01 ng/mL, or without TPO for seven minutes and then analyzed by Western blot for phosphorylation of JAK2 and STAT3, demonstrating increased JAK2/STAT3 phosphorylation in response to TPO in 32D MPLW515L cells as compared with 32D-MPLWT cells. (D) Phosphorylation of JAK2, TYK2, STAT3, STAT5, AKT, and ERK in response to seven-minute stimulation with TPO (50ng/mL). 32D MPLWT or 32 MPLW515L cells were deprived of cytokine overnight and then stimulated with TPO 50 ng/mL for seven minutes and then analyzed by Western blot for phosphorylation of JAK2, TYK2, STAT3, STAT5, AKT, and ERK, demonstrating increased phosphorylation of these proteins in response to TPO in 32D MPLW515L cells as compared with 32D MPLWT cells.

Techniques Used: Expressing, Cell Culture, Concentration Assay, Standard Deviation, Western Blot

MPLW515L Mutation Is Found in JAK2V617F-Negative MF and Causes Cytokine-Independent Growth in 32D and UT7 Cells, and Constitutively Activates the JAK-STAT Signaling Pathway (A) Forward (middle trace) and reverse (lower trace) sequence traces demonstrating a heterozygous guanine to thymine substitution (arrows) present in granulocyte DNA from a patient with MF. The mutation is not present in buccal DNA from the same patient (upper trace). (B) DNA sequence and protein translation for both the wild-type and mutant MPL alleles. The mutation results in a tryptophan-to-leucine substitution at codon 515. (C) Upper: 32D cells transduced with MPLW515L exhibit cytokine-independent growth compared with MPLWT (left). Cell lines grown in the presence of IL3 show equal rates of growth (right). Error bars denote the standard deviation for each sample measured in triplicate. Lower: UT7 cells transformed with MPLW515L exhibit cytokine-independent growth compared with MPLWT (left). Cell lines grown in the presence of TPO (5 ng/mL) show equal rates of growth (right). Error bars denote the standard deviation for each sample measured in triplicate. (D) 32D cells, 32D MPLWT cells, and 32D MPLW515L cells were deprived of cytokines and then analyzed by Western blots, demonstrating phosphorylation of JAK2, STAT5, STAT3, AKT, and ERK in MPLW515L compared with MPLWT.
Figure Legend Snippet: MPLW515L Mutation Is Found in JAK2V617F-Negative MF and Causes Cytokine-Independent Growth in 32D and UT7 Cells, and Constitutively Activates the JAK-STAT Signaling Pathway (A) Forward (middle trace) and reverse (lower trace) sequence traces demonstrating a heterozygous guanine to thymine substitution (arrows) present in granulocyte DNA from a patient with MF. The mutation is not present in buccal DNA from the same patient (upper trace). (B) DNA sequence and protein translation for both the wild-type and mutant MPL alleles. The mutation results in a tryptophan-to-leucine substitution at codon 515. (C) Upper: 32D cells transduced with MPLW515L exhibit cytokine-independent growth compared with MPLWT (left). Cell lines grown in the presence of IL3 show equal rates of growth (right). Error bars denote the standard deviation for each sample measured in triplicate. Lower: UT7 cells transformed with MPLW515L exhibit cytokine-independent growth compared with MPLWT (left). Cell lines grown in the presence of TPO (5 ng/mL) show equal rates of growth (right). Error bars denote the standard deviation for each sample measured in triplicate. (D) 32D cells, 32D MPLWT cells, and 32D MPLW515L cells were deprived of cytokines and then analyzed by Western blots, demonstrating phosphorylation of JAK2, STAT5, STAT3, AKT, and ERK in MPLW515L compared with MPLWT.

Techniques Used: Mutagenesis, Sequencing, Transduction, Standard Deviation, Transformation Assay, Western Blot

MPLW515L Increases the Number of Megakaryocyte and Myeloid Colonies in Spleen, without Affecting Megakaryocyte Ploidy, and Causes Cytokine-Independent Myeloid Colony Growth (A) Megakaryocyte colony–forming assay in the presence of TPO, IL3, IL11, and IL6 demonstrates similar numbers of megakaryocyte colonies obtained from MPLW515L-expressing bone marrow and an increase in the number of megakaryocyte colonies from spleen cells compared with MPLWT. (B) Acetylcholinesterase staining of megakaryocyte colonies derived from bone marrow demonstrates much larger colony size in MPLW515L as compared with MPLWT megakaryocyte colonies. (C) Megakaryocyte ploidy analysis shows the same distribution for MPLW515L-expressing cells and MPLWT-expressing cells. (D and E) Total myeloid colony formation from bone marrow cells (D) and spleen cells (E) demonstrates cytokine-independent colony formation in MPLW515L bone marrow and spleen. There is no difference in colony distribution between MPLWT- and MPLW515L-expressing cells. Colony counts reflect only positively identifiable colonies, with thorough megakaryocyte colony analysis done in MegaCult assay ( Figure 6 A), and thus are excluded from methylcellulose colony analysis. Colony numbers represent a total of three representative mice per group, in duplicate.
Figure Legend Snippet: MPLW515L Increases the Number of Megakaryocyte and Myeloid Colonies in Spleen, without Affecting Megakaryocyte Ploidy, and Causes Cytokine-Independent Myeloid Colony Growth (A) Megakaryocyte colony–forming assay in the presence of TPO, IL3, IL11, and IL6 demonstrates similar numbers of megakaryocyte colonies obtained from MPLW515L-expressing bone marrow and an increase in the number of megakaryocyte colonies from spleen cells compared with MPLWT. (B) Acetylcholinesterase staining of megakaryocyte colonies derived from bone marrow demonstrates much larger colony size in MPLW515L as compared with MPLWT megakaryocyte colonies. (C) Megakaryocyte ploidy analysis shows the same distribution for MPLW515L-expressing cells and MPLWT-expressing cells. (D and E) Total myeloid colony formation from bone marrow cells (D) and spleen cells (E) demonstrates cytokine-independent colony formation in MPLW515L bone marrow and spleen. There is no difference in colony distribution between MPLWT- and MPLW515L-expressing cells. Colony counts reflect only positively identifiable colonies, with thorough megakaryocyte colony analysis done in MegaCult assay ( Figure 6 A), and thus are excluded from methylcellulose colony analysis. Colony numbers represent a total of three representative mice per group, in duplicate.

Techniques Used: Expressing, Staining, Derivative Assay, Megacult Assay, Mouse Assay

32D MPLW515L Cells Are Sensitive to JAK Inhibitor I (A) Dose-dependent inhibition of growth of 32D MPLW515L and 32D MPLWT cells but not 32D FIP1L1-PDGFRA cells with increasing doses of JAK Inhibitor I. (B) Reduction in STAT3 phosphorylation in 32D MPLW515L cells but not 32D FIP1L1-PDGFRA cells with increasing doses of JAK Inhibitor I. Cells were incubated with varying drug concentrations for four hours and then collected for Western blot analysis.
Figure Legend Snippet: 32D MPLW515L Cells Are Sensitive to JAK Inhibitor I (A) Dose-dependent inhibition of growth of 32D MPLW515L and 32D MPLWT cells but not 32D FIP1L1-PDGFRA cells with increasing doses of JAK Inhibitor I. (B) Reduction in STAT3 phosphorylation in 32D MPLW515L cells but not 32D FIP1L1-PDGFRA cells with increasing doses of JAK Inhibitor I. Cells were incubated with varying drug concentrations for four hours and then collected for Western blot analysis.

Techniques Used: Inhibition, Incubation, Western Blot

Bone Marrow Transplant with MPLW515L-Transduced Bone Marrow Causes a Rapid Myeloproliferative Disease (A) Kaplan-Meier survival plot of Balb/C mice transduced with MPLW515L ( n = 9) and MPLWT ( n = 6) showing death of all MPLW515L mice between 17 and 32 days post-BMT compared with MPLWT ( n = 6), which were sacrificed for endpoint analysis without evidence of disease. (B) Complete blood counts show leukocytosis and thrombocytosis in MPLW515L BMT model compared with MPLWT. There is no difference in hematocrits. Standard deviation is indicated. (C) Spleen weights of MPLW515L and MPLWT mice shows splenomegaly in MPLW515L mice but not in MPLWT mice with average spleen weight equal to 1,171 mg. (D) Liver weights of MPLW515L and MPLWT mice shows hepatomegaly in MPLW515L mice but not in MPLWT mice, with average liver weight equal to 2,390 mg (compared with 1,222 mg in MPLWT mice). (E) Bone marrow shows significantly increased bone marrow fibrosis by reticulin staining at 17 days post-BMT in MPLW515L mice, but not in MPLWT-expressing mice.
Figure Legend Snippet: Bone Marrow Transplant with MPLW515L-Transduced Bone Marrow Causes a Rapid Myeloproliferative Disease (A) Kaplan-Meier survival plot of Balb/C mice transduced with MPLW515L ( n = 9) and MPLWT ( n = 6) showing death of all MPLW515L mice between 17 and 32 days post-BMT compared with MPLWT ( n = 6), which were sacrificed for endpoint analysis without evidence of disease. (B) Complete blood counts show leukocytosis and thrombocytosis in MPLW515L BMT model compared with MPLWT. There is no difference in hematocrits. Standard deviation is indicated. (C) Spleen weights of MPLW515L and MPLWT mice shows splenomegaly in MPLW515L mice but not in MPLWT mice with average spleen weight equal to 1,171 mg. (D) Liver weights of MPLW515L and MPLWT mice shows hepatomegaly in MPLW515L mice but not in MPLWT mice, with average liver weight equal to 2,390 mg (compared with 1,222 mg in MPLWT mice). (E) Bone marrow shows significantly increased bone marrow fibrosis by reticulin staining at 17 days post-BMT in MPLW515L mice, but not in MPLWT-expressing mice.

Techniques Used: Mouse Assay, Transduction, Standard Deviation, Staining, Expressing

MPLW515L and MPLWT Bone Marrow Transplant Model Histopathology Histology of MPLW515L-transduced and MPLWT-transduced Balb/C mice showing images of peripheral blood (A and B) and histopathology in representative sections of bone marrow (C and D), spleen (E–H), and liver (I–L). Peripheral blood smear (B) (600×, Wright-Giemsa) of a representative MPLWT animal displays an unremarkable white blood cell and platelet count. In contrast, peripheral blood smear (A) (600×, Wright-Giemsa) of a representative MPLW515L mutant animal reveals marked thrombocytosis and leukocytosis comprising a predominant population of maturing myeloid cells as well as frequent nucleated erythroid forms. Bone marrow images from MPLWT animals display preserved marrow architecture with maturing trilineage hematopoiesis (D) (600×, hematoxylin and eosin [H E]). Comparatively, bone marrow sections from MPLW515L mutant animals demonstrate marrow elements comprising a prominent population of maturing myeloid cells with increased numbers of megakaryocytes including atypical and dysplastic forms occurring in frequent clusters (C) (600×, H E) and showing emperipolesis of neutrophils in megakaryocyte cytoplasm. Spleen sections from MPLW515L mice display complete effacement of normal splenic architecture (E) (40×, H E) with a marked expansion of red pulp that is composed of an admixture of maturing myeloid and erythroid elements and numerous numbers of atypical megakaryocytes (G) (600×, H E) compared with MPLWT spleens (F and H) (40× and 600×, H E), which display a relative preservation of normal spleen architecture and the presence of only maturing erythroid forms in the red pulp. Liver images from MPLW515L mice illustrate evidence of extensive extramedullary hematopoiesis in a perivascular and sinusoidal distribution (I) (100×, H E) composed predominantly of a population of maturing erythroid elements with frequent large atypical megakaryocytes and occasional admixed myeloid forms (K) (600×, H E). In comparison, only small, focal areas of nucleated erythroid cells were observed in livers from MPLWT animals (J and L) (100× and 600×, H E).
Figure Legend Snippet: MPLW515L and MPLWT Bone Marrow Transplant Model Histopathology Histology of MPLW515L-transduced and MPLWT-transduced Balb/C mice showing images of peripheral blood (A and B) and histopathology in representative sections of bone marrow (C and D), spleen (E–H), and liver (I–L). Peripheral blood smear (B) (600×, Wright-Giemsa) of a representative MPLWT animal displays an unremarkable white blood cell and platelet count. In contrast, peripheral blood smear (A) (600×, Wright-Giemsa) of a representative MPLW515L mutant animal reveals marked thrombocytosis and leukocytosis comprising a predominant population of maturing myeloid cells as well as frequent nucleated erythroid forms. Bone marrow images from MPLWT animals display preserved marrow architecture with maturing trilineage hematopoiesis (D) (600×, hematoxylin and eosin [H E]). Comparatively, bone marrow sections from MPLW515L mutant animals demonstrate marrow elements comprising a prominent population of maturing myeloid cells with increased numbers of megakaryocytes including atypical and dysplastic forms occurring in frequent clusters (C) (600×, H E) and showing emperipolesis of neutrophils in megakaryocyte cytoplasm. Spleen sections from MPLW515L mice display complete effacement of normal splenic architecture (E) (40×, H E) with a marked expansion of red pulp that is composed of an admixture of maturing myeloid and erythroid elements and numerous numbers of atypical megakaryocytes (G) (600×, H E) compared with MPLWT spleens (F and H) (40× and 600×, H E), which display a relative preservation of normal spleen architecture and the presence of only maturing erythroid forms in the red pulp. Liver images from MPLW515L mice illustrate evidence of extensive extramedullary hematopoiesis in a perivascular and sinusoidal distribution (I) (100×, H E) composed predominantly of a population of maturing erythroid elements with frequent large atypical megakaryocytes and occasional admixed myeloid forms (K) (600×, H E). In comparison, only small, focal areas of nucleated erythroid cells were observed in livers from MPLWT animals (J and L) (100× and 600×, H E).

Techniques Used: Histopathology, Mouse Assay, Mutagenesis, Preserving

20) Product Images from "Genomic Responses from the Estrogen-responsive Element-dependent Signaling Pathway Mediated by Estrogen Receptor ? Are Required to Elicit Cellular Alterations *Genomic Responses from the Estrogen-responsive Element-dependent Signaling Pathway Mediated by Estrogen Receptor ? Are Required to Elicit Cellular Alterations * S⃞"

Article Title: Genomic Responses from the Estrogen-responsive Element-dependent Signaling Pathway Mediated by Estrogen Receptor ? Are Required to Elicit Cellular Alterations *Genomic Responses from the Estrogen-responsive Element-dependent Signaling Pathway Mediated by Estrogen Receptor ? Are Required to Elicit Cellular Alterations * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M900365200

Functional ER synthesis in infected MDA-MB-231 cells. A, cells were infected with the parent recombinant adenovirus ( Ad5 ) at m.o.i. 150, a recombinant adenovirus bearing cDNA for ERα (α) at 50 m.o.i., ERα 203/4/11 (α
Figure Legend Snippet: Functional ER synthesis in infected MDA-MB-231 cells. A, cells were infected with the parent recombinant adenovirus ( Ad5 ) at m.o.i. 150, a recombinant adenovirus bearing cDNA for ERα (α) at 50 m.o.i., ERα 203/4/11 (α

Techniques Used: Functional Assay, Infection, Multiple Displacement Amplification, Recombinant

Effects of ER α proteins on cell cycle distribution and the proliferation of infected MDA-MB-231 cells. A, to examine the effects of ERs on cell cycle distribution, MDA-MB-231 cells were infected with recombinant adenoviruses in the absence
Figure Legend Snippet: Effects of ER α proteins on cell cycle distribution and the proliferation of infected MDA-MB-231 cells. A, to examine the effects of ERs on cell cycle distribution, MDA-MB-231 cells were infected with recombinant adenoviruses in the absence

Techniques Used: Infection, Multiple Displacement Amplification, Recombinant

Effects of ERs on endogenous gene expression. A, summary of E2-ER-responsive genes identified with microarrays. MDA-MB-231 cells were infected with recombinant adenoviruses in the absence of E2 for 48 h. Cells were then treated with 10 -9 m E2 for 6
Figure Legend Snippet: Effects of ERs on endogenous gene expression. A, summary of E2-ER-responsive genes identified with microarrays. MDA-MB-231 cells were infected with recombinant adenoviruses in the absence of E2 for 48 h. Cells were then treated with 10 -9 m E2 for 6

Techniques Used: Expressing, Multiple Displacement Amplification, Infection, Recombinant

Effects of ER α and ER α EBD on proliferation and cycle distribution of U-2OS cells. A, synthesis of ERs. Cells were infected for 48 h with the parent recombinant adenovirus ( Ad5 ) at m.o.i. 50, a recombinant adenovirus bearing cDNA for
Figure Legend Snippet: Effects of ER α and ER α EBD on proliferation and cycle distribution of U-2OS cells. A, synthesis of ERs. Cells were infected for 48 h with the parent recombinant adenovirus ( Ad5 ) at m.o.i. 50, a recombinant adenovirus bearing cDNA for

Techniques Used: Infection, Recombinant

21) Product Images from "Crystal and Solution Studies Reveal That the Transcriptional Regulator AcnR of Corynebacterium glutamicum Is Regulated by Citrate-Mg2+"

Article Title: Crystal and Solution Studies Reveal That the Transcriptional Regulator AcnR of Corynebacterium glutamicum Is Regulated by Citrate-Mg2+

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.462440

Growth of C. glutamicum Δ acnR harboring different AcnR variants on glucose ( A ) and citrate ( B ) . After precultivation in brain-heart infusion broth and CGXII glucose medium, cells of C. glutamicum Δ acnR carrying pEKEx2 plasmids encoding
Figure Legend Snippet: Growth of C. glutamicum Δ acnR harboring different AcnR variants on glucose ( A ) and citrate ( B ) . After precultivation in brain-heart infusion broth and CGXII glucose medium, cells of C. glutamicum Δ acnR carrying pEKEx2 plasmids encoding

Techniques Used:

22) Product Images from "Arbovirus high fidelity variant loses fitness in mosquitoes and mice"

Article Title: Arbovirus high fidelity variant loses fitness in mosquitoes and mice

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1111650108

Competition assays comparing relative fitness of CHIKV WT and C483Y. ( A ) Direct assays: Viruses were mixed at a 1:1 ratio and inoculated in triplicate into BHK or C6/36 at 0.1 pfu/cell for three passages, at which point nsP4 483 was sequenced. The abundance
Figure Legend Snippet: Competition assays comparing relative fitness of CHIKV WT and C483Y. ( A ) Direct assays: Viruses were mixed at a 1:1 ratio and inoculated in triplicate into BHK or C6/36 at 0.1 pfu/cell for three passages, at which point nsP4 483 was sequenced. The abundance

Techniques Used:

23) Product Images from "Role of Acetyl-Phosphate in Activation of the Rrp2-RpoN-RpoS Pathway in Borrelia burgdorferi"

Article Title: Role of Acetyl-Phosphate in Activation of the Rrp2-RpoN-RpoS Pathway in Borrelia burgdorferi

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001104

Influence of overexpression of wild-type or mutated version of the Rrp2 N-terminal receiver domain on Rrp2 activation. ( A ) Schematic diagram of predicted Rrp2 domain structure and various versions of overexpressed N-terminal receiver domains. D52 is the putative phosphorylation site. ( B ) Immunoblot of wild-type strain (lane 1), the strain carrying the shuttle vector only (lane 2), the strain with overexpression of Rrp2-N (lane 3), the strain with overexpression of Rrp2-N(D52A) (lane 4), and the strain with overexpression of Rrp2-N(D52E) (lane 5). Cultures were grown to late logarithmic phase at 35°C. Pooled antibodies/antisera against Rrp2, FlaB, and OspC were used. Bands corresponding to each protein were labeled on the right. ( C ) qRT-PCR analysis of ospC expression in various strains shown in ( B ). Levels of ospC transcript were normalized per 1000 copies of flaB in each sample.
Figure Legend Snippet: Influence of overexpression of wild-type or mutated version of the Rrp2 N-terminal receiver domain on Rrp2 activation. ( A ) Schematic diagram of predicted Rrp2 domain structure and various versions of overexpressed N-terminal receiver domains. D52 is the putative phosphorylation site. ( B ) Immunoblot of wild-type strain (lane 1), the strain carrying the shuttle vector only (lane 2), the strain with overexpression of Rrp2-N (lane 3), the strain with overexpression of Rrp2-N(D52A) (lane 4), and the strain with overexpression of Rrp2-N(D52E) (lane 5). Cultures were grown to late logarithmic phase at 35°C. Pooled antibodies/antisera against Rrp2, FlaB, and OspC were used. Bands corresponding to each protein were labeled on the right. ( C ) qRT-PCR analysis of ospC expression in various strains shown in ( B ). Levels of ospC transcript were normalized per 1000 copies of flaB in each sample.

Techniques Used: Over Expression, Activation Assay, Plasmid Preparation, Labeling, Quantitative RT-PCR, Expressing

Acetyl∼P plays an important role in Rrp2 activation under in vitro cultivation conditions. ( A ) Diagram of the ACK-PTA pathway in B. burgdorferi . ack ( bb0622 ) encodes acetate kinase (Ack), which converts acetate to the intermediate acetyl∼P, while pta (bb0589) encodes phosphate acetyltransferase (Pta), which synthesizes acetyl-CoA from acetyl∼P and CoASH [17] . In B. burgdorferi , the Ack-Pta pathway appears to be the sole pathway for biosynthesis of acetyl-CoA, a molecule required for cell membrane biosynthesis (see Results and Discussion for details). ( B ) Acetate induces activation of the Rrp2-RpoN-RpoS pathway. Wild-type B. burgdorferi strain B31-A3 was cultivated in the BSK-H medium supplemented with 0–90 mM NaOAc with a final media pH value of 7.0. Cells were harvested at the early-logarithmic phase (5×10 6 spirochetes/ml). Cell lysates were subjected to SDS-PAGE (top panel) or immunoblot (bottom panels) analysis. The bands corresponding to OspC, RpoS and FlaB were labeled on the right. ( C ) Overexpression of Pta reduces acetate-induced Rrp2 activation. Wild-type B. burgdorferi strain B31 13A (-) or the strain carrying flaBp-pta (+) were cultivated in the BSK-H medium supplemented with 15 mM NaOAc at pH 7. Cells were harvested at the cell density of 5×10 6 and then subjected to immunoblot analyses with a mixture of antibodies against RpoS, OspC, or FlaB (internal control). The bands corresponding to each protein are indicated on the right. ( D ) Overexpression of Pta reduces temperature and cell density-induced activation of the Rrp2-RpoN-RpoS pathway. Wild-type B. burgdorferi strain B31 13A (-) or the strain carrying flaBp-pta (+) were cultivated either at 23 or 35°C in the standard BSK-H medium. Cells were harvested at the late-logarithmic growth phase (5×10 7 spirochetes/ml) and then subjected to immunoblot analyses. ( E ) In vitro phosphorylation of recombinant Rrp2 by acetyl∼P. Different quantities of purified recombinant Rrp2 or various versions of Rrp2-N were incubated with [ 32 P]acetyl phosphate and the reactions were terminated at 15 or 30 min. The reaction mixtures were separated by SDS-PAGE followed by exposure on Kodak X-ray film.
Figure Legend Snippet: Acetyl∼P plays an important role in Rrp2 activation under in vitro cultivation conditions. ( A ) Diagram of the ACK-PTA pathway in B. burgdorferi . ack ( bb0622 ) encodes acetate kinase (Ack), which converts acetate to the intermediate acetyl∼P, while pta (bb0589) encodes phosphate acetyltransferase (Pta), which synthesizes acetyl-CoA from acetyl∼P and CoASH [17] . In B. burgdorferi , the Ack-Pta pathway appears to be the sole pathway for biosynthesis of acetyl-CoA, a molecule required for cell membrane biosynthesis (see Results and Discussion for details). ( B ) Acetate induces activation of the Rrp2-RpoN-RpoS pathway. Wild-type B. burgdorferi strain B31-A3 was cultivated in the BSK-H medium supplemented with 0–90 mM NaOAc with a final media pH value of 7.0. Cells were harvested at the early-logarithmic phase (5×10 6 spirochetes/ml). Cell lysates were subjected to SDS-PAGE (top panel) or immunoblot (bottom panels) analysis. The bands corresponding to OspC, RpoS and FlaB were labeled on the right. ( C ) Overexpression of Pta reduces acetate-induced Rrp2 activation. Wild-type B. burgdorferi strain B31 13A (-) or the strain carrying flaBp-pta (+) were cultivated in the BSK-H medium supplemented with 15 mM NaOAc at pH 7. Cells were harvested at the cell density of 5×10 6 and then subjected to immunoblot analyses with a mixture of antibodies against RpoS, OspC, or FlaB (internal control). The bands corresponding to each protein are indicated on the right. ( D ) Overexpression of Pta reduces temperature and cell density-induced activation of the Rrp2-RpoN-RpoS pathway. Wild-type B. burgdorferi strain B31 13A (-) or the strain carrying flaBp-pta (+) were cultivated either at 23 or 35°C in the standard BSK-H medium. Cells were harvested at the late-logarithmic growth phase (5×10 7 spirochetes/ml) and then subjected to immunoblot analyses. ( E ) In vitro phosphorylation of recombinant Rrp2 by acetyl∼P. Different quantities of purified recombinant Rrp2 or various versions of Rrp2-N were incubated with [ 32 P]acetyl phosphate and the reactions were terminated at 15 or 30 min. The reaction mixtures were separated by SDS-PAGE followed by exposure on Kodak X-ray film.

Techniques Used: Activation Assay, In Vitro, SDS Page, Labeling, Over Expression, Recombinant, Purification, Incubation

24) Product Images from "Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in "Streptomyces carzinostaticus""

Article Title: Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in "Streptomyces carzinostaticus"

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.48.9.3468-3476.2004

Structures of “ S. carzinostaticus ” products, neocarzinostatin chromophore and NCZs.
Figure Legend Snippet: Structures of “ S. carzinostaticus ” products, neocarzinostatin chromophore and NCZs.

Techniques Used:

25) Product Images from "Effects of ranolazine on wild-type and mutant hNav1.7 channels and on DRG neuron excitability"

Article Title: Effects of ranolazine on wild-type and mutant hNav1.7 channels and on DRG neuron excitability

Journal: Molecular Pain

doi: 10.1186/1744-8069-6-35

Frequency-dependence of use-dependent block of Na v 1 .7 currents by ranolazine. Trains of twenty 30 msec duration pulses to -10 mV from a holding potential of -120 mV were applied at five different frequencies and were performed both before and after exposure to 10 μM ranolazine. Example traces from a HEK 293 cell expressing WT channels are shown in panels A and B. The peak for each pulse is normalized to the peak of the first pulse, and the values are plotted in panel C. The use-dependent block, defined as the ratio of the peak from the 20 th pulse normalized to the peak of the first pulse, was determined for each cell and the averages are plotted with the basal responses shown in grey and the ranolazine responses shown in solid bars. In each panel the two datasets are shown to compare the response to ranolazine in cells expressing WT (panel D, n = 5), the IEM mutant L858H (panel E, n = 4), or the PEPD mutant V1298F (panel F, n = 7), channels.
Figure Legend Snippet: Frequency-dependence of use-dependent block of Na v 1 .7 currents by ranolazine. Trains of twenty 30 msec duration pulses to -10 mV from a holding potential of -120 mV were applied at five different frequencies and were performed both before and after exposure to 10 μM ranolazine. Example traces from a HEK 293 cell expressing WT channels are shown in panels A and B. The peak for each pulse is normalized to the peak of the first pulse, and the values are plotted in panel C. The use-dependent block, defined as the ratio of the peak from the 20 th pulse normalized to the peak of the first pulse, was determined for each cell and the averages are plotted with the basal responses shown in grey and the ranolazine responses shown in solid bars. In each panel the two datasets are shown to compare the response to ranolazine in cells expressing WT (panel D, n = 5), the IEM mutant L858H (panel E, n = 4), or the PEPD mutant V1298F (panel F, n = 7), channels.

Techniques Used: Blocking Assay, Expressing, Mutagenesis

Effect of ranolazine on the excitability of DRG neurons expressing WT, L858H or V1298F channels . Action potentials were recorded in current-clamp mode from DRG neurons transfected with WT (panel A), the IEM mutant L858H (panel B), or the PEPD mutant V1298F (panel C), channels as described in the Methods section. The number of action potentials elicited during current injections of 1-sec duration ranging from 50 pA to 1000 pA in 50 pA increments are counted both before and then again after exposure to 10 μM ranolazine. In this figure, cells whose response did not exceed 5 spikes were removed to compare just the high firing cells. The average number of action potentials elicited at each current injection level are plotted before (grey) and after 10 μM ranolazine (solid) exposure. In each panel the two datasets are shown to compare the response to ranolazine in cells expressing WT (panel A, n = 10), the IEM mutant L858H (panel B, n = 4), or the PEPD mutant V1298F (panel C, n = 5), channels.
Figure Legend Snippet: Effect of ranolazine on the excitability of DRG neurons expressing WT, L858H or V1298F channels . Action potentials were recorded in current-clamp mode from DRG neurons transfected with WT (panel A), the IEM mutant L858H (panel B), or the PEPD mutant V1298F (panel C), channels as described in the Methods section. The number of action potentials elicited during current injections of 1-sec duration ranging from 50 pA to 1000 pA in 50 pA increments are counted both before and then again after exposure to 10 μM ranolazine. In this figure, cells whose response did not exceed 5 spikes were removed to compare just the high firing cells. The average number of action potentials elicited at each current injection level are plotted before (grey) and after 10 μM ranolazine (solid) exposure. In each panel the two datasets are shown to compare the response to ranolazine in cells expressing WT (panel A, n = 10), the IEM mutant L858H (panel B, n = 4), or the PEPD mutant V1298F (panel C, n = 5), channels.

Techniques Used: Expressing, Transfection, Mutagenesis, Size-exclusion Chromatography, Injection

Voltage-dependence of ranolazine block: Example traces . The voltage-dependence of ranolazine block was determined by utilizing a ten-second conditioning pulse protocol as described in Methods. After the protocol was performed once (black traces), the cells were exposed to a single concentration of ranolazine and then the protocol was repeated (red traces). These data traces were obtained from an HEK + hNav1.7r-L858H expressing cell treated with 10 μM Ranolazine. The fraction of ranolazine block was determined at each conditioning potential by dividing the peak current in the presence of ranolazine by the baseline peak current.
Figure Legend Snippet: Voltage-dependence of ranolazine block: Example traces . The voltage-dependence of ranolazine block was determined by utilizing a ten-second conditioning pulse protocol as described in Methods. After the protocol was performed once (black traces), the cells were exposed to a single concentration of ranolazine and then the protocol was repeated (red traces). These data traces were obtained from an HEK + hNav1.7r-L858H expressing cell treated with 10 μM Ranolazine. The fraction of ranolazine block was determined at each conditioning potential by dividing the peak current in the presence of ranolazine by the baseline peak current.

Techniques Used: Blocking Assay, Concentration Assay, Expressing

Voltage-dependence of ranolazine block of hNav1 .7r-WT and L858H and V1298F mutants: Dose-response curve fits. The extent of ranolazine block for each cell was determined for conditioning potentials ranging from the holding potential of -120 mV (resting block) to Vcond of -40 mV (inactivated block). Each cell was exposed to only one concentration of ranolazine. The dose-response curve for each conditioning potential (see legend symbols) was obtained by fitting a single-site binding curve to the average current block (3-9 cells/point).
Figure Legend Snippet: Voltage-dependence of ranolazine block of hNav1 .7r-WT and L858H and V1298F mutants: Dose-response curve fits. The extent of ranolazine block for each cell was determined for conditioning potentials ranging from the holding potential of -120 mV (resting block) to Vcond of -40 mV (inactivated block). Each cell was exposed to only one concentration of ranolazine. The dose-response curve for each conditioning potential (see legend symbols) was obtained by fitting a single-site binding curve to the average current block (3-9 cells/point).

Techniques Used: Blocking Assay, Concentration Assay, Binding Assay

Ranolazine block of ramp currents . The response to slow gradual depolarization (ramp) stimulus protocol (-100 mV to +20 mV over 600 msec) from representative cells is normalized to the peak inward current for that cell as described in Methods. Separate cells were exposed to either 0.1% HCl vehicle control (grey line) or 10 μM ranolazine (bold line). In each panel the two traces are overlaid to compare the response to ranolazine. (A) HEK 293 cells expressing WT show a peak ramp response of 0.58 ± 0.15% and the voltage peaks at -43.0 ± 1.2 mV in the presence of 0.1% HCl vehicle control (n = 3). In the presence of 10 μM ranolazine (n = 4), the peak ramp response was 0.79 ± 0.22% and the voltage peaks at -48.0 ± 3.9 mV. (B) HEK 293 cells expressing, the IEM mutant L858H show a peak ramp response of 4.48 ± 1.13% and the voltage peaks at -55.8 ± 2.2 mV in the presence of 0.1% HCl vehicle control (n = 5). In the presence of 10 μM ranolazine (n = 4), the peak ramp response was 3.31 ± 0.60% and the voltage peaks at -54.1 ± 2.0 mV. (C) HEK 293 cells expressing the PEPD mutant V1298F show a peak ramp response of 1.05 ± 0.21% and the voltage peaks at -48.5 ± 2.2 mV in the presence of 0.1% HCl vehicle control (n = 8). In the presence of 10 μM ranolazine (n = 7), the peak ramp response was 0.84 ± 0.11% and the voltage peaks at -49.7 ± 2.7 mV.
Figure Legend Snippet: Ranolazine block of ramp currents . The response to slow gradual depolarization (ramp) stimulus protocol (-100 mV to +20 mV over 600 msec) from representative cells is normalized to the peak inward current for that cell as described in Methods. Separate cells were exposed to either 0.1% HCl vehicle control (grey line) or 10 μM ranolazine (bold line). In each panel the two traces are overlaid to compare the response to ranolazine. (A) HEK 293 cells expressing WT show a peak ramp response of 0.58 ± 0.15% and the voltage peaks at -43.0 ± 1.2 mV in the presence of 0.1% HCl vehicle control (n = 3). In the presence of 10 μM ranolazine (n = 4), the peak ramp response was 0.79 ± 0.22% and the voltage peaks at -48.0 ± 3.9 mV. (B) HEK 293 cells expressing, the IEM mutant L858H show a peak ramp response of 4.48 ± 1.13% and the voltage peaks at -55.8 ± 2.2 mV in the presence of 0.1% HCl vehicle control (n = 5). In the presence of 10 μM ranolazine (n = 4), the peak ramp response was 3.31 ± 0.60% and the voltage peaks at -54.1 ± 2.0 mV. (C) HEK 293 cells expressing the PEPD mutant V1298F show a peak ramp response of 1.05 ± 0.21% and the voltage peaks at -48.5 ± 2.2 mV in the presence of 0.1% HCl vehicle control (n = 8). In the presence of 10 μM ranolazine (n = 7), the peak ramp response was 0.84 ± 0.11% and the voltage peaks at -49.7 ± 2.7 mV.

Techniques Used: Blocking Assay, Expressing, Mutagenesis

Voltage-dependence of WT and the L858H and V1298F mutant channels . (A) Superimposed activation traces recorded from a representative HEK + hNav1.7r-WT expressing cell. (Average peak current is -4.2 ± 0.7 nA, n = 12) (B) Superimposed fast-inactivation traces recorded from the same cell as in (A). (C) Superimposed activation traces recorded from a representative HEK + hNav1.7r-L858H expressing cell. (Average peak current is -2.3 ± 0.3 nA, n = 12). (D) Superimposed fast-inactivation traces recorded from the same cell as in (C). (E) Superimposed activation traces recorded from a representative HEK + hNav1.7r-V1298F expressing cell (Average peak current is -1.9 ± 0.3 nA, n = 18). (F) Superimposed fast-inactivation traces from the same cell as in (E). Insets illustrate the voltage pulse protocols for activation and fast-inactivation. The bold bars indicate the portion of the data sweeps displayed in this figure. (G) Normalized conductance-voltage (G-V) curves are constructed from the averages of individual HEK 293 cells expressing WT (black squares, n = 12), the IEM mutation L858H (red circles, n = 12), or the PEPD mutation V1298F (blue triangles, n = 18). (H) Normalized fast-inactivation curves are constructed from the averages of the same cells as in panel G.
Figure Legend Snippet: Voltage-dependence of WT and the L858H and V1298F mutant channels . (A) Superimposed activation traces recorded from a representative HEK + hNav1.7r-WT expressing cell. (Average peak current is -4.2 ± 0.7 nA, n = 12) (B) Superimposed fast-inactivation traces recorded from the same cell as in (A). (C) Superimposed activation traces recorded from a representative HEK + hNav1.7r-L858H expressing cell. (Average peak current is -2.3 ± 0.3 nA, n = 12). (D) Superimposed fast-inactivation traces recorded from the same cell as in (C). (E) Superimposed activation traces recorded from a representative HEK + hNav1.7r-V1298F expressing cell (Average peak current is -1.9 ± 0.3 nA, n = 18). (F) Superimposed fast-inactivation traces from the same cell as in (E). Insets illustrate the voltage pulse protocols for activation and fast-inactivation. The bold bars indicate the portion of the data sweeps displayed in this figure. (G) Normalized conductance-voltage (G-V) curves are constructed from the averages of individual HEK 293 cells expressing WT (black squares, n = 12), the IEM mutation L858H (red circles, n = 12), or the PEPD mutation V1298F (blue triangles, n = 18). (H) Normalized fast-inactivation curves are constructed from the averages of the same cells as in panel G.

Techniques Used: Mutagenesis, Activation Assay, Expressing, Construct

26) Product Images from "NEIL1 Responds and Binds to Psoralen-induced DNA Interstrand Crosslinks *"

Article Title: NEIL1 Responds and Binds to Psoralen-induced DNA Interstrand Crosslinks *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.456087

Intracellular localization of NEIL1. A , HeLa cells were transfected with either pAcGFP1-N1 or the pAcGFP-NEIL1 plasmid, fixed and stained with DAPI. Cells were then visualized and imaged for GFP or DAPI. Merge and merge plus brightfield are shown. B ,
Figure Legend Snippet: Intracellular localization of NEIL1. A , HeLa cells were transfected with either pAcGFP1-N1 or the pAcGFP-NEIL1 plasmid, fixed and stained with DAPI. Cells were then visualized and imaged for GFP or DAPI. Merge and merge plus brightfield are shown. B ,

Techniques Used: Transfection, Plasmid Preparation, Staining

Recruitment and retention of NEIL1 at sites of microirradiation in live cells, with or without psoralen or angelicin treatment. HeLa cells were transfected with the pAcGFP-NEIL1 plasmid, and incubated where indicated with psoralen ( Pso ) or angelicin (
Figure Legend Snippet: Recruitment and retention of NEIL1 at sites of microirradiation in live cells, with or without psoralen or angelicin treatment. HeLa cells were transfected with the pAcGFP-NEIL1 plasmid, and incubated where indicated with psoralen ( Pso ) or angelicin (

Techniques Used: Transfection, Plasmid Preparation, Incubation

Effect of NAC or XPC on NEIL1 recruitment. A , NAC. Following transfection of HeLa cells with pAcGFP-NEIL1, cells were incubated with NAC for 30 min prior to targeted microirradiation at either 2.7% or 1.7%/Pso. Recruitment and dispersion of GFP-NEIL1
Figure Legend Snippet: Effect of NAC or XPC on NEIL1 recruitment. A , NAC. Following transfection of HeLa cells with pAcGFP-NEIL1, cells were incubated with NAC for 30 min prior to targeted microirradiation at either 2.7% or 1.7%/Pso. Recruitment and dispersion of GFP-NEIL1

Techniques Used: Transfection, Incubation

27) Product Images from "Identification of four novel phosphorylation sites in estrogen receptor ?: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2"

Article Title: Identification of four novel phosphorylation sites in estrogen receptor ?: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-10-36

Phosphorylation of endogenous ERα in ERα (+) cell lines at S282, S294, and S559 . ( A) 10 7 MCF-7 and MCF-7-(LCC2) breast cancer and Ishikawa endometrial adenocarcinoma cell lines were cultured in medium supplemented with 10% FBS. Cells were lysed and total ERα was immunoprecipitated with α-p-S282, α-p-S294, or α-p-S559 antibodies for 3 hours. Immunoprecipitates were analyzed by Western blot for total ERα ( B) S282 and S559 are phosphorylated following incubation of MCF-7 breast cancer cells with estradiol. MCF-7 breast cancer cells were cultured for 48 hours in phenol red free medium supplemented with 10% charcoal-stripped FBS. Cells were serum starved overnight prior to incubation with vehicle (veh) or 10 -8 M estradiol (E2). ERα was immunoprecipitated from lysates and Western blot analysis performed with α-pS282, α-pS294, or α-pS559, and with α-ERα. Substantial ligand-induced phosphorylation was detected at S282 and S559, with only modest ligand induced phosphorylation at S294.
Figure Legend Snippet: Phosphorylation of endogenous ERα in ERα (+) cell lines at S282, S294, and S559 . ( A) 10 7 MCF-7 and MCF-7-(LCC2) breast cancer and Ishikawa endometrial adenocarcinoma cell lines were cultured in medium supplemented with 10% FBS. Cells were lysed and total ERα was immunoprecipitated with α-p-S282, α-p-S294, or α-p-S559 antibodies for 3 hours. Immunoprecipitates were analyzed by Western blot for total ERα ( B) S282 and S559 are phosphorylated following incubation of MCF-7 breast cancer cells with estradiol. MCF-7 breast cancer cells were cultured for 48 hours in phenol red free medium supplemented with 10% charcoal-stripped FBS. Cells were serum starved overnight prior to incubation with vehicle (veh) or 10 -8 M estradiol (E2). ERα was immunoprecipitated from lysates and Western blot analysis performed with α-pS282, α-pS294, or α-pS559, and with α-ERα. Substantial ligand-induced phosphorylation was detected at S282 and S559, with only modest ligand induced phosphorylation at S294.

Techniques Used: Cell Culture, Immunoprecipitation, Western Blot, Incubation

Confirmation of the specificity of ER phospho-antibodies . (A) Serine to alanine mutations at ERα phosphorylation sites inhibit reactivity of phospho-specific antibodies. COS-1 cells cultured in DMEM growth supplemented with 10% FBS were transiently transfected with 500 ng of either wt ERα or serine to alanine substituted ERα expression plasmids (S47A, S282A, S294A, or S559A). 18 hours post-transfection, cells were lysed and subjected to Western immunoblot analysis utilizing custom polyclonal antibodies directed toward the individual phosphorylated ERα residues (S47, S282, S294, or S559) or monoclonal ERα antibody as indicated. α-p-S282, α-p-S294, or α-p-S559 antibodies did not recognize S282A, S294A, or S559A, respectively, indicating phospho-antibody specificity. Mutation of S47 failed to eliminate immunoreactivity of αp-S47. (B) In vitro λ phosphatase treatment of ERα inhibits immunoreactivity of ERα phospho-antibodies. Baculovirus expressed ERα was subjected to dephosphorylation by λ phosphatase for 30 minutes at 30°C and analyzed by Western immunoblot with antibodies against p-S282, p-S294, p-S559, and total ERα. Dephosphorylation inhibited immunoreactivity of, α-p-S282, α-p-S294, and α-p-S559 without impacting immunoreactivity of total ERα antibody.
Figure Legend Snippet: Confirmation of the specificity of ER phospho-antibodies . (A) Serine to alanine mutations at ERα phosphorylation sites inhibit reactivity of phospho-specific antibodies. COS-1 cells cultured in DMEM growth supplemented with 10% FBS were transiently transfected with 500 ng of either wt ERα or serine to alanine substituted ERα expression plasmids (S47A, S282A, S294A, or S559A). 18 hours post-transfection, cells were lysed and subjected to Western immunoblot analysis utilizing custom polyclonal antibodies directed toward the individual phosphorylated ERα residues (S47, S282, S294, or S559) or monoclonal ERα antibody as indicated. α-p-S282, α-p-S294, or α-p-S559 antibodies did not recognize S282A, S294A, or S559A, respectively, indicating phospho-antibody specificity. Mutation of S47 failed to eliminate immunoreactivity of αp-S47. (B) In vitro λ phosphatase treatment of ERα inhibits immunoreactivity of ERα phospho-antibodies. Baculovirus expressed ERα was subjected to dephosphorylation by λ phosphatase for 30 minutes at 30°C and analyzed by Western immunoblot with antibodies against p-S282, p-S294, p-S559, and total ERα. Dephosphorylation inhibited immunoreactivity of, α-p-S282, α-p-S294, and α-p-S559 without impacting immunoreactivity of total ERα antibody.

Techniques Used: Cell Culture, Transfection, Expressing, Western Blot, Mutagenesis, In Vitro, De-Phosphorylation Assay

Estrogen receptor α (ERα) phosphorylation sites . The schematic in Figure 1 depicts both previously identified and novel ERα phosphorylation sites with relative locations within the ERα functional domains. Serines 104, 106, 118, and 167 constitute phosphorylation sites within the ligand-independent activation function-1 (AF-1) domain of ERα. S236 is the first phosphorylation site within the DNA binding domain of ERα. Serine 305, threonine 311 and tyrosine 537 are phosphorylation sites identified within the ligand-dependent activation function-2 (AF-2) domain. Indicated in bold italicized type are newly characterized phosphorylation sites of ERα: S46/47, S282, S294 and S559. S46/47 constitutes an additional site of phosphorylation within the AF-1 domain. Serines 282 and 294 are located in the hinge domain of ERα proximal to the DNA binding domain. Of note, S559 is the first phosphorylation site identified in the extreme C-terminal F domain of ERα and other steroid receptors. S154, S212, S294, S554, and S559 have been recently identified or independently confirmed by mass spectrophotometry (11).
Figure Legend Snippet: Estrogen receptor α (ERα) phosphorylation sites . The schematic in Figure 1 depicts both previously identified and novel ERα phosphorylation sites with relative locations within the ERα functional domains. Serines 104, 106, 118, and 167 constitute phosphorylation sites within the ligand-independent activation function-1 (AF-1) domain of ERα. S236 is the first phosphorylation site within the DNA binding domain of ERα. Serine 305, threonine 311 and tyrosine 537 are phosphorylation sites identified within the ligand-dependent activation function-2 (AF-2) domain. Indicated in bold italicized type are newly characterized phosphorylation sites of ERα: S46/47, S282, S294 and S559. S46/47 constitutes an additional site of phosphorylation within the AF-1 domain. Serines 282 and 294 are located in the hinge domain of ERα proximal to the DNA binding domain. Of note, S559 is the first phosphorylation site identified in the extreme C-terminal F domain of ERα and other steroid receptors. S154, S212, S294, S554, and S559 have been recently identified or independently confirmed by mass spectrophotometry (11).

Techniques Used: Functional Assay, Activation Assay, Binding Assay, Spectrophotometry

28) Product Images from "Evidence for Horizontal Transfer of SsuDAT1I Restriction-Modification Genes to the Streptococcus suis Genome"

Article Title: Evidence for Horizontal Transfer of SsuDAT1I Restriction-Modification Genes to the Streptococcus suis Genome

Journal: Journal of Bacteriology

doi: 10.1128/JB.183.2.500-511.2001

(A) Methylation of plasmid DNAs containing the cloned methylation gene(s) of the Ssu DAT1I system. The plasmids recovered from E. coli SCS110 were digested with restriction endonucleases and analyzed by agarose gel electrophoresis. Lane 1, pSUMA1; lane 2, pSUMAB14; lane 3, pSUMB1. D, digested with Dpn I; M, digested with Mbo I; S, 1-kb ladder size standards (GIBCO/BRL). (B) DNA cleavage activities of crude extracts prepared from E. coli strains carrying the following plasmids using unmethylated pUC19 as the substrate: lane 1, pSURA1; lane 2, pSURA2; lane 3, pSURA3; lane 4, pSURA4; lane 5, pSURB1; lane 6, pSURB2; lane 7, pSURB3; lane 8, pSURB4; lane 9, pSURB5. M, pUC19 digested with Mbo I; S, 100-bp ladder size standards (GIBCO/BRL).
Figure Legend Snippet: (A) Methylation of plasmid DNAs containing the cloned methylation gene(s) of the Ssu DAT1I system. The plasmids recovered from E. coli SCS110 were digested with restriction endonucleases and analyzed by agarose gel electrophoresis. Lane 1, pSUMA1; lane 2, pSUMAB14; lane 3, pSUMB1. D, digested with Dpn I; M, digested with Mbo I; S, 1-kb ladder size standards (GIBCO/BRL). (B) DNA cleavage activities of crude extracts prepared from E. coli strains carrying the following plasmids using unmethylated pUC19 as the substrate: lane 1, pSURA1; lane 2, pSURA2; lane 3, pSURA3; lane 4, pSURA4; lane 5, pSURB1; lane 6, pSURB2; lane 7, pSURB3; lane 8, pSURB4; lane 9, pSURB5. M, pUC19 digested with Mbo I; S, 100-bp ladder size standards (GIBCO/BRL).

Techniques Used: Methylation, Plasmid Preparation, Clone Assay, Agarose Gel Electrophoresis

29) Product Images from "Identification of four novel phosphorylation sites in estrogen receptor ?: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2"

Article Title: Identification of four novel phosphorylation sites in estrogen receptor ?: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-10-36

Phosphorylation of endogenous ERα in ERα (+) cell lines at S282, S294, and S559 . ( A) 10 7 MCF-7 and MCF-7-(LCC2) breast cancer and Ishikawa endometrial adenocarcinoma cell lines were cultured in medium supplemented with 10% FBS. Cells were lysed and total ERα was immunoprecipitated with α-p-S282, α-p-S294, or α-p-S559 antibodies for 3 hours. Immunoprecipitates were analyzed by Western blot for total ERα ( B) S282 and S559 are phosphorylated following incubation of MCF-7 breast cancer cells with estradiol. MCF-7 breast cancer cells were cultured for 48 hours in phenol red free medium supplemented with 10% charcoal-stripped FBS. Cells were serum starved overnight prior to incubation with vehicle (veh) or 10 -8 M estradiol (E2). ERα was immunoprecipitated from lysates and Western blot analysis performed with α-pS282, α-pS294, or α-pS559, and with α-ERα. Substantial ligand-induced phosphorylation was detected at S282 and S559, with only modest ligand induced phosphorylation at S294.
Figure Legend Snippet: Phosphorylation of endogenous ERα in ERα (+) cell lines at S282, S294, and S559 . ( A) 10 7 MCF-7 and MCF-7-(LCC2) breast cancer and Ishikawa endometrial adenocarcinoma cell lines were cultured in medium supplemented with 10% FBS. Cells were lysed and total ERα was immunoprecipitated with α-p-S282, α-p-S294, or α-p-S559 antibodies for 3 hours. Immunoprecipitates were analyzed by Western blot for total ERα ( B) S282 and S559 are phosphorylated following incubation of MCF-7 breast cancer cells with estradiol. MCF-7 breast cancer cells were cultured for 48 hours in phenol red free medium supplemented with 10% charcoal-stripped FBS. Cells were serum starved overnight prior to incubation with vehicle (veh) or 10 -8 M estradiol (E2). ERα was immunoprecipitated from lysates and Western blot analysis performed with α-pS282, α-pS294, or α-pS559, and with α-ERα. Substantial ligand-induced phosphorylation was detected at S282 and S559, with only modest ligand induced phosphorylation at S294.

Techniques Used: Cell Culture, Immunoprecipitation, Western Blot, Incubation

Protein Kinase CK2 phosphorylates of S282 and S559 . (A) 400 ng baculovirus expressed ERα was incubated in CK2 kinase buffer supplemented with 10 mM ATP, in the presence or absence of 200 ng recombinant catalytic α subunit of CK2. Reactions were stopped with Laemmli buffer, subjected to Western blot analysis, and probed with α-pS282, α-pS559, α-pS118 or αER. These studies show that the CK2α catalytic subunit specifically phosphorylates ERα at S282 and S559. Western blot for phosphorylation of S118, a site that exhibits strong phosphorylation in baculovirus expressed ERα, is shown for comparison to demonstrate absence of nonspecific phosphorylation by CK2 on other ERα phosphorylation sites. (B) 10 6 MCF7 breast cancer cells were pretreated with DMAT (4 uM) for 90 minutes, followed by 30 minutes with estradiol (10 -8 M) or vehicle. Immunoprecipitation of S282 or S559 was performed using phosphoantibodies and Western blot for total ERα. DMAT inhibited phosphorylation at both sites, indicating that CK2 phosphorylates these sites in vivo .
Figure Legend Snippet: Protein Kinase CK2 phosphorylates of S282 and S559 . (A) 400 ng baculovirus expressed ERα was incubated in CK2 kinase buffer supplemented with 10 mM ATP, in the presence or absence of 200 ng recombinant catalytic α subunit of CK2. Reactions were stopped with Laemmli buffer, subjected to Western blot analysis, and probed with α-pS282, α-pS559, α-pS118 or αER. These studies show that the CK2α catalytic subunit specifically phosphorylates ERα at S282 and S559. Western blot for phosphorylation of S118, a site that exhibits strong phosphorylation in baculovirus expressed ERα, is shown for comparison to demonstrate absence of nonspecific phosphorylation by CK2 on other ERα phosphorylation sites. (B) 10 6 MCF7 breast cancer cells were pretreated with DMAT (4 uM) for 90 minutes, followed by 30 minutes with estradiol (10 -8 M) or vehicle. Immunoprecipitation of S282 or S559 was performed using phosphoantibodies and Western blot for total ERα. DMAT inhibited phosphorylation at both sites, indicating that CK2 phosphorylates these sites in vivo .

Techniques Used: Incubation, Recombinant, Western Blot, Immunoprecipitation, In Vivo

Mutation of serine residues to alanine eliminates specific phosphorylation of peptides . To confirm the identity of phosphorylated serine residues within peptides A, B, C, and D, serine to alanine mutations were introduced into wt ERα (S47A, S282A, S294A, or S559A). 12 plates of COS-1 cells (4 × 10 7 /plate) were transfected with 500 ng/plate of wt ERα, S47A, S282A, S294A, or S559A expression plasmids. 18 hours post-transfection, cells were phosphate-depleted, labeled with 4 mCi [ 32 P]H 3 PO 4 and incubated with 10 -8 M estradiol overnight. ERα was immunopurified and tryptic peptides were separated by HPLC using a C-18 reversed phase column. Fractions were collected and electrophoresed on a 40% alkaline polyacrylamide gel followed by autoradiography. (A) Peptide map of wt ERα displaying 4 novel phosphopeptides A-D. (B) S294A resulted in loss of peptide C. C) S559A resulted in loss of peptide B. (D) S47A resulted in a modest decrease in peptide D compared to wt ERα. E) S47A/S104A/S106A/S118A resulted in loss of peptide D. (F) Mutation of S282 to alanine reduces ERα protein following 24 h incubation with estradiol . 10 6 COS-1 monkey embryonic kidney cells which had been cultured in phenol-red free DMEM supplemented with 10% fetal bovine serum were transfected with 2.5 μg of wt ERα or S282A expression plasmid. 24 hours after transfection, cells were incubated with vehicle (veh) or estradiol (10 -8 M) for an additional 24 hours. Cell lysates were collected and ERα protein levels determined, using α-tubulin as a loading control.
Figure Legend Snippet: Mutation of serine residues to alanine eliminates specific phosphorylation of peptides . To confirm the identity of phosphorylated serine residues within peptides A, B, C, and D, serine to alanine mutations were introduced into wt ERα (S47A, S282A, S294A, or S559A). 12 plates of COS-1 cells (4 × 10 7 /plate) were transfected with 500 ng/plate of wt ERα, S47A, S282A, S294A, or S559A expression plasmids. 18 hours post-transfection, cells were phosphate-depleted, labeled with 4 mCi [ 32 P]H 3 PO 4 and incubated with 10 -8 M estradiol overnight. ERα was immunopurified and tryptic peptides were separated by HPLC using a C-18 reversed phase column. Fractions were collected and electrophoresed on a 40% alkaline polyacrylamide gel followed by autoradiography. (A) Peptide map of wt ERα displaying 4 novel phosphopeptides A-D. (B) S294A resulted in loss of peptide C. C) S559A resulted in loss of peptide B. (D) S47A resulted in a modest decrease in peptide D compared to wt ERα. E) S47A/S104A/S106A/S118A resulted in loss of peptide D. (F) Mutation of S282 to alanine reduces ERα protein following 24 h incubation with estradiol . 10 6 COS-1 monkey embryonic kidney cells which had been cultured in phenol-red free DMEM supplemented with 10% fetal bovine serum were transfected with 2.5 μg of wt ERα or S282A expression plasmid. 24 hours after transfection, cells were incubated with vehicle (veh) or estradiol (10 -8 M) for an additional 24 hours. Cell lysates were collected and ERα protein levels determined, using α-tubulin as a loading control.

Techniques Used: Mutagenesis, Transfection, Expressing, Labeling, Incubation, High Performance Liquid Chromatography, Autoradiography, Cell Culture, Plasmid Preparation

Confirmation of the specificity of ER phospho-antibodies . (A) Serine to alanine mutations at ERα phosphorylation sites inhibit reactivity of phospho-specific antibodies. COS-1 cells cultured in DMEM growth supplemented with 10% FBS were transiently transfected with 500 ng of either wt ERα or serine to alanine substituted ERα expression plasmids (S47A, S282A, S294A, or S559A). 18 hours post-transfection, cells were lysed and subjected to Western immunoblot analysis utilizing custom polyclonal antibodies directed toward the individual phosphorylated ERα residues (S47, S282, S294, or S559) or monoclonal ERα antibody as indicated. α-p-S282, α-p-S294, or α-p-S559 antibodies did not recognize S282A, S294A, or S559A, respectively, indicating phospho-antibody specificity. Mutation of S47 failed to eliminate immunoreactivity of αp-S47. (B) In vitro λ phosphatase treatment of ERα inhibits immunoreactivity of ERα phospho-antibodies. Baculovirus expressed ERα was subjected to dephosphorylation by λ phosphatase for 30 minutes at 30°C and analyzed by Western immunoblot with antibodies against p-S282, p-S294, p-S559, and total ERα. Dephosphorylation inhibited immunoreactivity of, α-p-S282, α-p-S294, and α-p-S559 without impacting immunoreactivity of total ERα antibody.
Figure Legend Snippet: Confirmation of the specificity of ER phospho-antibodies . (A) Serine to alanine mutations at ERα phosphorylation sites inhibit reactivity of phospho-specific antibodies. COS-1 cells cultured in DMEM growth supplemented with 10% FBS were transiently transfected with 500 ng of either wt ERα or serine to alanine substituted ERα expression plasmids (S47A, S282A, S294A, or S559A). 18 hours post-transfection, cells were lysed and subjected to Western immunoblot analysis utilizing custom polyclonal antibodies directed toward the individual phosphorylated ERα residues (S47, S282, S294, or S559) or monoclonal ERα antibody as indicated. α-p-S282, α-p-S294, or α-p-S559 antibodies did not recognize S282A, S294A, or S559A, respectively, indicating phospho-antibody specificity. Mutation of S47 failed to eliminate immunoreactivity of αp-S47. (B) In vitro λ phosphatase treatment of ERα inhibits immunoreactivity of ERα phospho-antibodies. Baculovirus expressed ERα was subjected to dephosphorylation by λ phosphatase for 30 minutes at 30°C and analyzed by Western immunoblot with antibodies against p-S282, p-S294, p-S559, and total ERα. Dephosphorylation inhibited immunoreactivity of, α-p-S282, α-p-S294, and α-p-S559 without impacting immunoreactivity of total ERα antibody.

Techniques Used: Cell Culture, Transfection, Expressing, Western Blot, Mutagenesis, In Vitro, De-Phosphorylation Assay

Estrogen receptor α (ERα) phosphorylation sites . The schematic in Figure 1 depicts both previously identified and novel ERα phosphorylation sites with relative locations within the ERα functional domains. Serines 104, 106, 118, and 167 constitute phosphorylation sites within the ligand-independent activation function-1 (AF-1) domain of ERα. S236 is the first phosphorylation site within the DNA binding domain of ERα. Serine 305, threonine 311 and tyrosine 537 are phosphorylation sites identified within the ligand-dependent activation function-2 (AF-2) domain. Indicated in bold italicized type are newly characterized phosphorylation sites of ERα: S46/47, S282, S294 and S559. S46/47 constitutes an additional site of phosphorylation within the AF-1 domain. Serines 282 and 294 are located in the hinge domain of ERα proximal to the DNA binding domain. Of note, S559 is the first phosphorylation site identified in the extreme C-terminal F domain of ERα and other steroid receptors. S154, S212, S294, S554, and S559 have been recently identified or independently confirmed by mass spectrophotometry (11).
Figure Legend Snippet: Estrogen receptor α (ERα) phosphorylation sites . The schematic in Figure 1 depicts both previously identified and novel ERα phosphorylation sites with relative locations within the ERα functional domains. Serines 104, 106, 118, and 167 constitute phosphorylation sites within the ligand-independent activation function-1 (AF-1) domain of ERα. S236 is the first phosphorylation site within the DNA binding domain of ERα. Serine 305, threonine 311 and tyrosine 537 are phosphorylation sites identified within the ligand-dependent activation function-2 (AF-2) domain. Indicated in bold italicized type are newly characterized phosphorylation sites of ERα: S46/47, S282, S294 and S559. S46/47 constitutes an additional site of phosphorylation within the AF-1 domain. Serines 282 and 294 are located in the hinge domain of ERα proximal to the DNA binding domain. Of note, S559 is the first phosphorylation site identified in the extreme C-terminal F domain of ERα and other steroid receptors. S154, S212, S294, S554, and S559 have been recently identified or independently confirmed by mass spectrophotometry (11).

Techniques Used: Functional Assay, Activation Assay, Binding Assay, Spectrophotometry

30) Product Images from "Identification of bdm-1, a gene involved in G protein ?-subunit function and ?-subunit accumulation"

Article Title: Identification of bdm-1, a gene involved in G protein ?-subunit function and ?-subunit accumulation

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Complementation mapping and disruption of bdm-1. ( A1 ) Restriction map of cosmid clone rescued from the chromosome of a bdmA complemented transformant. ( A2 ) Detailed organization of bdm-1 in the context of the 6.5-kb Kpn I bdmA complementing fragment. The bdm-1 promoter and terminator are indicated by P bdm-1 and T bdm-1 , respectively. The protein-coding regions are represented by boxes. Thick solid lines denote 5′ and 3′ noncoding region. Two introns are marked by vertical arrows. ( A3 ) DNA fragment used to disrupt bdm-1 . The Bam HI– Sma I fragment within the bdm-1 structural gene was replaced with a hygromycin-resistance gene ( hph ) cassette that contained the A. nidulans TrpC promoter and terminator, P trpC and T trpC ). A 7.3-kb fragment released by digestion with Kpn I was used directly for transformation of C. parasitica strain EP155 spheroplasts. ( B ) Southern blot analysis of Pst I-digested genomic DNA (10 μg) isolated from wild-type C. parasitica strain EP155, Gβ disruptant Δ cpgb-1 , bdmA , and bdm-1 disruptant Δ bdm-1 by using probe 1 (specific for the hph gene) shown in A . Sizes of expected hybridization bands are indicated at the left. ( C ) Southern blot analysis of the same strains listed in B by using probe 2 shown in A ), which is specific for bdm-1. ( D ) Northern blot analysis of RNA (15 μg) isolated from the same strains shown in C and the cpg-1 disruptant Δ cpg-1 by using the entire coding region of bdm-1 .
Figure Legend Snippet: Complementation mapping and disruption of bdm-1. ( A1 ) Restriction map of cosmid clone rescued from the chromosome of a bdmA complemented transformant. ( A2 ) Detailed organization of bdm-1 in the context of the 6.5-kb Kpn I bdmA complementing fragment. The bdm-1 promoter and terminator are indicated by P bdm-1 and T bdm-1 , respectively. The protein-coding regions are represented by boxes. Thick solid lines denote 5′ and 3′ noncoding region. Two introns are marked by vertical arrows. ( A3 ) DNA fragment used to disrupt bdm-1 . The Bam HI– Sma I fragment within the bdm-1 structural gene was replaced with a hygromycin-resistance gene ( hph ) cassette that contained the A. nidulans TrpC promoter and terminator, P trpC and T trpC ). A 7.3-kb fragment released by digestion with Kpn I was used directly for transformation of C. parasitica strain EP155 spheroplasts. ( B ) Southern blot analysis of Pst I-digested genomic DNA (10 μg) isolated from wild-type C. parasitica strain EP155, Gβ disruptant Δ cpgb-1 , bdmA , and bdm-1 disruptant Δ bdm-1 by using probe 1 (specific for the hph gene) shown in A . Sizes of expected hybridization bands are indicated at the left. ( C ) Southern blot analysis of the same strains listed in B by using probe 2 shown in A ), which is specific for bdm-1. ( D ) Northern blot analysis of RNA (15 μg) isolated from the same strains shown in C and the cpg-1 disruptant Δ cpg-1 by using the entire coding region of bdm-1 .

Techniques Used: Transformation Assay, Southern Blot, Isolation, Hybridization, Northern Blot

31) Product Images from "Sulf-2, a Proangiogenic Heparan Sulfate Endosulfatase, Is Upregulated in Breast Cancer 1"

Article Title: Sulf-2, a Proangiogenic Heparan Sulfate Endosulfatase, Is Upregulated in Breast Cancer 1

Journal:

doi:

Sulf-2 mRNA in human breast carcinoma cell lines. RT-PCR was performed on cDNA that were prepared from eight human breast carcinoma cell lines with primer pairs for Sulf-2 and β-actin primer. A 314-bp HSulf-2 cDNA product (indicated by an arrow)
Figure Legend Snippet: Sulf-2 mRNA in human breast carcinoma cell lines. RT-PCR was performed on cDNA that were prepared from eight human breast carcinoma cell lines with primer pairs for Sulf-2 and β-actin primer. A 314-bp HSulf-2 cDNA product (indicated by an arrow)

Techniques Used: Reverse Transcription Polymerase Chain Reaction

Arylsulfatase activity of Sulf-2 in MCF-7 CM. Different volumes of CM obtained from MCF-7 cells or HSulf-2–transfected CHO cells were incubated with Sulf-2 antibody (H2.3) or rabbit IgG coupled to protein A beads. Bead-bound material was tested
Figure Legend Snippet: Arylsulfatase activity of Sulf-2 in MCF-7 CM. Different volumes of CM obtained from MCF-7 cells or HSulf-2–transfected CHO cells were incubated with Sulf-2 antibody (H2.3) or rabbit IgG coupled to protein A beads. Bead-bound material was tested

Techniques Used: Activity Assay, Transfection, Incubation

32) Product Images from "Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1"

Article Title: Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.2435

Binding of PHF1 to H3K36me3 decreases PRC2-mediated deposition of H3K27me3. ( a ) ChIP assays on chromatin from HEK293T cells fixed and harvested 48 hours after transfection with either empty vector, wild-type HA-PHF1 or W41A or Y47A mutants. The levels of HA-PHF1, H3K27me3, H3K36me3 and H3 were probed across the MYT1 promoter using 7 primer sets. The top panel shows occupancy of wild type and mutant HA-PHF1 and the bottom two panels show H3K27me3 and H3K36me3 levels normalized to H3 (H3K27me3:H3). All data are relative to the PCLB4 non-target control gene. Error bars represent SD based on three experiments. ( b ) ChIP assays on chromatin harvested from mouse ES cells transduced with empty vector (pRTF, blue) or vector containing Flag-PHF1 (PHF1, red). Using quantitative PCR (qPCR), the levels of H3K27me3 and H3 were probed at the Oct4 , Nanog , HoxA4 and HoxA11 promoters. Data are presented as the ratio of H3K27me3 to total H3 signal to correct for possible differences in nucleosome density at the different loci examined. Error bars represent SD based on two experiments. ( c ) A model for inhibition of EZH2-PRC2 activity through recognition of H3K36me3 by the Tudor domain of PHF1.
Figure Legend Snippet: Binding of PHF1 to H3K36me3 decreases PRC2-mediated deposition of H3K27me3. ( a ) ChIP assays on chromatin from HEK293T cells fixed and harvested 48 hours after transfection with either empty vector, wild-type HA-PHF1 or W41A or Y47A mutants. The levels of HA-PHF1, H3K27me3, H3K36me3 and H3 were probed across the MYT1 promoter using 7 primer sets. The top panel shows occupancy of wild type and mutant HA-PHF1 and the bottom two panels show H3K27me3 and H3K36me3 levels normalized to H3 (H3K27me3:H3). All data are relative to the PCLB4 non-target control gene. Error bars represent SD based on three experiments. ( b ) ChIP assays on chromatin harvested from mouse ES cells transduced with empty vector (pRTF, blue) or vector containing Flag-PHF1 (PHF1, red). Using quantitative PCR (qPCR), the levels of H3K27me3 and H3 were probed at the Oct4 , Nanog , HoxA4 and HoxA11 promoters. Data are presented as the ratio of H3K27me3 to total H3 signal to correct for possible differences in nucleosome density at the different loci examined. Error bars represent SD based on two experiments. ( c ) A model for inhibition of EZH2-PRC2 activity through recognition of H3K36me3 by the Tudor domain of PHF1.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Mutagenesis, Transduction, Real-time Polymerase Chain Reaction, Inhibition, Activity Assay

Recognition of H3K36me3 by PHF1 inhibits PRC2 methyltransferase activity. ( a ) Western analysis of wild type and mutated Flag-PHF1 and EZH2 in the PRC2 complexes. ( b ) HMT assays with PHF1-PRC2 complexes purified from HEK293T cells on native wild type chromatin (wt SON, blue) and chromatin lacking the H3K36me mark (Δ set2 SON, red). Error bars represent SD based on three experiments. ( c ) Western analysis of whole cell extract from HEK293T cells 48 hours after transfection with wild type HA-PHF1 or W41A or Y47A mutants. Empty vector is control. (d) Western analysis of K562 cells stably expressing Flag-PHF1.
Figure Legend Snippet: Recognition of H3K36me3 by PHF1 inhibits PRC2 methyltransferase activity. ( a ) Western analysis of wild type and mutated Flag-PHF1 and EZH2 in the PRC2 complexes. ( b ) HMT assays with PHF1-PRC2 complexes purified from HEK293T cells on native wild type chromatin (wt SON, blue) and chromatin lacking the H3K36me mark (Δ set2 SON, red). Error bars represent SD based on three experiments. ( c ) Western analysis of whole cell extract from HEK293T cells 48 hours after transfection with wild type HA-PHF1 or W41A or Y47A mutants. Empty vector is control. (d) Western analysis of K562 cells stably expressing Flag-PHF1.

Techniques Used: Activity Assay, Western Blot, HMT Assay, Purification, Transfection, Plasmid Preparation, Stable Transfection, Expressing

Tudor-dependent accumulation and retention of PHF1 at laser-irradiated sites of DSBs. Co-localization of GFP-PHF1 wild type ( a ), GFP-PHF1 W41A ( b ) and GFP-PHF1 Y47A ( c ) at the DSB sites in U2OS cells 3 min after irradiation. Scale bars correspond to 10 μm. ( d ) Kinetic analysis of GFP-PHF1 intensity at laser irradiated sites. GFP-PHF1 wild type (closed diamond), GFP-PHF1 W41A (X) and GFP-PHF1 Y47A (open circle). Error bars represent SD based on three experiments.
Figure Legend Snippet: Tudor-dependent accumulation and retention of PHF1 at laser-irradiated sites of DSBs. Co-localization of GFP-PHF1 wild type ( a ), GFP-PHF1 W41A ( b ) and GFP-PHF1 Y47A ( c ) at the DSB sites in U2OS cells 3 min after irradiation. Scale bars correspond to 10 μm. ( d ) Kinetic analysis of GFP-PHF1 intensity at laser irradiated sites. GFP-PHF1 wild type (closed diamond), GFP-PHF1 W41A (X) and GFP-PHF1 Y47A (open circle). Error bars represent SD based on three experiments.

Techniques Used: Irradiation

33) Product Images from "The human ERG1 channel polymorphism, K897T, creates a phosphorylation site that inhibits channel activity"

Article Title: The human ERG1 channel polymorphism, K897T, creates a phosphorylation site that inhibits channel activity

Journal:

doi: 10.1073/pnas.0802250105

The hERG1 polymorphism, K897T, alters channel regulation by thyroid hormone. ( A ) hERG1 currents were elicited from cell-attached patches on CHO cells that were held at 0 mV in equimolar potassium by transiently repolarizing the patch to negative voltages
Figure Legend Snippet: The hERG1 polymorphism, K897T, alters channel regulation by thyroid hormone. ( A ) hERG1 currents were elicited from cell-attached patches on CHO cells that were held at 0 mV in equimolar potassium by transiently repolarizing the patch to negative voltages

Techniques Used:

Thyroid hormone stimulates hERG1–897K channels through dephosphorylation of T895. ( A ) hERG1 currents were elicited from cell-attached patches on CHO cells that were held at 0 mV in equimolar potassium by transiently repolarizing the patch to negative
Figure Legend Snippet: Thyroid hormone stimulates hERG1–897K channels through dephosphorylation of T895. ( A ) hERG1 currents were elicited from cell-attached patches on CHO cells that were held at 0 mV in equimolar potassium by transiently repolarizing the patch to negative

Techniques Used: De-Phosphorylation Assay

Thyroid hormone regulates different isoforms through different signaling branches downstream of PI3K. ( A ) Summary of signaling cascades downstream of phosphatidylinositol 3,4,5 Tris phosphate (PIP 3 ). ( B ) Percentage change in hERG1 current in response
Figure Legend Snippet: Thyroid hormone regulates different isoforms through different signaling branches downstream of PI3K. ( A ) Summary of signaling cascades downstream of phosphatidylinositol 3,4,5 Tris phosphate (PIP 3 ). ( B ) Percentage change in hERG1 current in response

Techniques Used:

Thyroid hormone inhibits hERG1–897T through Akt. ( A ) Sequence alignment of the predicted Akt consensus site in hERG1–897K, hERG1–897T, and the glycogen synthase kinase, GSK-3. The Glu residues in the Akt active site, which coordinate
Figure Legend Snippet: Thyroid hormone inhibits hERG1–897T through Akt. ( A ) Sequence alignment of the predicted Akt consensus site in hERG1–897K, hERG1–897T, and the glycogen synthase kinase, GSK-3. The Glu residues in the Akt active site, which coordinate

Techniques Used: Sequencing

34) Product Images from "A Gβ protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes"

Article Title: A Gβ protein and the TupA Co-Regulator Bind to Protein Kinase A Tpk2 to Act as Antagonistic Molecular Switches of Fungal Morphological Changes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0136866

PbTupA induces hyperfilamentous growth that can be repressed by PbGpb1. The S . cerevisiae diploid strain MLY61a/α (WT) and its TPK2Δ mutant XPY5a/α (XPY) were transformed with the PbTUPA , ScTUP1 , PBTPK2 and PbGPB1 as indicated. To allow selection, generally, constructs for the expression of the PbTupA-mRFP, ScTup1-mRFP, PbTpk2-GFP and PbGpb1-GFP fusion proteins were used and the transformants, selected on the basis of their green and/or red fluorescence. (A) The cells were analysed for pseudohyphal growth in SLAD agar containing 50 μM (upper panel) or 200 μM (lower panel) ammonium sulfate. Single colonies from the agar plate were observed at 20x magnification in an Eclipse E-400 microscope. The scale bar is 50μm. WT cells expressing PbTupA were hyperfilamentous; whilst those expressing ScTup1 did not produce pseudohyphae. XPY cells expressing PbTupA produced few pseudohyphae; whilst those expressing PbTupA with PbTpk2, but not a kinase defective K301R derivative, were hyperfilamentous, indicating the requirement for a functional PKA. The co-expression of PbGpb1 with PbTupA repressed the filamentous growth of the XPY/ PBTPK2 but not the WT cells, indicating that PbGpb1 specifically inhibits PbTpk2. (B) A bar chart showing FLO11 transcript levels for the indicated transformants. The measured quantity of the FLO11 mRNA in each of the treated samples is the relative abundance to the value for actin . The data represent the average of 3 measurements. (C) The S . cerevisiae diploid strain XPY95a/α, a FLO8Δ mutant, was transformed with the PbTUPA-mRFP and the transformants selected on the basis of their red fluorescence (lower panel), and analysed for pseudohyphal growth in SLAD agar containing 50 μM (upper panel) ammonium sulfate. PbTupA was in capable of inducing either the development of pseudohyphae (upper panel) or invasive growth (lower panel), which are the hallmarks of the parent strain MLY61a/α (WT) transformed with PbTUPA-mRFP . This data suggests that PbTupA works up-stream of Flo8 to induce hyperfilamentous growth.
Figure Legend Snippet: PbTupA induces hyperfilamentous growth that can be repressed by PbGpb1. The S . cerevisiae diploid strain MLY61a/α (WT) and its TPK2Δ mutant XPY5a/α (XPY) were transformed with the PbTUPA , ScTUP1 , PBTPK2 and PbGPB1 as indicated. To allow selection, generally, constructs for the expression of the PbTupA-mRFP, ScTup1-mRFP, PbTpk2-GFP and PbGpb1-GFP fusion proteins were used and the transformants, selected on the basis of their green and/or red fluorescence. (A) The cells were analysed for pseudohyphal growth in SLAD agar containing 50 μM (upper panel) or 200 μM (lower panel) ammonium sulfate. Single colonies from the agar plate were observed at 20x magnification in an Eclipse E-400 microscope. The scale bar is 50μm. WT cells expressing PbTupA were hyperfilamentous; whilst those expressing ScTup1 did not produce pseudohyphae. XPY cells expressing PbTupA produced few pseudohyphae; whilst those expressing PbTupA with PbTpk2, but not a kinase defective K301R derivative, were hyperfilamentous, indicating the requirement for a functional PKA. The co-expression of PbGpb1 with PbTupA repressed the filamentous growth of the XPY/ PBTPK2 but not the WT cells, indicating that PbGpb1 specifically inhibits PbTpk2. (B) A bar chart showing FLO11 transcript levels for the indicated transformants. The measured quantity of the FLO11 mRNA in each of the treated samples is the relative abundance to the value for actin . The data represent the average of 3 measurements. (C) The S . cerevisiae diploid strain XPY95a/α, a FLO8Δ mutant, was transformed with the PbTUPA-mRFP and the transformants selected on the basis of their red fluorescence (lower panel), and analysed for pseudohyphal growth in SLAD agar containing 50 μM (upper panel) ammonium sulfate. PbTupA was in capable of inducing either the development of pseudohyphae (upper panel) or invasive growth (lower panel), which are the hallmarks of the parent strain MLY61a/α (WT) transformed with PbTUPA-mRFP . This data suggests that PbTupA works up-stream of Flo8 to induce hyperfilamentous growth.

Techniques Used: Mutagenesis, Transformation Assay, Selection, Construct, Expressing, Fluorescence, Microscopy, Produced, Functional Assay

(A) P . brasiliensis TPK2 complements the growth defect of the S . cerevisiae ΔTPK2 Ts mutant strain SGY446. The S . cerevisiae haploid strain SGY446 that cannot grow at 37°C was transformed with p426MET25 (a), p426MET25-GFP (b), and the p426MET25-GFP constructs for the expression of GFP-fusion proteins of the N-terminal domain (PbTpk2 (1–225) ) (c), C-terminal domain (PbTpk2 (226–583) ) (d)) and full-length (PbTpk2 (1–583) ) PbTpk2 (e) from P . brasiliensis and incubated at 25°C and 37°C. The cells expressing the C-terminal domain (d) and full-length PbTpk2 (e) were able to grow at 37°C. (B) P . brasiliensis PbTPK2 complements the defect in the ability of the S . cerevisiae ΔTPK2 XPY5a/α strain to form pseudohyphae. The S . cerevisiae diploid strain XPY5a/α was transformed with constructs for the expression of GFP (GFP), the the N-terminal domain (PbTpk2 (1–225) ), C-terminal domain (PbTpk2 (226–583) and PbTpk2 (265–583) ), full-length (PbTpk2 (1–583) ) PbTpk2 and the K301R full-length PbTpk2 derivative from P . brasiliensis and the transformants were analysed for pseudohyphal growth in SLAD agar, containing 50 μM (upper panel) or 200 μM (lower panel) ammonium sulfate. Single colonies from the agar plate were observed at 20x magnification in an Eclipse E-400 microscope. The scale bar is 50 μm. The cells expressing the C-terminal domains and full-length PbTpk2 were able to form pseudohyphae, but cells expressing the full-length K301R PbTpk2 derivative, defective in kinase activity, were unable to form pseudohyphae.
Figure Legend Snippet: (A) P . brasiliensis TPK2 complements the growth defect of the S . cerevisiae ΔTPK2 Ts mutant strain SGY446. The S . cerevisiae haploid strain SGY446 that cannot grow at 37°C was transformed with p426MET25 (a), p426MET25-GFP (b), and the p426MET25-GFP constructs for the expression of GFP-fusion proteins of the N-terminal domain (PbTpk2 (1–225) ) (c), C-terminal domain (PbTpk2 (226–583) ) (d)) and full-length (PbTpk2 (1–583) ) PbTpk2 (e) from P . brasiliensis and incubated at 25°C and 37°C. The cells expressing the C-terminal domain (d) and full-length PbTpk2 (e) were able to grow at 37°C. (B) P . brasiliensis PbTPK2 complements the defect in the ability of the S . cerevisiae ΔTPK2 XPY5a/α strain to form pseudohyphae. The S . cerevisiae diploid strain XPY5a/α was transformed with constructs for the expression of GFP (GFP), the the N-terminal domain (PbTpk2 (1–225) ), C-terminal domain (PbTpk2 (226–583) and PbTpk2 (265–583) ), full-length (PbTpk2 (1–583) ) PbTpk2 and the K301R full-length PbTpk2 derivative from P . brasiliensis and the transformants were analysed for pseudohyphal growth in SLAD agar, containing 50 μM (upper panel) or 200 μM (lower panel) ammonium sulfate. Single colonies from the agar plate were observed at 20x magnification in an Eclipse E-400 microscope. The scale bar is 50 μm. The cells expressing the C-terminal domains and full-length PbTpk2 were able to form pseudohyphae, but cells expressing the full-length K301R PbTpk2 derivative, defective in kinase activity, were unable to form pseudohyphae.

Techniques Used: Mutagenesis, Transformation Assay, Construct, Expressing, Incubation, Microscopy, Activity Assay

35) Product Images from "Identification of four novel phosphorylation sites in estrogen receptor ?: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2"

Article Title: Identification of four novel phosphorylation sites in estrogen receptor ?: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-10-36

Mutation of serine residues to alanine eliminates specific phosphorylation of peptides . To confirm the identity of phosphorylated serine residues within peptides A, B, C, and D, serine to alanine mutations were introduced into wt ERα (S47A, S282A, S294A, or S559A). 12 plates of COS-1 cells (4 × 10 7 /plate) were transfected with 500 ng/plate of wt ERα, S47A, S282A, S294A, or S559A expression plasmids. 18 hours post-transfection, cells were phosphate-depleted, labeled with 4 mCi [ 32 P]H 3 PO 4 and incubated with 10 -8 M estradiol overnight. ERα was immunopurified and tryptic peptides were separated by HPLC using a C-18 reversed phase column. Fractions were collected and electrophoresed on a 40% alkaline polyacrylamide gel followed by autoradiography. (A) Peptide map of wt ERα displaying 4 novel phosphopeptides A-D. (B) S294A resulted in loss of peptide C. C) S559A resulted in loss of peptide B. (D) S47A resulted in a modest decrease in peptide D compared to wt ERα. E) S47A/S104A/S106A/S118A resulted in loss of peptide D. (F) Mutation of S282 to alanine reduces ERα protein following 24 h incubation with estradiol . 10 6 COS-1 monkey embryonic kidney cells which had been cultured in phenol-red free DMEM supplemented with 10% fetal bovine serum were transfected with 2.5 μg of wt ERα or S282A expression plasmid. 24 hours after transfection, cells were incubated with vehicle (veh) or estradiol (10 -8 M) for an additional 24 hours. Cell lysates were collected and ERα protein levels determined, using α-tubulin as a loading control.
Figure Legend Snippet: Mutation of serine residues to alanine eliminates specific phosphorylation of peptides . To confirm the identity of phosphorylated serine residues within peptides A, B, C, and D, serine to alanine mutations were introduced into wt ERα (S47A, S282A, S294A, or S559A). 12 plates of COS-1 cells (4 × 10 7 /plate) were transfected with 500 ng/plate of wt ERα, S47A, S282A, S294A, or S559A expression plasmids. 18 hours post-transfection, cells were phosphate-depleted, labeled with 4 mCi [ 32 P]H 3 PO 4 and incubated with 10 -8 M estradiol overnight. ERα was immunopurified and tryptic peptides were separated by HPLC using a C-18 reversed phase column. Fractions were collected and electrophoresed on a 40% alkaline polyacrylamide gel followed by autoradiography. (A) Peptide map of wt ERα displaying 4 novel phosphopeptides A-D. (B) S294A resulted in loss of peptide C. C) S559A resulted in loss of peptide B. (D) S47A resulted in a modest decrease in peptide D compared to wt ERα. E) S47A/S104A/S106A/S118A resulted in loss of peptide D. (F) Mutation of S282 to alanine reduces ERα protein following 24 h incubation with estradiol . 10 6 COS-1 monkey embryonic kidney cells which had been cultured in phenol-red free DMEM supplemented with 10% fetal bovine serum were transfected with 2.5 μg of wt ERα or S282A expression plasmid. 24 hours after transfection, cells were incubated with vehicle (veh) or estradiol (10 -8 M) for an additional 24 hours. Cell lysates were collected and ERα protein levels determined, using α-tubulin as a loading control.

Techniques Used: Mutagenesis, Transfection, Expressing, Labeling, Incubation, High Performance Liquid Chromatography, Autoradiography, Cell Culture, Plasmid Preparation

Confirmation of the specificity of ER phospho-antibodies . (A) Serine to alanine mutations at ERα phosphorylation sites inhibit reactivity of phospho-specific antibodies. COS-1 cells cultured in DMEM growth supplemented with 10% FBS were transiently transfected with 500 ng of either wt ERα or serine to alanine substituted ERα expression plasmids (S47A, S282A, S294A, or S559A). 18 hours post-transfection, cells were lysed and subjected to Western immunoblot analysis utilizing custom polyclonal antibodies directed toward the individual phosphorylated ERα residues (S47, S282, S294, or S559) or monoclonal ERα antibody as indicated. α-p-S282, α-p-S294, or α-p-S559 antibodies did not recognize S282A, S294A, or S559A, respectively, indicating phospho-antibody specificity. Mutation of S47 failed to eliminate immunoreactivity of αp-S47. (B) In vitro λ phosphatase treatment of ERα inhibits immunoreactivity of ERα phospho-antibodies. Baculovirus expressed ERα was subjected to dephosphorylation by λ phosphatase for 30 minutes at 30°C and analyzed by Western immunoblot with antibodies against p-S282, p-S294, p-S559, and total ERα. Dephosphorylation inhibited immunoreactivity of, α-p-S282, α-p-S294, and α-p-S559 without impacting immunoreactivity of total ERα antibody.
Figure Legend Snippet: Confirmation of the specificity of ER phospho-antibodies . (A) Serine to alanine mutations at ERα phosphorylation sites inhibit reactivity of phospho-specific antibodies. COS-1 cells cultured in DMEM growth supplemented with 10% FBS were transiently transfected with 500 ng of either wt ERα or serine to alanine substituted ERα expression plasmids (S47A, S282A, S294A, or S559A). 18 hours post-transfection, cells were lysed and subjected to Western immunoblot analysis utilizing custom polyclonal antibodies directed toward the individual phosphorylated ERα residues (S47, S282, S294, or S559) or monoclonal ERα antibody as indicated. α-p-S282, α-p-S294, or α-p-S559 antibodies did not recognize S282A, S294A, or S559A, respectively, indicating phospho-antibody specificity. Mutation of S47 failed to eliminate immunoreactivity of αp-S47. (B) In vitro λ phosphatase treatment of ERα inhibits immunoreactivity of ERα phospho-antibodies. Baculovirus expressed ERα was subjected to dephosphorylation by λ phosphatase for 30 minutes at 30°C and analyzed by Western immunoblot with antibodies against p-S282, p-S294, p-S559, and total ERα. Dephosphorylation inhibited immunoreactivity of, α-p-S282, α-p-S294, and α-p-S559 without impacting immunoreactivity of total ERα antibody.

Techniques Used: Cell Culture, Transfection, Expressing, Western Blot, Mutagenesis, In Vitro, De-Phosphorylation Assay

Phosphorylation of ERα impacts receptor transcriptional activity . (A) ERα (-) HeLa cells were cotransfected with 100 ng ERE 2 -TK-luciferase reporter and 200 ng wt ERα (wt), or serine to alanine mutants of ERα for each novel phosphorylation site (S47A, S282A, S294A, and S559A). 24 hours post transfection, cells were incubated with vehicle (veh) or estradiol (10 -8 M) overnight. Luciferase assays were performed and transcriptional activity was normalized to protein concentration and/or ERα expression by Western blot analysis. S47A exhibited similar transcriptional activity to wt ERα, whereas S294A resulted in suppressed transcriptional activity vehicle and estradiol. S282A and S559A displayed enhanced ligand independent transcriptional activity as compared to wt ERα. (B) HeLa cervical cancer cells were transfected with 500 ng of wt ERα or ERα phospho-mutant (S47A, S282A, S294A, S559A) expression plasmids. 24 hours post-transfection, cells were incubated with vehicle (veh), 10 -8 estradiol (E2), for 3 hours. pS2 expression was measured by real-time RT-PCR, relative to GAPDH. S47A resulted in suppression of estradiol-induced pS2 expression, whereas S559A exhibited ligand-independent activation of ER. S282A and S294A displayed no statistical differences in pS2 mRNA. (C) HeLa cells were transfected with 500 ng of wt ERα or ERα phospho-mutant (S47A, S282A, S294A, and S559A) expression plasmids and incubated for 3 hours with estradiol (1 -8 M) at 18-24 hours post transfection. Transfection and incubation with estradiol were performed in parallel with those for RT-PCR (panel B). A and B represent composite results for 6 identical experiments. Statistical significance was determined using ANOVA and Fisher's LSD post-hoc analysis, p ≤ 0.05.
Figure Legend Snippet: Phosphorylation of ERα impacts receptor transcriptional activity . (A) ERα (-) HeLa cells were cotransfected with 100 ng ERE 2 -TK-luciferase reporter and 200 ng wt ERα (wt), or serine to alanine mutants of ERα for each novel phosphorylation site (S47A, S282A, S294A, and S559A). 24 hours post transfection, cells were incubated with vehicle (veh) or estradiol (10 -8 M) overnight. Luciferase assays were performed and transcriptional activity was normalized to protein concentration and/or ERα expression by Western blot analysis. S47A exhibited similar transcriptional activity to wt ERα, whereas S294A resulted in suppressed transcriptional activity vehicle and estradiol. S282A and S559A displayed enhanced ligand independent transcriptional activity as compared to wt ERα. (B) HeLa cervical cancer cells were transfected with 500 ng of wt ERα or ERα phospho-mutant (S47A, S282A, S294A, S559A) expression plasmids. 24 hours post-transfection, cells were incubated with vehicle (veh), 10 -8 estradiol (E2), for 3 hours. pS2 expression was measured by real-time RT-PCR, relative to GAPDH. S47A resulted in suppression of estradiol-induced pS2 expression, whereas S559A exhibited ligand-independent activation of ER. S282A and S294A displayed no statistical differences in pS2 mRNA. (C) HeLa cells were transfected with 500 ng of wt ERα or ERα phospho-mutant (S47A, S282A, S294A, and S559A) expression plasmids and incubated for 3 hours with estradiol (1 -8 M) at 18-24 hours post transfection. Transfection and incubation with estradiol were performed in parallel with those for RT-PCR (panel B). A and B represent composite results for 6 identical experiments. Statistical significance was determined using ANOVA and Fisher's LSD post-hoc analysis, p ≤ 0.05.

Techniques Used: Activity Assay, Luciferase, Transfection, Incubation, Protein Concentration, Expressing, Western Blot, Mutagenesis, Quantitative RT-PCR, Activation Assay, Reverse Transcription Polymerase Chain Reaction

36) Product Images from "Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells"

Article Title: Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.01163

T595 and S597 phosphosites in DEF6 protein contribute to IP 3 R interaction and T cell activation. Plasmids encoding for DEF6-WT, DEF6 T595_S597 phospho-mutant (DEF6-2A), and DEF6 T595_S597 phospho-mimic (DEF6-2E) were generated. (A) HEK293 cells were cotransfected with indicated myc-tagged DEF6 constructs along with FLAG-tagged IP 3 R. Whole cell lysates (WCLs) were subjected to immunoprecipitation (IP) with anti-myc antibody (left), and aliquots of WCLs were kept untreated (right). Western blot antibodies are indicated. A representative experiment of 2 is shown. (B–E) Primary human Tcons were transiently transfected with plasmids encoding the indicated DEF6 protein or empty vector [EV; green fluorescent protein (GFP) in place of DEF6] for 8 h. Where indicated, cells were subsequently stimulated (Stim) with anti-CD3/anti-CD28 antibodies and processed as follows: (B) transfection efficiency based on GFP expression in EV-transfected cells was determined by flow cytometry. It was pre-gated on live lymphocytes based on fsc/ssc. Overlaid histogram of GFP expression in untransfected and EV-transfected Tcons is representative of five donors in two independent experiments. (C) Cells were stimulated for 5 min and analyzed by Western blot with antibodies against indicated targets (left). The ratio of levels of dephosphorylated NFAT1 and phosphorylated NFAT1 was calculated after quantification of the bands. Expression levels of myc-tagged DEF6 or total DEF6 and GAPDH served as transfection and loading controls, respectively. Blot is representative of five donors. Right: NFAT1 ratios were normalized to the corresponding NFAT1 ratio in unstimulated Tcons of the same donor which was set to 1. Individual NFAT1 ratios from n = 5 donors (represented by individual symbol per donor) from 2 independent experiments along with the mean values (line) are shown. IL2 (D) and IFNG (E) mRNA in transfected Tcons after 3 h of stimulation or without stimulation (Unstim), normalized to RPL13A mRNA. Results are presented as fold change compared to unstimulated Tcons of the same donor, which was set to 1. Left: mean ± SD of technical triplicates from quantitative RT-PCR is shown for one donor, representative of five donors from two independent experiments. Right: individual values of log 2 fold change of mRNA levels of the respective cytokines from five donors (represented by individual symbol) are shown along with mean values (lines). P values (C–E) were determined by paired, two-sided Student’s t -test (* P
Figure Legend Snippet: T595 and S597 phosphosites in DEF6 protein contribute to IP 3 R interaction and T cell activation. Plasmids encoding for DEF6-WT, DEF6 T595_S597 phospho-mutant (DEF6-2A), and DEF6 T595_S597 phospho-mimic (DEF6-2E) were generated. (A) HEK293 cells were cotransfected with indicated myc-tagged DEF6 constructs along with FLAG-tagged IP 3 R. Whole cell lysates (WCLs) were subjected to immunoprecipitation (IP) with anti-myc antibody (left), and aliquots of WCLs were kept untreated (right). Western blot antibodies are indicated. A representative experiment of 2 is shown. (B–E) Primary human Tcons were transiently transfected with plasmids encoding the indicated DEF6 protein or empty vector [EV; green fluorescent protein (GFP) in place of DEF6] for 8 h. Where indicated, cells were subsequently stimulated (Stim) with anti-CD3/anti-CD28 antibodies and processed as follows: (B) transfection efficiency based on GFP expression in EV-transfected cells was determined by flow cytometry. It was pre-gated on live lymphocytes based on fsc/ssc. Overlaid histogram of GFP expression in untransfected and EV-transfected Tcons is representative of five donors in two independent experiments. (C) Cells were stimulated for 5 min and analyzed by Western blot with antibodies against indicated targets (left). The ratio of levels of dephosphorylated NFAT1 and phosphorylated NFAT1 was calculated after quantification of the bands. Expression levels of myc-tagged DEF6 or total DEF6 and GAPDH served as transfection and loading controls, respectively. Blot is representative of five donors. Right: NFAT1 ratios were normalized to the corresponding NFAT1 ratio in unstimulated Tcons of the same donor which was set to 1. Individual NFAT1 ratios from n = 5 donors (represented by individual symbol per donor) from 2 independent experiments along with the mean values (line) are shown. IL2 (D) and IFNG (E) mRNA in transfected Tcons after 3 h of stimulation or without stimulation (Unstim), normalized to RPL13A mRNA. Results are presented as fold change compared to unstimulated Tcons of the same donor, which was set to 1. Left: mean ± SD of technical triplicates from quantitative RT-PCR is shown for one donor, representative of five donors from two independent experiments. Right: individual values of log 2 fold change of mRNA levels of the respective cytokines from five donors (represented by individual symbol) are shown along with mean values (lines). P values (C–E) were determined by paired, two-sided Student’s t -test (* P

Techniques Used: Activation Assay, Mutagenesis, Generated, Construct, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Cytometry, Quantitative RT-PCR

Tregs suppress T595_S597 phosphorylation of DEF6 in responder Tcons. (A) The peptide sequences of DEF6 containing the conserved phosphosites in respective organisms are shown. Ser (S) and Thr (T) with detected phosphorylation in ≥2/3 donors are highlighted in red. Phosphosites of interest (T595 and S597) are additionally highlighted in big letters. (B) log 2 of average ratio of T595_S597 phosphorylated DEF6 peptide in the given comparisons. (C) Representative FTMS spectrum of the indicated DEF6 phosphopeptide (phospho-T595_S597). Precursor areas of the three indicated samples are depicted. Shown is the spectrum from Donor 3 (from one out of three technical replicate mass spectrometry runs) representative of three donors, with following properties: quantified Ion: z = +3, Mono m / z = 744.31616 Da, and MH+ = 2,230.93393 Da. Filled circles are isotope pattern peaks used in calculating the quantification values for the different quantification channels, as opposed to unfilled circles. The bar chart (right) shows quantification of the respective spectrum.
Figure Legend Snippet: Tregs suppress T595_S597 phosphorylation of DEF6 in responder Tcons. (A) The peptide sequences of DEF6 containing the conserved phosphosites in respective organisms are shown. Ser (S) and Thr (T) with detected phosphorylation in ≥2/3 donors are highlighted in red. Phosphosites of interest (T595 and S597) are additionally highlighted in big letters. (B) log 2 of average ratio of T595_S597 phosphorylated DEF6 peptide in the given comparisons. (C) Representative FTMS spectrum of the indicated DEF6 phosphopeptide (phospho-T595_S597). Precursor areas of the three indicated samples are depicted. Shown is the spectrum from Donor 3 (from one out of three technical replicate mass spectrometry runs) representative of three donors, with following properties: quantified Ion: z = +3, Mono m / z = 744.31616 Da, and MH+ = 2,230.93393 Da. Filled circles are isotope pattern peaks used in calculating the quantification values for the different quantification channels, as opposed to unfilled circles. The bar chart (right) shows quantification of the respective spectrum.

Techniques Used: Mass Spectrometry

37) Product Images from "Effects of ranolazine on wild-type and mutant hNav1.7 channels and on DRG neuron excitability"

Article Title: Effects of ranolazine on wild-type and mutant hNav1.7 channels and on DRG neuron excitability

Journal: Molecular Pain

doi: 10.1186/1744-8069-6-35

Frequency-dependence of use-dependent block of Na v 1 .7 currents by ranolazine. Trains of twenty 30 msec duration pulses to -10 mV from a holding potential of -120 mV were applied at five different frequencies and were performed both before and after exposure to 10 μM ranolazine. Example traces from a HEK 293 cell expressing WT channels are shown in panels A and B. The peak for each pulse is normalized to the peak of the first pulse, and the values are plotted in panel C. The use-dependent block, defined as the ratio of the peak from the 20 th pulse normalized to the peak of the first pulse, was determined for each cell and the averages are plotted with the basal responses shown in grey and the ranolazine responses shown in solid bars. In each panel the two datasets are shown to compare the response to ranolazine in cells expressing WT (panel D, n = 5), the IEM mutant L858H (panel E, n = 4), or the PEPD mutant V1298F (panel F, n = 7), channels.
Figure Legend Snippet: Frequency-dependence of use-dependent block of Na v 1 .7 currents by ranolazine. Trains of twenty 30 msec duration pulses to -10 mV from a holding potential of -120 mV were applied at five different frequencies and were performed both before and after exposure to 10 μM ranolazine. Example traces from a HEK 293 cell expressing WT channels are shown in panels A and B. The peak for each pulse is normalized to the peak of the first pulse, and the values are plotted in panel C. The use-dependent block, defined as the ratio of the peak from the 20 th pulse normalized to the peak of the first pulse, was determined for each cell and the averages are plotted with the basal responses shown in grey and the ranolazine responses shown in solid bars. In each panel the two datasets are shown to compare the response to ranolazine in cells expressing WT (panel D, n = 5), the IEM mutant L858H (panel E, n = 4), or the PEPD mutant V1298F (panel F, n = 7), channels.

Techniques Used: Blocking Assay, Expressing, Mutagenesis

Effect of ranolazine on the excitability of DRG neurons expressing WT, L858H or V1298F channels . Action potentials were recorded in current-clamp mode from DRG neurons transfected with WT (panel A), the IEM mutant L858H (panel B), or the PEPD mutant V1298F (panel C), channels as described in the Methods section. The number of action potentials elicited during current injections of 1-sec duration ranging from 50 pA to 1000 pA in 50 pA increments are counted both before and then again after exposure to 10 μM ranolazine. In this figure, cells whose response did not exceed 5 spikes were removed to compare just the high firing cells. The average number of action potentials elicited at each current injection level are plotted before (grey) and after 10 μM ranolazine (solid) exposure. In each panel the two datasets are shown to compare the response to ranolazine in cells expressing WT (panel A, n = 10), the IEM mutant L858H (panel B, n = 4), or the PEPD mutant V1298F (panel C, n = 5), channels.
Figure Legend Snippet: Effect of ranolazine on the excitability of DRG neurons expressing WT, L858H or V1298F channels . Action potentials were recorded in current-clamp mode from DRG neurons transfected with WT (panel A), the IEM mutant L858H (panel B), or the PEPD mutant V1298F (panel C), channels as described in the Methods section. The number of action potentials elicited during current injections of 1-sec duration ranging from 50 pA to 1000 pA in 50 pA increments are counted both before and then again after exposure to 10 μM ranolazine. In this figure, cells whose response did not exceed 5 spikes were removed to compare just the high firing cells. The average number of action potentials elicited at each current injection level are plotted before (grey) and after 10 μM ranolazine (solid) exposure. In each panel the two datasets are shown to compare the response to ranolazine in cells expressing WT (panel A, n = 10), the IEM mutant L858H (panel B, n = 4), or the PEPD mutant V1298F (panel C, n = 5), channels.

Techniques Used: Expressing, Transfection, Mutagenesis, Size-exclusion Chromatography, Injection

Voltage-dependence of ranolazine block of hNav1 .7r-WT and L858H and V1298F mutants: Dose-response curve fits. The extent of ranolazine block for each cell was determined for conditioning potentials ranging from the holding potential of -120 mV (resting block) to Vcond of -40 mV (inactivated block). Each cell was exposed to only one concentration of ranolazine. The dose-response curve for each conditioning potential (see legend symbols) was obtained by fitting a single-site binding curve to the average current block (3-9 cells/point).
Figure Legend Snippet: Voltage-dependence of ranolazine block of hNav1 .7r-WT and L858H and V1298F mutants: Dose-response curve fits. The extent of ranolazine block for each cell was determined for conditioning potentials ranging from the holding potential of -120 mV (resting block) to Vcond of -40 mV (inactivated block). Each cell was exposed to only one concentration of ranolazine. The dose-response curve for each conditioning potential (see legend symbols) was obtained by fitting a single-site binding curve to the average current block (3-9 cells/point).

Techniques Used: Blocking Assay, Concentration Assay, Binding Assay

Ranolazine block of ramp currents . The response to slow gradual depolarization (ramp) stimulus protocol (-100 mV to +20 mV over 600 msec) from representative cells is normalized to the peak inward current for that cell as described in Methods. Separate cells were exposed to either 0.1% HCl vehicle control (grey line) or 10 μM ranolazine (bold line). In each panel the two traces are overlaid to compare the response to ranolazine. (A) HEK 293 cells expressing WT show a peak ramp response of 0.58 ± 0.15% and the voltage peaks at -43.0 ± 1.2 mV in the presence of 0.1% HCl vehicle control (n = 3). In the presence of 10 μM ranolazine (n = 4), the peak ramp response was 0.79 ± 0.22% and the voltage peaks at -48.0 ± 3.9 mV. (B) HEK 293 cells expressing, the IEM mutant L858H show a peak ramp response of 4.48 ± 1.13% and the voltage peaks at -55.8 ± 2.2 mV in the presence of 0.1% HCl vehicle control (n = 5). In the presence of 10 μM ranolazine (n = 4), the peak ramp response was 3.31 ± 0.60% and the voltage peaks at -54.1 ± 2.0 mV. (C) HEK 293 cells expressing the PEPD mutant V1298F show a peak ramp response of 1.05 ± 0.21% and the voltage peaks at -48.5 ± 2.2 mV in the presence of 0.1% HCl vehicle control (n = 8). In the presence of 10 μM ranolazine (n = 7), the peak ramp response was 0.84 ± 0.11% and the voltage peaks at -49.7 ± 2.7 mV.
Figure Legend Snippet: Ranolazine block of ramp currents . The response to slow gradual depolarization (ramp) stimulus protocol (-100 mV to +20 mV over 600 msec) from representative cells is normalized to the peak inward current for that cell as described in Methods. Separate cells were exposed to either 0.1% HCl vehicle control (grey line) or 10 μM ranolazine (bold line). In each panel the two traces are overlaid to compare the response to ranolazine. (A) HEK 293 cells expressing WT show a peak ramp response of 0.58 ± 0.15% and the voltage peaks at -43.0 ± 1.2 mV in the presence of 0.1% HCl vehicle control (n = 3). In the presence of 10 μM ranolazine (n = 4), the peak ramp response was 0.79 ± 0.22% and the voltage peaks at -48.0 ± 3.9 mV. (B) HEK 293 cells expressing, the IEM mutant L858H show a peak ramp response of 4.48 ± 1.13% and the voltage peaks at -55.8 ± 2.2 mV in the presence of 0.1% HCl vehicle control (n = 5). In the presence of 10 μM ranolazine (n = 4), the peak ramp response was 3.31 ± 0.60% and the voltage peaks at -54.1 ± 2.0 mV. (C) HEK 293 cells expressing the PEPD mutant V1298F show a peak ramp response of 1.05 ± 0.21% and the voltage peaks at -48.5 ± 2.2 mV in the presence of 0.1% HCl vehicle control (n = 8). In the presence of 10 μM ranolazine (n = 7), the peak ramp response was 0.84 ± 0.11% and the voltage peaks at -49.7 ± 2.7 mV.

Techniques Used: Blocking Assay, Expressing, Mutagenesis

Voltage-dependence of WT and the L858H and V1298F mutant channels . (A) Superimposed activation traces recorded from a representative HEK + hNav1.7r-WT expressing cell. (Average peak current is -4.2 ± 0.7 nA, n = 12) (B) Superimposed fast-inactivation traces recorded from the same cell as in (A). (C) Superimposed activation traces recorded from a representative HEK + hNav1.7r-L858H expressing cell. (Average peak current is -2.3 ± 0.3 nA, n = 12). (D) Superimposed fast-inactivation traces recorded from the same cell as in (C). (E) Superimposed activation traces recorded from a representative HEK + hNav1.7r-V1298F expressing cell (Average peak current is -1.9 ± 0.3 nA, n = 18). (F) Superimposed fast-inactivation traces from the same cell as in (E). Insets illustrate the voltage pulse protocols for activation and fast-inactivation. The bold bars indicate the portion of the data sweeps displayed in this figure. (G) Normalized conductance-voltage (G-V) curves are constructed from the averages of individual HEK 293 cells expressing WT (black squares, n = 12), the IEM mutation L858H (red circles, n = 12), or the PEPD mutation V1298F (blue triangles, n = 18). (H) Normalized fast-inactivation curves are constructed from the averages of the same cells as in panel G.
Figure Legend Snippet: Voltage-dependence of WT and the L858H and V1298F mutant channels . (A) Superimposed activation traces recorded from a representative HEK + hNav1.7r-WT expressing cell. (Average peak current is -4.2 ± 0.7 nA, n = 12) (B) Superimposed fast-inactivation traces recorded from the same cell as in (A). (C) Superimposed activation traces recorded from a representative HEK + hNav1.7r-L858H expressing cell. (Average peak current is -2.3 ± 0.3 nA, n = 12). (D) Superimposed fast-inactivation traces recorded from the same cell as in (C). (E) Superimposed activation traces recorded from a representative HEK + hNav1.7r-V1298F expressing cell (Average peak current is -1.9 ± 0.3 nA, n = 18). (F) Superimposed fast-inactivation traces from the same cell as in (E). Insets illustrate the voltage pulse protocols for activation and fast-inactivation. The bold bars indicate the portion of the data sweeps displayed in this figure. (G) Normalized conductance-voltage (G-V) curves are constructed from the averages of individual HEK 293 cells expressing WT (black squares, n = 12), the IEM mutation L858H (red circles, n = 12), or the PEPD mutation V1298F (blue triangles, n = 18). (H) Normalized fast-inactivation curves are constructed from the averages of the same cells as in panel G.

Techniques Used: Mutagenesis, Activation Assay, Expressing, Construct

38) Product Images from "Evidence for a role of vertebrate Disp1 in long-range Shh signaling"

Article Title: Evidence for a role of vertebrate Disp1 in long-range Shh signaling

Journal: Development (Cambridge, England)

doi: 10.1242/dev.043547

Long-range Shh signaling is reduced in Disp1 −/− EBs. To test whether Disp1 is required outside of Shh-expressing cells for a normal long-range response to cholesterol-modified Shh, wild-type (Wt) EBs expressing Shh were co-cultured for 36 or 48 hours with EBs with or without Disp1 function. Repression of Pax7 was used to measure the range of the Shh response. ( A , B ) In the absence of a Shh source, Pax7 is expressed throughout both Disp +/− and Disp −/− EBs. ( C ) The level of Shh present in the lysates and supernatants of Shh AB1 cells. Shh accumulated more efficiently in the medium when suramin (Sur) was present. ( D , F ) When Shh AB1 EBs were co-cultured with Disp1 +/− EBs, Pax7 was significantly reduced throughout the entire EB. ( E , G ) In Disp1 −/− EBs grown in contact with Shh-expressing EBs, Pax7 was only repressed in cells nearest to the Shh source. F and G are low magnification images of the cultures shown in D and E. Induction of HB9 was used to measure the range of Shh response. ( H , J ) In cases where Shh-expressing EBs contacted Disp1 +/− EBs, HB9 was induced close to the Shh source. ( I , K ) In Disp1 −/− EBs grown in contact with Shh-expressing EBs, very few HB9 positive cells were observed. J and K are lower magnification images of the cultures shown in H and I. As Disp1 is not required for the Shh response per se, these results suggest that Disp1 function is important for transmission of the Shh ligand through the responding tissue. Dashed lines, borders between EBs; asterisks, Shh-expressing EBs. Scale bars: 75 μm in A,B,D,E,H,I; 150 μm in F,G,J,K.
Figure Legend Snippet: Long-range Shh signaling is reduced in Disp1 −/− EBs. To test whether Disp1 is required outside of Shh-expressing cells for a normal long-range response to cholesterol-modified Shh, wild-type (Wt) EBs expressing Shh were co-cultured for 36 or 48 hours with EBs with or without Disp1 function. Repression of Pax7 was used to measure the range of the Shh response. ( A , B ) In the absence of a Shh source, Pax7 is expressed throughout both Disp +/− and Disp −/− EBs. ( C ) The level of Shh present in the lysates and supernatants of Shh AB1 cells. Shh accumulated more efficiently in the medium when suramin (Sur) was present. ( D , F ) When Shh AB1 EBs were co-cultured with Disp1 +/− EBs, Pax7 was significantly reduced throughout the entire EB. ( E , G ) In Disp1 −/− EBs grown in contact with Shh-expressing EBs, Pax7 was only repressed in cells nearest to the Shh source. F and G are low magnification images of the cultures shown in D and E. Induction of HB9 was used to measure the range of Shh response. ( H , J ) In cases where Shh-expressing EBs contacted Disp1 +/− EBs, HB9 was induced close to the Shh source. ( I , K ) In Disp1 −/− EBs grown in contact with Shh-expressing EBs, very few HB9 positive cells were observed. J and K are lower magnification images of the cultures shown in H and I. As Disp1 is not required for the Shh response per se, these results suggest that Disp1 function is important for transmission of the Shh ligand through the responding tissue. Dashed lines, borders between EBs; asterisks, Shh-expressing EBs. Scale bars: 75 μm in A,B,D,E,H,I; 150 μm in F,G,J,K.

Techniques Used: Expressing, Modification, Cell Culture, Transmission Assay

Ptch1 on neighboring cells mediates the transport of Shh secreted in a Disp1-dependent manner. ( A-F ) Shh-expressing wild-type (Wt) ES cells (blue, A,C,E) and Shh-expressing Disp1 −/− cells (blue, B,D,F) were co-cultured with Wt (A,B), Disp1 −/− (C,D) and Ptch1 −/− cells (E,F). Shh was visualized using directly conjugated 5E1 included in the live cultures for 1 hour, followed by fixation. When Ptch1 was absent from the surrounding cells more Shh could be detected on cells containing Disp1 (E), whereas this had no effect on the Shh present on cells without Disp1 (F). ( G ) Quantification of the amount of Shh present on cells cultured under the indicated conditions. Error bars indicate s.e.m. Only the amount of Shh present on Wt cells surrounded by Ptch1 −/− cells was significantly different from all others. ( H ) Analysis of the Wt and Disp1 −/− Shh-expressing ES cells. Shh was easily detected in lysates, but only a small amount of Shh was detected in the concentrated supernatant from the Wt cells, and not at all in the Disp1 −/− cells. Scale bar: 20 μm.
Figure Legend Snippet: Ptch1 on neighboring cells mediates the transport of Shh secreted in a Disp1-dependent manner. ( A-F ) Shh-expressing wild-type (Wt) ES cells (blue, A,C,E) and Shh-expressing Disp1 −/− cells (blue, B,D,F) were co-cultured with Wt (A,B), Disp1 −/− (C,D) and Ptch1 −/− cells (E,F). Shh was visualized using directly conjugated 5E1 included in the live cultures for 1 hour, followed by fixation. When Ptch1 was absent from the surrounding cells more Shh could be detected on cells containing Disp1 (E), whereas this had no effect on the Shh present on cells without Disp1 (F). ( G ) Quantification of the amount of Shh present on cells cultured under the indicated conditions. Error bars indicate s.e.m. Only the amount of Shh present on Wt cells surrounded by Ptch1 −/− cells was significantly different from all others. ( H ) Analysis of the Wt and Disp1 −/− Shh-expressing ES cells. Shh was easily detected in lysates, but only a small amount of Shh was detected in the concentrated supernatant from the Wt cells, and not at all in the Disp1 −/− cells. Scale bar: 20 μm.

Techniques Used: Expressing, Cell Culture

Disp1 is required for the basolateral secretion of Shh. MDCK cells were grown on 12 mm polyester transwell filters (0.4 μm pore size) and transfected with Shh (red) and either control Disp1 miRNA constructs (green, A,E) or Disp1 miRNA constructs (green, B,F,G). ( A , B ) Shh was visualized after inclusion of 5E1 in the compartment at the basolateral side of the MDCK cells. Secreted Shh is visible in the cells co-transfected with control Disp1 miRNA but is severely reduced in cells co-transfected with Disp1 miRNA1 or 2. ( C ) Shh staining was quantified by measuring staining intensity in transfected cells (retention) or in a 1.5-cell diameter wide ring around the transfected cells (secretion). Co-transfection of Disp1 miRNA1 or 2 caused significant increase in retention of Shh, whereas the secretion of Shh was significantly reduced. The y -axis is a relative measure of Shh staining. Error bars are s.e.m., P
Figure Legend Snippet: Disp1 is required for the basolateral secretion of Shh. MDCK cells were grown on 12 mm polyester transwell filters (0.4 μm pore size) and transfected with Shh (red) and either control Disp1 miRNA constructs (green, A,E) or Disp1 miRNA constructs (green, B,F,G). ( A , B ) Shh was visualized after inclusion of 5E1 in the compartment at the basolateral side of the MDCK cells. Secreted Shh is visible in the cells co-transfected with control Disp1 miRNA but is severely reduced in cells co-transfected with Disp1 miRNA1 or 2. ( C ) Shh staining was quantified by measuring staining intensity in transfected cells (retention) or in a 1.5-cell diameter wide ring around the transfected cells (secretion). Co-transfection of Disp1 miRNA1 or 2 caused significant increase in retention of Shh, whereas the secretion of Shh was significantly reduced. The y -axis is a relative measure of Shh staining. Error bars are s.e.m., P

Techniques Used: Transfection, Construct, Staining, Cotransfection

Disp1 is located basolaterally in polarized epithelial cells and assembles as trimers. ( A ) Blue-Native gel analysis of Disp1 transfected HEK 293T cells. The 480 kD band corresponds to the predicted size of Disp1 trimers. Disp1 mutants are indicated. ( B ) Diagram illustrating Disp1 deletion mutants. In addition to deletion of the entire CTD, a series of six ~60-amino-acid-deletion mutants covering the CTD were generated. The box to the right indicates how each mutant localizes relative to the three Disp1 forms shown in C-E, and trimer formation is shown in A. ( C-E ) Visualization of transiently transfected V5-tagged Disp1 (green) and ZO1 (red) in polarized MDCK cells. ZO1 marks the border between the apical and basolateral aspects of the polarized cells. Wild-type (Wt) Disp1 (C) is located basolaterally, Disp1ΔCTD (D) is located throughout the cell and Disp1 Del2 (E) is primarily located at or near the apical cell surface. x,z -reconstructions are shown on the top and at the left of each panel, yellow As and Bs indicate the apical and basolateral sides, respectively. Scale bar: 20 μm.
Figure Legend Snippet: Disp1 is located basolaterally in polarized epithelial cells and assembles as trimers. ( A ) Blue-Native gel analysis of Disp1 transfected HEK 293T cells. The 480 kD band corresponds to the predicted size of Disp1 trimers. Disp1 mutants are indicated. ( B ) Diagram illustrating Disp1 deletion mutants. In addition to deletion of the entire CTD, a series of six ~60-amino-acid-deletion mutants covering the CTD were generated. The box to the right indicates how each mutant localizes relative to the three Disp1 forms shown in C-E, and trimer formation is shown in A. ( C-E ) Visualization of transiently transfected V5-tagged Disp1 (green) and ZO1 (red) in polarized MDCK cells. ZO1 marks the border between the apical and basolateral aspects of the polarized cells. Wild-type (Wt) Disp1 (C) is located basolaterally, Disp1ΔCTD (D) is located throughout the cell and Disp1 Del2 (E) is primarily located at or near the apical cell surface. x,z -reconstructions are shown on the top and at the left of each panel, yellow As and Bs indicate the apical and basolateral sides, respectively. Scale bar: 20 μm.

Techniques Used: Transfection, Generated, Mutagenesis

Disp1 mutants act as dominant-negatives. MDCK cells were grown on transwell filters and transfected with Shh and wild-type Disp1 ( A ), Disp1ΔCTD ( B ) or Disp1AAA ( C ). The anti-Shh monoclonal antibody 5E1 was included overnight in the basolateral compartment and visualized. Both Disp1 mutants caused a reduction of Shh secretion and an increase in Shh retention. ( D ) Quantification of Shh secretion by measuring staining intensity in a 1.5-cell diameter wide ring around the transfected cells, and of Shh retention by measuring staining intensity in transfected cells. The mutant Disp1 co-transfected cells are significantly different from wild-type Disp1-transfected cells both for secretion and retention ( P
Figure Legend Snippet: Disp1 mutants act as dominant-negatives. MDCK cells were grown on transwell filters and transfected with Shh and wild-type Disp1 ( A ), Disp1ΔCTD ( B ) or Disp1AAA ( C ). The anti-Shh monoclonal antibody 5E1 was included overnight in the basolateral compartment and visualized. Both Disp1 mutants caused a reduction of Shh secretion and an increase in Shh retention. ( D ) Quantification of Shh secretion by measuring staining intensity in a 1.5-cell diameter wide ring around the transfected cells, and of Shh retention by measuring staining intensity in transfected cells. The mutant Disp1 co-transfected cells are significantly different from wild-type Disp1-transfected cells both for secretion and retention ( P

Techniques Used: Activated Clotting Time Assay, Transfection, Staining, Mutagenesis

Non-acylated Shh (C25S) is secreted in a Disp1-dependent manner. MDCK cells were grown on transwell filters and transfected with wild-type (Wt) Shh ( A ), Shh lacking cholesterol modification (C*) ( B ), Shh lacking amino terminal acyl modification (C25S) ( C ) and Shh with neither modification (C*/C25S) ( D ). The cells were grown overnight with 5E1 included in the basolateral compartment and visualized. The cholesterol moiety (present in A and C) mediates the retention of Shh outside the cells, whereas the loss of the acyl modification resulted in a shallow Shh gradient (C). Shh C25S was co-transfected either with Wt Disp1 ( E ) or Disp1AAA ( F ) into MDCK cells that were grown on transwell filters. Anti-Shh monoclonal 5E1 was included in the basolateral compartment for 16 hours and visualized. Disp1AAA caused significant inhibition of Shh secretion from, and a significant increase of Shh retention in, the transfected cells. ( G ) Quantification of secretion and retention of Shh. Error bars are s.e.m. Scale bar: 20 μm in A-D; 40 μm in E,F.
Figure Legend Snippet: Non-acylated Shh (C25S) is secreted in a Disp1-dependent manner. MDCK cells were grown on transwell filters and transfected with wild-type (Wt) Shh ( A ), Shh lacking cholesterol modification (C*) ( B ), Shh lacking amino terminal acyl modification (C25S) ( C ) and Shh with neither modification (C*/C25S) ( D ). The cells were grown overnight with 5E1 included in the basolateral compartment and visualized. The cholesterol moiety (present in A and C) mediates the retention of Shh outside the cells, whereas the loss of the acyl modification resulted in a shallow Shh gradient (C). Shh C25S was co-transfected either with Wt Disp1 ( E ) or Disp1AAA ( F ) into MDCK cells that were grown on transwell filters. Anti-Shh monoclonal 5E1 was included in the basolateral compartment for 16 hours and visualized. Disp1AAA caused significant inhibition of Shh secretion from, and a significant increase of Shh retention in, the transfected cells. ( G ) Quantification of secretion and retention of Shh. Error bars are s.e.m. Scale bar: 20 μm in A-D; 40 μm in E,F.

Techniques Used: Transfection, Modification, Inhibition

39) Product Images from "Vesicle Fusion Probability Is Determined by the Specific Interactions of Munc18 *"

Article Title: Vesicle Fusion Probability Is Determined by the Specific Interactions of Munc18 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.164038

Munc18 spatial distribution at the plasma membrane is unaffected by the mode of interaction. A , sequential frames of a single molecule of PA-mCherry-labeled munc18 on the plasma membrane demonstrating the quantal activation and bleaching characteristic of single fluorescent molecules. Each frame is a 300-ms integration with fluorophore activation by brief 405-nm illumination preceding the first frame. Scale bar , 1 μm. B , intensity plot over time for representative molecule bleaching events. The 405-nm activation pulse immediately preceded the first time point. The relative intensity of each molecule and the period of time each molecule spends emitting photons are stochastic. Bleaching events are quantal. C , frames containing detectable fluorescence averaged and displayed as an intensity profile plot for the region. The peak in fluorescence equates to the size of the point spread function of the microscope. D , Munc18 ( upper ) and munc18[I127A] ( lower ) TIRFM image ( left ), TIRFM image showing NPY-EGFP-labeled secretory vesicles ( center ), and a rendered map of single munc18 molecules, in munc18-1-silenced PC-12 cells (KD43), within the boxed region at the plasma membrane ( right ).
Figure Legend Snippet: Munc18 spatial distribution at the plasma membrane is unaffected by the mode of interaction. A , sequential frames of a single molecule of PA-mCherry-labeled munc18 on the plasma membrane demonstrating the quantal activation and bleaching characteristic of single fluorescent molecules. Each frame is a 300-ms integration with fluorophore activation by brief 405-nm illumination preceding the first frame. Scale bar , 1 μm. B , intensity plot over time for representative molecule bleaching events. The 405-nm activation pulse immediately preceded the first time point. The relative intensity of each molecule and the period of time each molecule spends emitting photons are stochastic. Bleaching events are quantal. C , frames containing detectable fluorescence averaged and displayed as an intensity profile plot for the region. The peak in fluorescence equates to the size of the point spread function of the microscope. D , Munc18 ( upper ) and munc18[I127A] ( lower ) TIRFM image ( left ), TIRFM image showing NPY-EGFP-labeled secretory vesicles ( center ), and a rendered map of single munc18 molecules, in munc18-1-silenced PC-12 cells (KD43), within the boxed region at the plasma membrane ( right ).

Techniques Used: Labeling, Activation Assay, Mass Spectrometry, Fluorescence, Microscopy

Targeted disruption of mode 2/3 interaction both in vitro and in live cells. A , structural alignment of munc18 (based on crystal structure PDB 3C98 ) ( 27 ) bound to syntaxin (gray helices) with amino acid conservation are shown on a color-coded scale ( red , low; blue , high conservation; left panel). Amino acids Asp 112 , Glu 132 , and Ile 127 , predicted to disrupt N-terminal binding, are highlighted on an enlarged view ( right panel ). B , truncation of the N terminus [Syx7–261] (to inhibit mode 2/3 binding and/or removal of the ionic layer [Syx1–225] of syntaxin (to inhibit mode 1 binding) did not eliminate binding to native munc18. The combination of these truncations or removal of the entire SNARE helix of syntaxin [Syx1–213] eliminated detectable binding to munc18. Mode 2/3 binding to syntaxin [Syx1–255] was sensitive to a high ionic strength wash. Bound material was analyzed by SDS-PAGE and Coomassie staining or Western blotting. C , GST-Syx7–261 and GST-Syx1–225 were immobilized on glutathione-Sepharose beads and incubated with bacterial lysate containing His 6 -munc18 or its mutant forms. Bound material was analyzed by SDS-PAGE and Coomassie staining. D , EYFP-munc18–1[I127A] resulted in a significant change in the fluorescence co-variance with mCer-syntaxin. Wild type or EYFP-munc18-1[I127A] ( red ) and mCer-Syx ( green ) were expressed in munc18-1 silenced PC-12 cells (KD43) and imaged by confocal laser scanning microscopy and subsequent image data deconvolution. The merge image shows areas of coincidence in yellow hues . The two-dimensional histogram represents the intensity for each channel in each voxel with a color scale representing frequency. The residual map displays weighted residuals from the line fit to the histogram, thus indicating fluorescence channel co-variance. The hue is from −1 to 1 with cyan corresponding to a 0 residual. Scale bar , 5 μm. E , cerulean-Syx1–288, in the presence of EYFP-munc18, in munc18-1-silenced PC-12 cells (KD43), exhibited a plasma membrane and intracellular membrane distribution. The color scale represents the fluorescence lifetime, and brightness represents intensity. The weighted mean fluorescence lifetime values were plotted as a frequency distribution histogram ( right panels ) with a mean fluorescence lifetime of 1568 ± 131 ps (mean ± S.E., n = 6). Munc18[I127A] resulted in a quenching of the fluorescence lifetime to a lesser extent compared with wild type munc18 ( lower panels ). These data are plotted ( lower right panel ) and reveal a single lifetime of 2047 ± 133 ps (mean ± S.E., n = 5). The dashed line on both graphs represents the mean fluorescence lifetime of cerulean-Syx1–288 in the presence of unfused munc18 and EYFP (2321 ± 40 ps (mean ± S.E., n = 10; supplemental Fig. 1 B ). Scale bar , 5 μm, fluorescence lifetime color bar 1250 ps ( red )–2250 ps ( blue ).
Figure Legend Snippet: Targeted disruption of mode 2/3 interaction both in vitro and in live cells. A , structural alignment of munc18 (based on crystal structure PDB 3C98 ) ( 27 ) bound to syntaxin (gray helices) with amino acid conservation are shown on a color-coded scale ( red , low; blue , high conservation; left panel). Amino acids Asp 112 , Glu 132 , and Ile 127 , predicted to disrupt N-terminal binding, are highlighted on an enlarged view ( right panel ). B , truncation of the N terminus [Syx7–261] (to inhibit mode 2/3 binding and/or removal of the ionic layer [Syx1–225] of syntaxin (to inhibit mode 1 binding) did not eliminate binding to native munc18. The combination of these truncations or removal of the entire SNARE helix of syntaxin [Syx1–213] eliminated detectable binding to munc18. Mode 2/3 binding to syntaxin [Syx1–255] was sensitive to a high ionic strength wash. Bound material was analyzed by SDS-PAGE and Coomassie staining or Western blotting. C , GST-Syx7–261 and GST-Syx1–225 were immobilized on glutathione-Sepharose beads and incubated with bacterial lysate containing His 6 -munc18 or its mutant forms. Bound material was analyzed by SDS-PAGE and Coomassie staining. D , EYFP-munc18–1[I127A] resulted in a significant change in the fluorescence co-variance with mCer-syntaxin. Wild type or EYFP-munc18-1[I127A] ( red ) and mCer-Syx ( green ) were expressed in munc18-1 silenced PC-12 cells (KD43) and imaged by confocal laser scanning microscopy and subsequent image data deconvolution. The merge image shows areas of coincidence in yellow hues . The two-dimensional histogram represents the intensity for each channel in each voxel with a color scale representing frequency. The residual map displays weighted residuals from the line fit to the histogram, thus indicating fluorescence channel co-variance. The hue is from −1 to 1 with cyan corresponding to a 0 residual. Scale bar , 5 μm. E , cerulean-Syx1–288, in the presence of EYFP-munc18, in munc18-1-silenced PC-12 cells (KD43), exhibited a plasma membrane and intracellular membrane distribution. The color scale represents the fluorescence lifetime, and brightness represents intensity. The weighted mean fluorescence lifetime values were plotted as a frequency distribution histogram ( right panels ) with a mean fluorescence lifetime of 1568 ± 131 ps (mean ± S.E., n = 6). Munc18[I127A] resulted in a quenching of the fluorescence lifetime to a lesser extent compared with wild type munc18 ( lower panels ). These data are plotted ( lower right panel ) and reveal a single lifetime of 2047 ± 133 ps (mean ± S.E., n = 5). The dashed line on both graphs represents the mean fluorescence lifetime of cerulean-Syx1–288 in the presence of unfused munc18 and EYFP (2321 ± 40 ps (mean ± S.E., n = 10; supplemental Fig. 1 B ). Scale bar , 5 μm, fluorescence lifetime color bar 1250 ps ( red )–2250 ps ( blue ).

Techniques Used: In Vitro, Binding Assay, SDS Page, Staining, Western Blot, Incubation, Mutagenesis, Fluorescence, Confocal Laser Scanning Microscopy

N-terminal interactions increase the fusion likelihood in a specific pool of vesicles. A , total number of labeled vesicles at the beginning of the experiment ( left panel ) compared with the number of fusion events ( right panel ) is shown. Individual fusion events were detected by analyzing the rate of fluorescence intensity change during the recording. Arrows indicate single-vesicle fusion events within munc18-1-silenced PC-12 cells (KD43) detected throughout the entire recording period of 3 min. Rescue with munc18[I127A] elicits fewer fusion events compared with wild type munc18. B , exocytosis is reduced by knockdown of munc18 and is partly rescued by munc18[I127A], compared with wild type munc18. Fusion events were calculated as the percentage of the total number of vesicles visible at the start of the recording that underwent fusion after stimulation with 300 μ m ATP. Error bars are S.E. (one-way ANOVA, n = 4). C , FLIM was used to quantify interaction changes specifically at the plasma membrane upon disruption of N-terminal binding, correlated with vesicle position. FLIM maps reveal that munc18[I127A] decreases the amount of interaction detected, specifically within large areas at the plasma membrane ( right panels ). Pixels containing donor fluorescence lifetime values > 2 SD below the mean, noninteracting control values obtained ( supplemental Fig. 1 B ) are shown ( red ). Pixels containing donor fluorescence lifetime values consistent with noninteracting syntaxin are shown ( gray scale ). D , majority of nonfusing vesicles have a limited displacement distance whereas a second, more highly mobile pool of vesicles has an increased likelihood of exocytosis. Graph shows the measured vesicle displacements from wild type ( dark gray ) and munc18[I127A]-rescued cells ( light gray ), best fit by a double Gaussian function (wild type, green ; [I127A], red ). Both distributions are bimodal, with a significant decrease in the magnitude of the higher mobility, fusion-competent pool of vesicles in the mutant cells (mean ± S.E., n = 4 cells; sum of squares F -test, p
Figure Legend Snippet: N-terminal interactions increase the fusion likelihood in a specific pool of vesicles. A , total number of labeled vesicles at the beginning of the experiment ( left panel ) compared with the number of fusion events ( right panel ) is shown. Individual fusion events were detected by analyzing the rate of fluorescence intensity change during the recording. Arrows indicate single-vesicle fusion events within munc18-1-silenced PC-12 cells (KD43) detected throughout the entire recording period of 3 min. Rescue with munc18[I127A] elicits fewer fusion events compared with wild type munc18. B , exocytosis is reduced by knockdown of munc18 and is partly rescued by munc18[I127A], compared with wild type munc18. Fusion events were calculated as the percentage of the total number of vesicles visible at the start of the recording that underwent fusion after stimulation with 300 μ m ATP. Error bars are S.E. (one-way ANOVA, n = 4). C , FLIM was used to quantify interaction changes specifically at the plasma membrane upon disruption of N-terminal binding, correlated with vesicle position. FLIM maps reveal that munc18[I127A] decreases the amount of interaction detected, specifically within large areas at the plasma membrane ( right panels ). Pixels containing donor fluorescence lifetime values > 2 SD below the mean, noninteracting control values obtained ( supplemental Fig. 1 B ) are shown ( red ). Pixels containing donor fluorescence lifetime values consistent with noninteracting syntaxin are shown ( gray scale ). D , majority of nonfusing vesicles have a limited displacement distance whereas a second, more highly mobile pool of vesicles has an increased likelihood of exocytosis. Graph shows the measured vesicle displacements from wild type ( dark gray ) and munc18[I127A]-rescued cells ( light gray ), best fit by a double Gaussian function (wild type, green ; [I127A], red ). Both distributions are bimodal, with a significant decrease in the magnitude of the higher mobility, fusion-competent pool of vesicles in the mutant cells (mean ± S.E., n = 4 cells; sum of squares F -test, p

Techniques Used: Labeling, Fluorescence, Binding Assay, Mutagenesis

Vesicle dynamics at the plasma membrane are modulated by munc18-syntaxin-N-terminal interaction. A and B , fluorescent vesicles were tracked under TIRF illumination in munc18-silenced PC-12 cells (KD43) expressing wild type munc18 ( A ) or munc18[I127A] ( B ). Individual vesicle trajectories are shown as tracks with color corresponding to time during acquisition ( blue (start) to red (end)). Examples most closely matching the mean track length for each condition are shown underneath each trajectory map. The dashed circles correspond to a vesicle diameter of 400 nm. C , individual tracks were measured for track length, mean vesicle speed, and vesicle track displacement ( > 250 tracks/cell, n = 5 cells). Rescue with munc18[I127A] resulted in reduced average track length, displacement and speed, indicative of a tighter tethering, compared with wild type munc18. Error bars are S.E. ( p
Figure Legend Snippet: Vesicle dynamics at the plasma membrane are modulated by munc18-syntaxin-N-terminal interaction. A and B , fluorescent vesicles were tracked under TIRF illumination in munc18-silenced PC-12 cells (KD43) expressing wild type munc18 ( A ) or munc18[I127A] ( B ). Individual vesicle trajectories are shown as tracks with color corresponding to time during acquisition ( blue (start) to red (end)). Examples most closely matching the mean track length for each condition are shown underneath each trajectory map. The dashed circles correspond to a vesicle diameter of 400 nm. C , individual tracks were measured for track length, mean vesicle speed, and vesicle track displacement ( > 250 tracks/cell, n = 5 cells). Rescue with munc18[I127A] resulted in reduced average track length, displacement and speed, indicative of a tighter tethering, compared with wild type munc18. Error bars are S.E. ( p

Techniques Used: Expressing

40) Product Images from "FBXW7-mutated colorectal cancer cells exhibit aberrant expression of phosphorylated-p53 at Serine-15"

Article Title: FBXW7-mutated colorectal cancer cells exhibit aberrant expression of phosphorylated-p53 at Serine-15

Journal: Oncotarget

doi:

phospho-p53(Ser15) is regulated by FBXW7 but not through direct interaction (A) FLAG-tagged versions of the indicated p53 mutant and GFP-FBXW7 proteins were expressed in HEK293T cells, and lysates were subjected to immunoblotting with anti-FLAG, anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (B) HEK293T cells were treated with cycloheximide (CHX) for the indicated time points. Lysates were examined by Western blotting anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (C) Western blot analysis of whole-cell lysates (input) (left-panel) and immunoprecipitates (IP) with GFP (middle-panel) and FLAG-p53 (right-panel) derived from 293T cells transfected with GFP-FBXW7 together with the FLAG-p53 constructs. Thirty hours after transfection, cells were pretreated with 10 μM MG132 for 4 h to block the proteasome pathway before cell collection. Both IPs was probed by anti-GFP and anti-FLAG antibodies simultaneously. (D) HCT116 and DLD-1 cells lacking or expressing FBXW7 were subjected to immunoblotting with anti-CK1α, pChk2 (S516), pChk2 (T66), phospho-p44/42 MAPK (pERK1/2) and p44/42 MAPK (ERK1/2). β-actin was loaded as a loading control. All experiments were repeated at least three times. (E) Immunofluorescent staining of anti-CK1α of HCT116 FBXW7(+/+) versus HCT116 FBXW7(−/−) cells. (F) Schematic of molecular changes may occur in the HCT116 FBXW7(−/−) compared with parental HCT116 FBXW7(+/+) cells treated with the oxaliplatin.
Figure Legend Snippet: phospho-p53(Ser15) is regulated by FBXW7 but not through direct interaction (A) FLAG-tagged versions of the indicated p53 mutant and GFP-FBXW7 proteins were expressed in HEK293T cells, and lysates were subjected to immunoblotting with anti-FLAG, anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (B) HEK293T cells were treated with cycloheximide (CHX) for the indicated time points. Lysates were examined by Western blotting anti-p-p53(Ser15) and anti-GFP. β-actin was used as a loading control. (C) Western blot analysis of whole-cell lysates (input) (left-panel) and immunoprecipitates (IP) with GFP (middle-panel) and FLAG-p53 (right-panel) derived from 293T cells transfected with GFP-FBXW7 together with the FLAG-p53 constructs. Thirty hours after transfection, cells were pretreated with 10 μM MG132 for 4 h to block the proteasome pathway before cell collection. Both IPs was probed by anti-GFP and anti-FLAG antibodies simultaneously. (D) HCT116 and DLD-1 cells lacking or expressing FBXW7 were subjected to immunoblotting with anti-CK1α, pChk2 (S516), pChk2 (T66), phospho-p44/42 MAPK (pERK1/2) and p44/42 MAPK (ERK1/2). β-actin was loaded as a loading control. All experiments were repeated at least three times. (E) Immunofluorescent staining of anti-CK1α of HCT116 FBXW7(+/+) versus HCT116 FBXW7(−/−) cells. (F) Schematic of molecular changes may occur in the HCT116 FBXW7(−/−) compared with parental HCT116 FBXW7(+/+) cells treated with the oxaliplatin.

Techniques Used: Mutagenesis, Western Blot, Derivative Assay, Transfection, Construct, Blocking Assay, Expressing, Staining

Induction in the level of the of murine phospho-p53(Ser18) in the intestine of fbxw7 ΔG mice (A) Schematic shows the floxed Fbxw7 allele ( fbxw 7 fl/fl ) before and after villin -Cre recombination to generate gut-specific conditional Fbxw7 inactivation ( fbxw 7 ΔG ) mice. (B) Western blot analysis of fbxw 7 fl/fl and fbxw 7 ΔG intestinal proteins using antibodies against Fbxw7, p53, phospho-p53(Ser15), and the loading control β-actin. Experiments were performed on at least two independent occasions.
Figure Legend Snippet: Induction in the level of the of murine phospho-p53(Ser18) in the intestine of fbxw7 ΔG mice (A) Schematic shows the floxed Fbxw7 allele ( fbxw 7 fl/fl ) before and after villin -Cre recombination to generate gut-specific conditional Fbxw7 inactivation ( fbxw 7 ΔG ) mice. (B) Western blot analysis of fbxw 7 fl/fl and fbxw 7 ΔG intestinal proteins using antibodies against Fbxw7, p53, phospho-p53(Ser15), and the loading control β-actin. Experiments were performed on at least two independent occasions.

Techniques Used: Mouse Assay, Western Blot

p53 levels remained unchanged in FBXW7 -mutant human CRC-tissues (A-D) IHC for p53 in human CRC with (C, D) and without (A, B) FBXW7 mutations. Boxed line indicates magnified tissue area. Scale bars; 50 μm. (E) Western blotting analysis of p53 expression in HCT116 and DLD-1 cell lines expressing or lacking FBXW7. β-actin was blotted for loading control. All experiments were repeated for two times.
Figure Legend Snippet: p53 levels remained unchanged in FBXW7 -mutant human CRC-tissues (A-D) IHC for p53 in human CRC with (C, D) and without (A, B) FBXW7 mutations. Boxed line indicates magnified tissue area. Scale bars; 50 μm. (E) Western blotting analysis of p53 expression in HCT116 and DLD-1 cell lines expressing or lacking FBXW7. β-actin was blotted for loading control. All experiments were repeated for two times.

Techniques Used: Mutagenesis, Immunohistochemistry, Western Blot, Expressing

Related Articles

Clone Assay:

Article Title: Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs ▿
Article Snippet: .. Plasmids containing the HIV-1 wild-type RT or env sequence were generated by amplifying the RT region from p83-2 or the env region from p83-10, BaL, or HE, using PfuTurbo DNA polymerase (Stratagene, Amsterdam, The Netherlands), and subsequently cloning the sequence by use of a Topo XL PCR cloning kit (Invitrogen). .. These constructs were used as templates in site-directed mutagenesis experiments to generate the following reference plasmids: p83-10-gp41-D36G (wild type for ENF), p83-10-gp41-D36G-V38M, envBaL-gp41-V38M, envHE-gp41-V38M, RT-Q151M, RT-K70R, RT-K65R, and RT-A62V-S68G-V75I-I77L-F116Y-Q151M.

Article Title: Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1 [OA]
Article Snippet: .. The calmodulin-insensitive AtCPSF30 mutant was generated from the pMALC2-AtCPSF30 clone using the QuickChange XL site-directed mutagenesis kit (Stratagene) and the oligonucleotides indicated in ; several independent clones were isolated, the mutation confirmed by DNA sequencing, and further analysis performed as indicated in the following. .. To produce MBP fusion proteins, 200 mL of LB media were inoculated with 10 mL of an overnight culture of transformed Rosetta (D3) cells and the 200-mL cultures grown at 37°C for 3 to 4 h. Expression of the fusion protein gene was induced by addition of 200 μ L of 1 m isopropylthio- β -galactoside.

Mutagenesis:

Article Title: Discovery and Characterization of Novel Subtype-Selective Allosteric Agonists for the Investigation of M1 Receptor Function in the Central Nervous System
Article Snippet: .. EE397/401DD, E397D, E401D, and E401A M1 receptor mutants were all generated from a pcDNA3.1+ vector containing WT rM1 cDNA template using Stratagene’s QuikChange XL site-directed mutagenesis kit (La Jolla, CA). .. For each PCR reaction, 10 ng of dsDNA template was amplified with 125 ng each of forward and reverse primer in a total reaction volume of 50 μL according to Stratagene’s protocol.

Article Title: The neuronal RhoA GEF, Tech, interacts with the synaptic multi-PDZ-domain-containing protein, MUPP1
Article Snippet: .. A mutant version of the dyad MUPP1 fragment containing domains 10 and 11, MUPP1(m10 & 11), was also constructed using the Stratagene Quikchange II XL kit, using MUPP1(10 & 11) as template. ..

Article Title: Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia
Article Snippet: .. The construct encoding the AS160-S588D mutation was generated in this plasmid with the QuikChange XL site-directed mutagenesis kit from Stratagene, and the mutations were verified by DNA sequencing. .. The AS160 WT-FLAG and S588D-FLAG constructs were transfected into the AS160 KD MDCK cell line with Lipofectamine 2000 (Invitrogen).

Article Title: The factor VIII C1 domain contributes to platelet binding
Article Snippet: .. Codon 2296 in C1C2 was changed from Ser (TCT) to Cys (TGT) using the QuickChange XL Site-Directed Mutagenesis Kit (Stratagene). ..

Article Title: Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1 [OA]
Article Snippet: .. The calmodulin-insensitive AtCPSF30 mutant was generated from the pMALC2-AtCPSF30 clone using the QuickChange XL site-directed mutagenesis kit (Stratagene) and the oligonucleotides indicated in ; several independent clones were isolated, the mutation confirmed by DNA sequencing, and further analysis performed as indicated in the following. .. To produce MBP fusion proteins, 200 mL of LB media were inoculated with 10 mL of an overnight culture of transformed Rosetta (D3) cells and the 200-mL cultures grown at 37°C for 3 to 4 h. Expression of the fusion protein gene was induced by addition of 200 μ L of 1 m isopropylthio- β -galactoside.

Article Title: A role for planar cell polarity signaling in angiogenesis
Article Snippet: .. K446M-Dvl2 was generated by introducing a point mutation into HA-Dvl2 using the QuikChange II XL Site-Directed Mutagenesis Kit (Cat#: 200521, Stratagene). .. Co-transfections with a given construct of interest and GFP-expressing construct were done at a respective 10:1 ratio (total, 6 μg DNA per 10-cm plate) using Lipofectamine 2000 (Invitrogen).

Isolation:

Article Title: Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1 [OA]
Article Snippet: .. The calmodulin-insensitive AtCPSF30 mutant was generated from the pMALC2-AtCPSF30 clone using the QuickChange XL site-directed mutagenesis kit (Stratagene) and the oligonucleotides indicated in ; several independent clones were isolated, the mutation confirmed by DNA sequencing, and further analysis performed as indicated in the following. .. To produce MBP fusion proteins, 200 mL of LB media were inoculated with 10 mL of an overnight culture of transformed Rosetta (D3) cells and the 200-mL cultures grown at 37°C for 3 to 4 h. Expression of the fusion protein gene was induced by addition of 200 μ L of 1 m isopropylthio- β -galactoside.

Construct:

Article Title: The neuronal RhoA GEF, Tech, interacts with the synaptic multi-PDZ-domain-containing protein, MUPP1
Article Snippet: .. A mutant version of the dyad MUPP1 fragment containing domains 10 and 11, MUPP1(m10 & 11), was also constructed using the Stratagene Quikchange II XL kit, using MUPP1(10 & 11) as template. ..

Article Title: Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia
Article Snippet: .. The construct encoding the AS160-S588D mutation was generated in this plasmid with the QuikChange XL site-directed mutagenesis kit from Stratagene, and the mutations were verified by DNA sequencing. .. The AS160 WT-FLAG and S588D-FLAG constructs were transfected into the AS160 KD MDCK cell line with Lipofectamine 2000 (Invitrogen).

Sequencing:

Article Title: Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs ▿
Article Snippet: .. Plasmids containing the HIV-1 wild-type RT or env sequence were generated by amplifying the RT region from p83-2 or the env region from p83-10, BaL, or HE, using PfuTurbo DNA polymerase (Stratagene, Amsterdam, The Netherlands), and subsequently cloning the sequence by use of a Topo XL PCR cloning kit (Invitrogen). .. These constructs were used as templates in site-directed mutagenesis experiments to generate the following reference plasmids: p83-10-gp41-D36G (wild type for ENF), p83-10-gp41-D36G-V38M, envBaL-gp41-V38M, envHE-gp41-V38M, RT-Q151M, RT-K70R, RT-K65R, and RT-A62V-S68G-V75I-I77L-F116Y-Q151M.

Generated:

Article Title: Discovery and Characterization of Novel Subtype-Selective Allosteric Agonists for the Investigation of M1 Receptor Function in the Central Nervous System
Article Snippet: .. EE397/401DD, E397D, E401D, and E401A M1 receptor mutants were all generated from a pcDNA3.1+ vector containing WT rM1 cDNA template using Stratagene’s QuikChange XL site-directed mutagenesis kit (La Jolla, CA). .. For each PCR reaction, 10 ng of dsDNA template was amplified with 125 ng each of forward and reverse primer in a total reaction volume of 50 μL according to Stratagene’s protocol.

Article Title: Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia
Article Snippet: .. The construct encoding the AS160-S588D mutation was generated in this plasmid with the QuikChange XL site-directed mutagenesis kit from Stratagene, and the mutations were verified by DNA sequencing. .. The AS160 WT-FLAG and S588D-FLAG constructs were transfected into the AS160 KD MDCK cell line with Lipofectamine 2000 (Invitrogen).

Article Title: Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs ▿
Article Snippet: .. Plasmids containing the HIV-1 wild-type RT or env sequence were generated by amplifying the RT region from p83-2 or the env region from p83-10, BaL, or HE, using PfuTurbo DNA polymerase (Stratagene, Amsterdam, The Netherlands), and subsequently cloning the sequence by use of a Topo XL PCR cloning kit (Invitrogen). .. These constructs were used as templates in site-directed mutagenesis experiments to generate the following reference plasmids: p83-10-gp41-D36G (wild type for ENF), p83-10-gp41-D36G-V38M, envBaL-gp41-V38M, envHE-gp41-V38M, RT-Q151M, RT-K70R, RT-K65R, and RT-A62V-S68G-V75I-I77L-F116Y-Q151M.

Article Title: Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1 [OA]
Article Snippet: .. The calmodulin-insensitive AtCPSF30 mutant was generated from the pMALC2-AtCPSF30 clone using the QuickChange XL site-directed mutagenesis kit (Stratagene) and the oligonucleotides indicated in ; several independent clones were isolated, the mutation confirmed by DNA sequencing, and further analysis performed as indicated in the following. .. To produce MBP fusion proteins, 200 mL of LB media were inoculated with 10 mL of an overnight culture of transformed Rosetta (D3) cells and the 200-mL cultures grown at 37°C for 3 to 4 h. Expression of the fusion protein gene was induced by addition of 200 μ L of 1 m isopropylthio- β -galactoside.

Article Title: A role for planar cell polarity signaling in angiogenesis
Article Snippet: .. K446M-Dvl2 was generated by introducing a point mutation into HA-Dvl2 using the QuikChange II XL Site-Directed Mutagenesis Kit (Cat#: 200521, Stratagene). .. Co-transfections with a given construct of interest and GFP-expressing construct were done at a respective 10:1 ratio (total, 6 μg DNA per 10-cm plate) using Lipofectamine 2000 (Invitrogen).

DNA Sequencing:

Article Title: Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia
Article Snippet: .. The construct encoding the AS160-S588D mutation was generated in this plasmid with the QuikChange XL site-directed mutagenesis kit from Stratagene, and the mutations were verified by DNA sequencing. .. The AS160 WT-FLAG and S588D-FLAG constructs were transfected into the AS160 KD MDCK cell line with Lipofectamine 2000 (Invitrogen).

Article Title: Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1Calmodulin Interacts with and Regulates the RNA-Binding Activity of an Arabidopsis Polyadenylation Factor Subunit 1 [OA]
Article Snippet: .. The calmodulin-insensitive AtCPSF30 mutant was generated from the pMALC2-AtCPSF30 clone using the QuickChange XL site-directed mutagenesis kit (Stratagene) and the oligonucleotides indicated in ; several independent clones were isolated, the mutation confirmed by DNA sequencing, and further analysis performed as indicated in the following. .. To produce MBP fusion proteins, 200 mL of LB media were inoculated with 10 mL of an overnight culture of transformed Rosetta (D3) cells and the 200-mL cultures grown at 37°C for 3 to 4 h. Expression of the fusion protein gene was induced by addition of 200 μ L of 1 m isopropylthio- β -galactoside.

Polymerase Chain Reaction:

Article Title: Novel Recombinant Virus Assay for Measuring Susceptibility of Human Immunodeficiency Virus Type 1 Group M Subtypes To Clinically Approved Drugs ▿
Article Snippet: .. Plasmids containing the HIV-1 wild-type RT or env sequence were generated by amplifying the RT region from p83-2 or the env region from p83-10, BaL, or HE, using PfuTurbo DNA polymerase (Stratagene, Amsterdam, The Netherlands), and subsequently cloning the sequence by use of a Topo XL PCR cloning kit (Invitrogen). .. These constructs were used as templates in site-directed mutagenesis experiments to generate the following reference plasmids: p83-10-gp41-D36G (wild type for ENF), p83-10-gp41-D36G-V38M, envBaL-gp41-V38M, envHE-gp41-V38M, RT-Q151M, RT-K70R, RT-K65R, and RT-A62V-S68G-V75I-I77L-F116Y-Q151M.

Plasmid Preparation:

Article Title: Discovery and Characterization of Novel Subtype-Selective Allosteric Agonists for the Investigation of M1 Receptor Function in the Central Nervous System
Article Snippet: .. EE397/401DD, E397D, E401D, and E401A M1 receptor mutants were all generated from a pcDNA3.1+ vector containing WT rM1 cDNA template using Stratagene’s QuikChange XL site-directed mutagenesis kit (La Jolla, CA). .. For each PCR reaction, 10 ng of dsDNA template was amplified with 125 ng each of forward and reverse primer in a total reaction volume of 50 μL according to Stratagene’s protocol.

Article Title: Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia
Article Snippet: .. The construct encoding the AS160-S588D mutation was generated in this plasmid with the QuikChange XL site-directed mutagenesis kit from Stratagene, and the mutations were verified by DNA sequencing. .. The AS160 WT-FLAG and S588D-FLAG constructs were transfected into the AS160 KD MDCK cell line with Lipofectamine 2000 (Invitrogen).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 89
    Stratagene e coli strain xl 1 blue
    Selection of Phabs binding to TouRΔA. The number of phage bound to ELISA plates coated with 6xhis TouRΔA after each panning round is indicated. The bound phage were eluted from the plates by incubation with 0.1 M glycine (pH 2.5) as explained in Materials and Methods. After recovery, the titers of these phage were determined on E . coli <t>XL-1</t> Blue cells and selected for AMP resistance. In each panning round, the number of input phage was kept constant at 2 × 10 11 PFU and the phage that did not bind 6xhis TouRΔA were removed by 40 washing steps with PBS. The increase in the number of bound phage after the second round of panning is indicative of a preferential amplification of Phab clones binding to 6xhis TouRΔA.
    E Coli Strain Xl 1 Blue, supplied by Stratagene, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli strain xl 1 blue/product/Stratagene
    Average 89 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    e coli strain xl 1 blue - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    86
    Stratagene xl 10 gold ultra competent e coli
    Selection of Phabs binding to TouRΔA. The number of phage bound to ELISA plates coated with 6xhis TouRΔA after each panning round is indicated. The bound phage were eluted from the plates by incubation with 0.1 M glycine (pH 2.5) as explained in Materials and Methods. After recovery, the titers of these phage were determined on E . coli <t>XL-1</t> Blue cells and selected for AMP resistance. In each panning round, the number of input phage was kept constant at 2 × 10 11 PFU and the phage that did not bind 6xhis TouRΔA were removed by 40 washing steps with PBS. The increase in the number of bound phage after the second round of panning is indicative of a preferential amplification of Phab clones binding to 6xhis TouRΔA.
    Xl 10 Gold Ultra Competent E Coli, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xl 10 gold ultra competent e coli/product/Stratagene
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    xl 10 gold ultra competent e coli - by Bioz Stars, 2020-09
    86/100 stars
      Buy from Supplier

    85
    Stratagene recombination deficient competent xl 10 gold bacteria
    Selection of Phabs binding to TouRΔA. The number of phage bound to ELISA plates coated with 6xhis TouRΔA after each panning round is indicated. The bound phage were eluted from the plates by incubation with 0.1 M glycine (pH 2.5) as explained in Materials and Methods. After recovery, the titers of these phage were determined on E . coli <t>XL-1</t> Blue cells and selected for AMP resistance. In each panning round, the number of input phage was kept constant at 2 × 10 11 PFU and the phage that did not bind 6xhis TouRΔA were removed by 40 washing steps with PBS. The increase in the number of bound phage after the second round of panning is indicative of a preferential amplification of Phab clones binding to 6xhis TouRΔA.
    Recombination Deficient Competent Xl 10 Gold Bacteria, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombination deficient competent xl 10 gold bacteria/product/Stratagene
    Average 85 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    recombination deficient competent xl 10 gold bacteria - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    88
    Stratagene escherichia coli xl 10 gold
    Selection of Phabs binding to TouRΔA. The number of phage bound to ELISA plates coated with 6xhis TouRΔA after each panning round is indicated. The bound phage were eluted from the plates by incubation with 0.1 M glycine (pH 2.5) as explained in Materials and Methods. After recovery, the titers of these phage were determined on E . coli <t>XL-1</t> Blue cells and selected for AMP resistance. In each panning round, the number of input phage was kept constant at 2 × 10 11 PFU and the phage that did not bind 6xhis TouRΔA were removed by 40 washing steps with PBS. The increase in the number of bound phage after the second round of panning is indicative of a preferential amplification of Phab clones binding to 6xhis TouRΔA.
    Escherichia Coli Xl 10 Gold, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/escherichia coli xl 10 gold/product/Stratagene
    Average 88 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    escherichia coli xl 10 gold - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    Image Search Results


    Selection of Phabs binding to TouRΔA. The number of phage bound to ELISA plates coated with 6xhis TouRΔA after each panning round is indicated. The bound phage were eluted from the plates by incubation with 0.1 M glycine (pH 2.5) as explained in Materials and Methods. After recovery, the titers of these phage were determined on E . coli XL-1 Blue cells and selected for AMP resistance. In each panning round, the number of input phage was kept constant at 2 × 10 11 PFU and the phage that did not bind 6xhis TouRΔA were removed by 40 washing steps with PBS. The increase in the number of bound phage after the second round of panning is indicative of a preferential amplification of Phab clones binding to 6xhis TouRΔA.

    Journal: Journal of Bacteriology

    Article Title: Monitoring Intracellular Levels of XylR in Pseudomonas putida with a Single-Chain Antibody Specific for Aromatic-Responsive Enhancer-Binding Proteins

    doi: 10.1128/JB.183.19.5571-5579.2001

    Figure Lengend Snippet: Selection of Phabs binding to TouRΔA. The number of phage bound to ELISA plates coated with 6xhis TouRΔA after each panning round is indicated. The bound phage were eluted from the plates by incubation with 0.1 M glycine (pH 2.5) as explained in Materials and Methods. After recovery, the titers of these phage were determined on E . coli XL-1 Blue cells and selected for AMP resistance. In each panning round, the number of input phage was kept constant at 2 × 10 11 PFU and the phage that did not bind 6xhis TouRΔA were removed by 40 washing steps with PBS. The increase in the number of bound phage after the second round of panning is indicative of a preferential amplification of Phab clones binding to 6xhis TouRΔA.

    Article Snippet: The E. coli strain XL-1 Blue ( recA1 gyrA96 relA1 endA1 hsdR17 supE44 thi1 lac [F′ proAB lacI q lacZ ΔM15 Tn 10 ] Tcr ; Stratagene) was used as host for bacteriophages and phagemids.

    Techniques: Selection, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Amplification, Clone Assay

    Amino acid sequence of scFv B7. The amino acid sequence of the scFv B7 polypeptide encoded by the phagemid is shown. The positions of the N-terminal signal peptide, the V H domain, the (Gly 4 Ser) 3 linker peptide, the V L domain, and the E tag are indicated. The complementarity-determining regions (CDR) of the V H and V L domains are labeled and underlined. The site of cleavage of the bacterial signal peptidase is marked by an arrow. The five amino acid changes found in the scFv B9 are marked below the sequence of scFv B7. When produced in E . coli XL-1 Blue cells ( supE ), these scFvs are also synthesized as hybrids with protein 3 of M13. The location of the suppressed stop codon (amber), which is placed between the scFv and protein 3 coding sequences, is indicated.

    Journal: Journal of Bacteriology

    Article Title: Monitoring Intracellular Levels of XylR in Pseudomonas putida with a Single-Chain Antibody Specific for Aromatic-Responsive Enhancer-Binding Proteins

    doi: 10.1128/JB.183.19.5571-5579.2001

    Figure Lengend Snippet: Amino acid sequence of scFv B7. The amino acid sequence of the scFv B7 polypeptide encoded by the phagemid is shown. The positions of the N-terminal signal peptide, the V H domain, the (Gly 4 Ser) 3 linker peptide, the V L domain, and the E tag are indicated. The complementarity-determining regions (CDR) of the V H and V L domains are labeled and underlined. The site of cleavage of the bacterial signal peptidase is marked by an arrow. The five amino acid changes found in the scFv B9 are marked below the sequence of scFv B7. When produced in E . coli XL-1 Blue cells ( supE ), these scFvs are also synthesized as hybrids with protein 3 of M13. The location of the suppressed stop codon (amber), which is placed between the scFv and protein 3 coding sequences, is indicated.

    Article Snippet: The E. coli strain XL-1 Blue ( recA1 gyrA96 relA1 endA1 hsdR17 supE44 thi1 lac [F′ proAB lacI q lacZ ΔM15 Tn 10 ] Tcr ; Stratagene) was used as host for bacteriophages and phagemids.

    Techniques: Sequencing, Labeling, Produced, Synthesized