Structured Review

Promega bsa
( a ) <t>p22</t> binding to the DIC column is blocked by p150 Glued . Two identical DIC affinity columns were constructed. One column was blocked with excess p150 Glued ( fifth through seventh lanes ), whereas the other was blocked with <t>BSA</t> as a control ( second through fourth lanes ). Rat brain cytosol (1 ml) was then loaded ( first lane ), and the 1 M NaCl eluates ( fourth and seventh lanes ) were analyzed by Western blotting. Amounts of sample loaded in load and flow through lanes are equivalent (4 μl each), as are wash and 1 M eluate lanes (20 μl each from a total of 50 μl TCA precipitate). The results show that, like other dynactin subunits, p22 does not bind to a DIC column that is preblocked with p150 Glued . ( b and c ) p22 antibody immunoprecipitates the dynactin complex. Immunoprecipitations were carried out using antibodies to p150 Glued (1.5 ml), p22 (0.7 ml), and beads only (control) on rat brain cytosol (1 ml each). After thorough washing with RIPA buffer, the precipitates were eluted with 100 μl 1× Laemmli sample buffer. 1 μl each of control and anti-p150 Glued precipitate and 2 μl of anti-p22 precipitate were loaded and analyzed by SDS-PAGE ( b ) followed by Western blotting ( c ). A panel of dynactin subunit was used. The results demonstrate that p22 antibody coprecipitates the same subunits coprecipitated by anti-p150 Glued antibody.
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1) Product Images from "Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells "

Article Title: Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells

Journal: The Journal of Cell Biology

doi:

( a ) p22 binding to the DIC column is blocked by p150 Glued . Two identical DIC affinity columns were constructed. One column was blocked with excess p150 Glued ( fifth through seventh lanes ), whereas the other was blocked with BSA as a control ( second through fourth lanes ). Rat brain cytosol (1 ml) was then loaded ( first lane ), and the 1 M NaCl eluates ( fourth and seventh lanes ) were analyzed by Western blotting. Amounts of sample loaded in load and flow through lanes are equivalent (4 μl each), as are wash and 1 M eluate lanes (20 μl each from a total of 50 μl TCA precipitate). The results show that, like other dynactin subunits, p22 does not bind to a DIC column that is preblocked with p150 Glued . ( b and c ) p22 antibody immunoprecipitates the dynactin complex. Immunoprecipitations were carried out using antibodies to p150 Glued (1.5 ml), p22 (0.7 ml), and beads only (control) on rat brain cytosol (1 ml each). After thorough washing with RIPA buffer, the precipitates were eluted with 100 μl 1× Laemmli sample buffer. 1 μl each of control and anti-p150 Glued precipitate and 2 μl of anti-p22 precipitate were loaded and analyzed by SDS-PAGE ( b ) followed by Western blotting ( c ). A panel of dynactin subunit was used. The results demonstrate that p22 antibody coprecipitates the same subunits coprecipitated by anti-p150 Glued antibody.
Figure Legend Snippet: ( a ) p22 binding to the DIC column is blocked by p150 Glued . Two identical DIC affinity columns were constructed. One column was blocked with excess p150 Glued ( fifth through seventh lanes ), whereas the other was blocked with BSA as a control ( second through fourth lanes ). Rat brain cytosol (1 ml) was then loaded ( first lane ), and the 1 M NaCl eluates ( fourth and seventh lanes ) were analyzed by Western blotting. Amounts of sample loaded in load and flow through lanes are equivalent (4 μl each), as are wash and 1 M eluate lanes (20 μl each from a total of 50 μl TCA precipitate). The results show that, like other dynactin subunits, p22 does not bind to a DIC column that is preblocked with p150 Glued . ( b and c ) p22 antibody immunoprecipitates the dynactin complex. Immunoprecipitations were carried out using antibodies to p150 Glued (1.5 ml), p22 (0.7 ml), and beads only (control) on rat brain cytosol (1 ml each). After thorough washing with RIPA buffer, the precipitates were eluted with 100 μl 1× Laemmli sample buffer. 1 μl each of control and anti-p150 Glued precipitate and 2 μl of anti-p22 precipitate were loaded and analyzed by SDS-PAGE ( b ) followed by Western blotting ( c ). A panel of dynactin subunit was used. The results demonstrate that p22 antibody coprecipitates the same subunits coprecipitated by anti-p150 Glued antibody.

Techniques Used: Binding Assay, Construct, Western Blot, Flow Cytometry, SDS Page

p22 binds to the p150 Glued subunit of dynactin. ATP-extract (500 μl) prepared from five rat brains was incubated with PBS alone (control) or with recombinant p50 (dynamitin) and subjected to linear density sucrose gradient (5–20%) for 18 h at 4°C in a Beckman SW41.Ti rotor at 32K rpm. Approximately 0.9-ml fractions were collected, and the fractions were analyzed by SDS-PAGE followed by Western blotting using antibodies to p150 Glued , Arp1, p50, and p22. In a , all subunits probed peak exclusively at fraction 5, corresponding to the 20- S peak. However, incubation of ATP-extract with recombinant p50 partially disrupts the dynactin complex as indicated by the presence of p150 Glued at fractions 9–11 ( b ). Interestingly, p22 is also found at the second peak at fraction 10. Note that the heavy p50 staining in b is due to excess recombinant p50 used for dynactin disruption. ( c ) A p150 Glued affinity column and a BSA control column were constructed and loaded with in vitro–translated and radio-labeled recombinant p22. The columns were extensively washed and eluted with 1 M NaCl. The loaded material ( load ), flow-through ( F.T. ), wash, and the eluate samples were analyzed by SDS-PAGE followed by autoradiography.
Figure Legend Snippet: p22 binds to the p150 Glued subunit of dynactin. ATP-extract (500 μl) prepared from five rat brains was incubated with PBS alone (control) or with recombinant p50 (dynamitin) and subjected to linear density sucrose gradient (5–20%) for 18 h at 4°C in a Beckman SW41.Ti rotor at 32K rpm. Approximately 0.9-ml fractions were collected, and the fractions were analyzed by SDS-PAGE followed by Western blotting using antibodies to p150 Glued , Arp1, p50, and p22. In a , all subunits probed peak exclusively at fraction 5, corresponding to the 20- S peak. However, incubation of ATP-extract with recombinant p50 partially disrupts the dynactin complex as indicated by the presence of p150 Glued at fractions 9–11 ( b ). Interestingly, p22 is also found at the second peak at fraction 10. Note that the heavy p50 staining in b is due to excess recombinant p50 used for dynactin disruption. ( c ) A p150 Glued affinity column and a BSA control column were constructed and loaded with in vitro–translated and radio-labeled recombinant p22. The columns were extensively washed and eluted with 1 M NaCl. The loaded material ( load ), flow-through ( F.T. ), wash, and the eluate samples were analyzed by SDS-PAGE followed by autoradiography.

Techniques Used: Incubation, Recombinant, SDS Page, Western Blot, Staining, Affinity Column, Construct, In Vitro, Labeling, Flow Cytometry, Autoradiography

2) Product Images from "RBM24 stabilizes hepatitis B virus pregenomic RNA but inhibits core protein translation by targeting the terminal redundancy sequence"

Article Title: RBM24 stabilizes hepatitis B virus pregenomic RNA but inhibits core protein translation by targeting the terminal redundancy sequence

Journal: Emerging Microbes & Infections

doi: 10.1038/s41426-018-0091-4

RBM24 inhibits the translation of the core protein by binding to the 5′ TR of pgRNA and blocking 80S ribosome assembly on HBV pgRNA. a The HepG2.2.15 cell line was transfected with siNC or siRBM24 (left panels), HepG2 cells were co-transfected with pHY106 and pRBM24 or empty vector (right panels). Cell lysates were collected at 48 hpt, and the expression of core and RBM24 was detected by western blotting. b HepG2 cells were co-transfected with pHA-core and pRBM24 or empty vector, and the expression of core and RBM24 was detected by western blotting. c HepG2 cells were co-transfected with pTR-core and the indicated siRNA or plasmid and harvested at 48 hpt. The expression of core and RBM24 was detected by western blotting. d HepG2 cells were transfected with the indicated plasmids, and luciferase activity was determined with Steady-Glo ® . The relative luciferase activity (RLA) values were calculated and are shown in the bar graph on the left. The luciferase activity of 5′ TR-associated luciferase reporter plasmids in vitro was detected (bar graph on the right). e The 5′ TR-luciferase RNA together with rhRBM24 or BSA was incubated in rabbit reticulocyte lysate (RRL). The ribosome complexes were separated by sucrose gradient ultracentrifugation. The distribution of biotin-RNA was detected using a dot-blot assay
Figure Legend Snippet: RBM24 inhibits the translation of the core protein by binding to the 5′ TR of pgRNA and blocking 80S ribosome assembly on HBV pgRNA. a The HepG2.2.15 cell line was transfected with siNC or siRBM24 (left panels), HepG2 cells were co-transfected with pHY106 and pRBM24 or empty vector (right panels). Cell lysates were collected at 48 hpt, and the expression of core and RBM24 was detected by western blotting. b HepG2 cells were co-transfected with pHA-core and pRBM24 or empty vector, and the expression of core and RBM24 was detected by western blotting. c HepG2 cells were co-transfected with pTR-core and the indicated siRNA or plasmid and harvested at 48 hpt. The expression of core and RBM24 was detected by western blotting. d HepG2 cells were transfected with the indicated plasmids, and luciferase activity was determined with Steady-Glo ® . The relative luciferase activity (RLA) values were calculated and are shown in the bar graph on the left. The luciferase activity of 5′ TR-associated luciferase reporter plasmids in vitro was detected (bar graph on the right). e The 5′ TR-luciferase RNA together with rhRBM24 or BSA was incubated in rabbit reticulocyte lysate (RRL). The ribosome complexes were separated by sucrose gradient ultracentrifugation. The distribution of biotin-RNA was detected using a dot-blot assay

Techniques Used: Protein Binding, Blocking Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Luciferase, Activity Assay, In Vitro, Incubation, Dot Blot

3) Product Images from "Receptor-Mediated Activation of a Proinsulin-Transferrin Fusion Protein in Hepatoma Cells"

Article Title: Receptor-Mediated Activation of a Proinsulin-Transferrin Fusion Protein in Hepatoma Cells

Journal: Journal of controlled release : official journal of the Controlled Release Society

doi: 10.1016/j.jconrel.2011.06.029

Progressive decrease of ProINS-Tf fusion protein in H4IIE cells. (A) ProINS-Tf (100 pM), in the presence or absence of a 1000-fold excess of apo-Tf or BSA (ProINS-Tf+1000Tf or ProINS-Tf+1000BSA, respectively), were incubated either with H4IIE cell monolayers
Figure Legend Snippet: Progressive decrease of ProINS-Tf fusion protein in H4IIE cells. (A) ProINS-Tf (100 pM), in the presence or absence of a 1000-fold excess of apo-Tf or BSA (ProINS-Tf+1000Tf or ProINS-Tf+1000BSA, respectively), were incubated either with H4IIE cell monolayers

Techniques Used: Incubation

Conversion of ProINS-Tf to irINS-Tf in H4IIE cells. (A) H4IIE cells were treated with 10 nM of ProINS, or 10 nM of ProINS-Tf in the presence or absence of 1000-fold excess of apo-Tf or BSA at 37 °C (ProINS-Tf+1000Tf or ProINS-Tf+1000BSA, respectively).
Figure Legend Snippet: Conversion of ProINS-Tf to irINS-Tf in H4IIE cells. (A) H4IIE cells were treated with 10 nM of ProINS, or 10 nM of ProINS-Tf in the presence or absence of 1000-fold excess of apo-Tf or BSA at 37 °C (ProINS-Tf+1000Tf or ProINS-Tf+1000BSA, respectively).

Techniques Used:

4) Product Images from "Prototype foamy virus intasome aggregation is mediated by outer protein domains and prevented by protocatechuic acid"

Article Title: Prototype foamy virus intasome aggregation is mediated by outer protein domains and prevented by protocatechuic acid

Journal: Scientific Reports

doi: 10.1038/s41598-018-36725-1

Rescue of PFV intasome activity by additives present during preincubation. PFV FL intasomes were incubated at 37 °C for 5 min. Following the preincubation, target DNA was added and reactions were incubated for an additional 5 min at 37 °C. Additives included in the reactions were: 5 mM PCA (PCA), 0.1 mg/ml acetylated BSA (BSA), 5% DMSO (DMSO), 10% glycerol (Gly), 10% sucrose (Suc), and 10% PEG 6000 (PEG). Control reactions include no preincubation (No preinc) or no addition of an additive (−). Fluorescent products were analyzed and expressed as the fraction of fluorescent signal in each lane (bottom graph). Experiments were performed at least three times with at least two independent intasome preparations. Error bars indicate standard deviation.
Figure Legend Snippet: Rescue of PFV intasome activity by additives present during preincubation. PFV FL intasomes were incubated at 37 °C for 5 min. Following the preincubation, target DNA was added and reactions were incubated for an additional 5 min at 37 °C. Additives included in the reactions were: 5 mM PCA (PCA), 0.1 mg/ml acetylated BSA (BSA), 5% DMSO (DMSO), 10% glycerol (Gly), 10% sucrose (Suc), and 10% PEG 6000 (PEG). Control reactions include no preincubation (No preinc) or no addition of an additive (−). Fluorescent products were analyzed and expressed as the fraction of fluorescent signal in each lane (bottom graph). Experiments were performed at least three times with at least two independent intasome preparations. Error bars indicate standard deviation.

Techniques Used: Activity Assay, Incubation, Standard Deviation

5) Product Images from "Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA"

Article Title: Molecular affinity rulers: systematic evaluation of DNA aptamers for their applicabilities in ELISA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkz688

Detection of the target proteins using aptamer variants as the primary detector agents in ELONA. The target was directly immobilized on the plate surface by an incubation with the target solution (100 μl per well) at each indicated concentration, in the presence of 1 μg/ml of BSA in 0.1 M carbonate buffer (pH 9.6), for 2 h. After the incubation, the plate surfaces were further coated with blocking solution (1% BSA in 1× d -PBS(–), 300 μl per well) for 2 h. After the blocking reaction, 100 μl of the detector solution (10 nM each aptamer derivative in 1× binding buffer) was added to each well and then the binding reaction was performed for 30 min. After the incubation, 100 μl of the secondary detector solution (50 ng/ml HRP-conjugated streptavidin in 1× binding buffer) was added to each well, and then incubated for 30 min. After washing the wells, the TMB reaction (100 μl per well) was performed for 5 min (left, VEGF 165 ) or 15 min (right, IFNγ). The sample size is two per each combination set, and the error bars represent one standard deviation. The bars with wavy lines indicate that at least one of the two sample wells showed overflow (OD 450 > 4.000).
Figure Legend Snippet: Detection of the target proteins using aptamer variants as the primary detector agents in ELONA. The target was directly immobilized on the plate surface by an incubation with the target solution (100 μl per well) at each indicated concentration, in the presence of 1 μg/ml of BSA in 0.1 M carbonate buffer (pH 9.6), for 2 h. After the incubation, the plate surfaces were further coated with blocking solution (1% BSA in 1× d -PBS(–), 300 μl per well) for 2 h. After the blocking reaction, 100 μl of the detector solution (10 nM each aptamer derivative in 1× binding buffer) was added to each well and then the binding reaction was performed for 30 min. After the incubation, 100 μl of the secondary detector solution (50 ng/ml HRP-conjugated streptavidin in 1× binding buffer) was added to each well, and then incubated for 30 min. After washing the wells, the TMB reaction (100 μl per well) was performed for 5 min (left, VEGF 165 ) or 15 min (right, IFNγ). The sample size is two per each combination set, and the error bars represent one standard deviation. The bars with wavy lines indicate that at least one of the two sample wells showed overflow (OD 450 > 4.000).

Techniques Used: Incubation, Concentration Assay, Blocking Assay, Binding Assay, Standard Deviation

6) Product Images from "Prototype foamy virus intasome aggregation is mediated by outer protein domains and prevented by protocatechuic acid"

Article Title: Prototype foamy virus intasome aggregation is mediated by outer protein domains and prevented by protocatechuic acid

Journal: Scientific Reports

doi: 10.1038/s41598-018-36725-1

Rescue of PFV intasome activity by additives present during preincubation. PFV FL intasomes were incubated at 37 °C for 5 min. Following the preincubation, target DNA was added and reactions were incubated for an additional 5 min at 37 °C. Additives included in the reactions were: 5 mM PCA (PCA), 0.1 mg/ml acetylated BSA (BSA), 5% DMSO (DMSO), 10% glycerol (Gly), 10% sucrose (Suc), and 10% PEG 6000 (PEG). Control reactions include no preincubation (No preinc) or no addition of an additive (−). Fluorescent products were analyzed and expressed as the fraction of fluorescent signal in each lane (bottom graph). Experiments were performed at least three times with at least two independent intasome preparations. Error bars indicate standard deviation.
Figure Legend Snippet: Rescue of PFV intasome activity by additives present during preincubation. PFV FL intasomes were incubated at 37 °C for 5 min. Following the preincubation, target DNA was added and reactions were incubated for an additional 5 min at 37 °C. Additives included in the reactions were: 5 mM PCA (PCA), 0.1 mg/ml acetylated BSA (BSA), 5% DMSO (DMSO), 10% glycerol (Gly), 10% sucrose (Suc), and 10% PEG 6000 (PEG). Control reactions include no preincubation (No preinc) or no addition of an additive (−). Fluorescent products were analyzed and expressed as the fraction of fluorescent signal in each lane (bottom graph). Experiments were performed at least three times with at least two independent intasome preparations. Error bars indicate standard deviation.

Techniques Used: Activity Assay, Incubation, Standard Deviation

7) Product Images from "IL-4 impairs wound healing potential in the skin by repressing fibronectin expression"

Article Title: IL-4 impairs wound healing potential in the skin by repressing fibronectin expression

Journal: The Journal of allergy and clinical immunology

doi: 10.1016/j.jaci.2016.07.012

IL-4-stimulated keratinocytes show delayed wound healing ( A ) Human keratinocytes (HK) were differentiated for 5 days with calcium chloride in the presence or absence of IL-4 before analysis of FN1 expression by qRT-PCR. (B) Cell lysates were collected and the expression of fibronectin and β-actin was determined by western blot ( C ) Gene expression was analyzed 3h after scratching using qRT–PCR. ( D ) Percentage of scratch closed after 24h of culture, and ( E ) microscopy of human keratinocytes differentiated with calcium chloride alone or calcium chloride plus IL-4 for 5 days in control or plates covered with a substratum of BSA or fibronectin. Dashed red lines represent original wounds. Pictures are representative of 4 fields analyzed over at least three independent experiments. Data represents the mean of three independent experiments. *p
Figure Legend Snippet: IL-4-stimulated keratinocytes show delayed wound healing ( A ) Human keratinocytes (HK) were differentiated for 5 days with calcium chloride in the presence or absence of IL-4 before analysis of FN1 expression by qRT-PCR. (B) Cell lysates were collected and the expression of fibronectin and β-actin was determined by western blot ( C ) Gene expression was analyzed 3h after scratching using qRT–PCR. ( D ) Percentage of scratch closed after 24h of culture, and ( E ) microscopy of human keratinocytes differentiated with calcium chloride alone or calcium chloride plus IL-4 for 5 days in control or plates covered with a substratum of BSA or fibronectin. Dashed red lines represent original wounds. Pictures are representative of 4 fields analyzed over at least three independent experiments. Data represents the mean of three independent experiments. *p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Microscopy

8) Product Images from "Smad1 transcription factor integrates BMP2 and Wnt3a signals in migrating cardiac progenitor cells"

Article Title: Smad1 transcription factor integrates BMP2 and Wnt3a signals in migrating cardiac progenitor cells

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

doi: 10.1073/pnas.1321764111

Active forms of Smad1 alter CPC migration trajectories. (A) Schematic representation of Smad, indicating C terminus Ser-Ser-X-Ser motif. (B) Schematic showing where HH5 embryos were grafted with BSA, BMP2 beads, or a Wnt3a pellet and immunostained for
Figure Legend Snippet: Active forms of Smad1 alter CPC migration trajectories. (A) Schematic representation of Smad, indicating C terminus Ser-Ser-X-Ser motif. (B) Schematic showing where HH5 embryos were grafted with BSA, BMP2 beads, or a Wnt3a pellet and immunostained for

Techniques Used: Migration

9) Product Images from "PKC? Is Activated in a Dietary Model of Steatohepatitis and Regulates Endoplasmic Reticulum Stress and Cell Death *"

Article Title: PKC? Is Activated in a Dietary Model of Steatohepatitis and Regulates Endoplasmic Reticulum Stress and Cell Death *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.168575

Triglyceride accumulation and ALT release in McA cells. McA cells were treated with control or MCD medium with palmitic ( PA ), oleic ( OA ), or linoleic ( LA ) acid or BSA ( Con ) for 16 h. Cells were homogenized and extracted to determine TG levels ( A ), or
Figure Legend Snippet: Triglyceride accumulation and ALT release in McA cells. McA cells were treated with control or MCD medium with palmitic ( PA ), oleic ( OA ), or linoleic ( LA ) acid or BSA ( Con ) for 16 h. Cells were homogenized and extracted to determine TG levels ( A ), or

Techniques Used:

Role of PKCδ and ER stress in MCD medium/palmitic acid-induced cell death in McA cells. McA cells expressing PKCδ shRNAs or a Luc control were treated with control or MCD medium with palmitic acid ( PA ) or BSA ( Con ) for 24 h. A , cell viability
Figure Legend Snippet: Role of PKCδ and ER stress in MCD medium/palmitic acid-induced cell death in McA cells. McA cells expressing PKCδ shRNAs or a Luc control were treated with control or MCD medium with palmitic acid ( PA ) or BSA ( Con ) for 24 h. A , cell viability

Techniques Used: Expressing

Chemical chaperone inhibition of MCD medium-induced ER stress and PKCδ activation in McA cells. McA cells were pretreated with TMAO as indicated for 6 h and then treated with control or MCD medium with palmitic acid ( P ) or BSA ( Con ) for 16 h.
Figure Legend Snippet: Chemical chaperone inhibition of MCD medium-induced ER stress and PKCδ activation in McA cells. McA cells were pretreated with TMAO as indicated for 6 h and then treated with control or MCD medium with palmitic acid ( P ) or BSA ( Con ) for 16 h.

Techniques Used: Inhibition, Activation Assay

10) Product Images from "RBM24 stabilizes hepatitis B virus pregenomic RNA but inhibits core protein translation by targeting the terminal redundancy sequence"

Article Title: RBM24 stabilizes hepatitis B virus pregenomic RNA but inhibits core protein translation by targeting the terminal redundancy sequence

Journal: Emerging Microbes & Infections

doi: 10.1038/s41426-018-0091-4

RBM24 inhibits the translation of the core protein by binding to the 5′ TR of pgRNA and blocking 80S ribosome assembly on HBV pgRNA. a The HepG2.2.15 cell line was transfected with siNC or siRBM24 (left panels), HepG2 cells were co-transfected with pHY106 and pRBM24 or empty vector (right panels). Cell lysates were collected at 48 hpt, and the expression of core and RBM24 was detected by western blotting. b HepG2 cells were co-transfected with pHA-core and pRBM24 or empty vector, and the expression of core and RBM24 was detected by western blotting. c HepG2 cells were co-transfected with pTR-core and the indicated siRNA or plasmid and harvested at 48 hpt. The expression of core and RBM24 was detected by western blotting. d HepG2 cells were transfected with the indicated plasmids, and luciferase activity was determined with Steady-Glo ® . The relative luciferase activity (RLA) values were calculated and are shown in the bar graph on the left. The luciferase activity of 5′ TR-associated luciferase reporter plasmids in vitro was detected (bar graph on the right). e The 5′ TR-luciferase RNA together with rhRBM24 or BSA was incubated in rabbit reticulocyte lysate (RRL). The ribosome complexes were separated by sucrose gradient ultracentrifugation. The distribution of biotin-RNA was detected using a dot-blot assay
Figure Legend Snippet: RBM24 inhibits the translation of the core protein by binding to the 5′ TR of pgRNA and blocking 80S ribosome assembly on HBV pgRNA. a The HepG2.2.15 cell line was transfected with siNC or siRBM24 (left panels), HepG2 cells were co-transfected with pHY106 and pRBM24 or empty vector (right panels). Cell lysates were collected at 48 hpt, and the expression of core and RBM24 was detected by western blotting. b HepG2 cells were co-transfected with pHA-core and pRBM24 or empty vector, and the expression of core and RBM24 was detected by western blotting. c HepG2 cells were co-transfected with pTR-core and the indicated siRNA or plasmid and harvested at 48 hpt. The expression of core and RBM24 was detected by western blotting. d HepG2 cells were transfected with the indicated plasmids, and luciferase activity was determined with Steady-Glo ® . The relative luciferase activity (RLA) values were calculated and are shown in the bar graph on the left. The luciferase activity of 5′ TR-associated luciferase reporter plasmids in vitro was detected (bar graph on the right). e The 5′ TR-luciferase RNA together with rhRBM24 or BSA was incubated in rabbit reticulocyte lysate (RRL). The ribosome complexes were separated by sucrose gradient ultracentrifugation. The distribution of biotin-RNA was detected using a dot-blot assay

Techniques Used: Protein Binding, Blocking Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Luciferase, Activity Assay, In Vitro, Incubation, Dot Blot

11) Product Images from "Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif"

Article Title: Pathogen recognition of a novel C-type lectin from Marsupenaeus japonicus reveals the divergent sugar-binding specificity of QAP motif

Journal: Scientific Reports

doi: 10.1038/srep45818

Bacterial agglutination activity of rMjGCTL with Gram-negative V. parahaemolyticus and Gram-positive S. agalactiae . Bacterial suspensions were incubated with rMjGCTL, BSA as the protein control, and TBS-Ca 2+ (TBSCa) as the negative control.
Figure Legend Snippet: Bacterial agglutination activity of rMjGCTL with Gram-negative V. parahaemolyticus and Gram-positive S. agalactiae . Bacterial suspensions were incubated with rMjGCTL, BSA as the protein control, and TBS-Ca 2+ (TBSCa) as the negative control.

Techniques Used: Agglutination, Activity Assay, Incubation, Negative Control

12) Product Images from "Substantial Histone Reduction Modulates Genomewide Nucleosomal Occupancy and Global Transcriptional OutputNot All Genes Are Equal; Shortage of Histones Affects Some Genes More Than Others"

Article Title: Substantial Histone Reduction Modulates Genomewide Nucleosomal Occupancy and Global Transcriptional OutputNot All Genes Are Equal; Shortage of Histones Affects Some Genes More Than Others

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1001086

HMGB1 promotes the assembly of chromatin in vitro. (A) Chromatin was assembled in vitro on linear DNA using purified histones, hNAP1, ACF, and increasing amounts of HMGB1 protein; then it was digested with MNase. The residual DNA after digestion was electrophoresed on a 1.5% agarose gel (upper panel) and quantified with PicoGreen (normalized to the reaction containing BSA, lower panel). Error bars, SD of three replicates. (B) Chromatin was assembled in the presence of a fixed amount of HMGB1 (1 µg/ml), or BSA as control, for the indicated time points. Electrophoresis (upper panel) and quantification by PicoGreen (lower panel) of the residual DNA after digestion with MNase are shown. Error bars, SD of three replicates. MW: 100 bp ladder.
Figure Legend Snippet: HMGB1 promotes the assembly of chromatin in vitro. (A) Chromatin was assembled in vitro on linear DNA using purified histones, hNAP1, ACF, and increasing amounts of HMGB1 protein; then it was digested with MNase. The residual DNA after digestion was electrophoresed on a 1.5% agarose gel (upper panel) and quantified with PicoGreen (normalized to the reaction containing BSA, lower panel). Error bars, SD of three replicates. (B) Chromatin was assembled in the presence of a fixed amount of HMGB1 (1 µg/ml), or BSA as control, for the indicated time points. Electrophoresis (upper panel) and quantification by PicoGreen (lower panel) of the residual DNA after digestion with MNase are shown. Error bars, SD of three replicates. MW: 100 bp ladder.

Techniques Used: In Vitro, Purification, Agarose Gel Electrophoresis, Electrophoresis

13) Product Images from "CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma"

Article Title: CD204 suppresses large heat shock protein-facilitated priming of tumor antigen gp100-specific T cells and chaperone vaccine activity against mouse melanoma

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1100703

SRA/CD204 suppresses large HSP-facilitated T cell activation in vitro A. Increased proliferation of gp100-specific T cells when co-cultured with chaperone vaccine-loaded SRA −/− DCs. DCs were pulsed with hsp110-gp100 complexes for 5 h and then cultured with naïve CD8 + T cells from pmel transgenic mice. Cell proliferation was assayed by incorporation of 3 H-TdR. B. Increased stimulation of pmel cells by chaperone vaccine-loaded SRA −/− DCs. Following DC-pmel cell co-culture, IL-2 levels in supernatant were measured by ELISA assays. C. DCs were incubated with BSA-gp100 or hsp110-gp100 complexes, and used to stimulate pmel cells. IL-2 levels in the media were assayed by ELISA. D. DCs were pulsed with hsp110-gp100 complexes or grp170-gp100 complexes, and assayed for their ability to stimulate gp100-specific T cells. E and F . SRA/CD204 silencing enhances large HSP-mediated T cell stimulation. BMDCs were infected with lentiviruses encoding scramble shRNA or SRA/CD204 shRNA. 2 days later, cells were incubated with hsp110-gp100 ( E ) or grp170-gp100 ( F ), and co-cultured with pmel cells at different ratios. Pmel cell proliferation was measured using 3 H-TdR incorporation assays. ShRNA-mediated downregulation of SRA/CD204 expression in DCs was confirmed using immunoblotting ( E ). (Values are means ± S.D., * p
Figure Legend Snippet: SRA/CD204 suppresses large HSP-facilitated T cell activation in vitro A. Increased proliferation of gp100-specific T cells when co-cultured with chaperone vaccine-loaded SRA −/− DCs. DCs were pulsed with hsp110-gp100 complexes for 5 h and then cultured with naïve CD8 + T cells from pmel transgenic mice. Cell proliferation was assayed by incorporation of 3 H-TdR. B. Increased stimulation of pmel cells by chaperone vaccine-loaded SRA −/− DCs. Following DC-pmel cell co-culture, IL-2 levels in supernatant were measured by ELISA assays. C. DCs were incubated with BSA-gp100 or hsp110-gp100 complexes, and used to stimulate pmel cells. IL-2 levels in the media were assayed by ELISA. D. DCs were pulsed with hsp110-gp100 complexes or grp170-gp100 complexes, and assayed for their ability to stimulate gp100-specific T cells. E and F . SRA/CD204 silencing enhances large HSP-mediated T cell stimulation. BMDCs were infected with lentiviruses encoding scramble shRNA or SRA/CD204 shRNA. 2 days later, cells were incubated with hsp110-gp100 ( E ) or grp170-gp100 ( F ), and co-cultured with pmel cells at different ratios. Pmel cell proliferation was measured using 3 H-TdR incorporation assays. ShRNA-mediated downregulation of SRA/CD204 expression in DCs was confirmed using immunoblotting ( E ). (Values are means ± S.D., * p

Techniques Used: Activation Assay, In Vitro, Cell Culture, Transgenic Assay, Mouse Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Incubation, Cell Stimulation, Infection, shRNA, Expressing

SRA/CD204 absence reduces binding and internalization of hsp110 A. SRA/CD204 directly interacts with exogenous hsp110. WT BMDCs were incubated with His-hsp110 at 4 °C for 30 min, and then extensively washed with PBS. Cell lysates were immunoprecipitated with antibodies for SRA/CD204 (top) or His-tag (bottom). The immunocomplexes were subjected to immunoblotting analyses using antibodies for His-tag (1:4000, top) or SRA/CD204 (1:3000, bottom). IgG serves as a negative control. B. BMDCs were incubated with His-hsp110 or His-gp100 protein. SRA/CD204 was immunoprecipitated and analyzed for its association with hsp110 or gp100 using His-tag antibodies. C . Competition assays. DCs were incubated with biotinylated-His-hsp110 (40 μg/ml) in the presence of 3× or 9× excess of His-gp100 or His-Hsp110. Immunocomplexes pulled down by SRA/CD204 antibodies were analyzed by immunoblot using Avidin-HRP or anti-SRA antibodies (left). Alternatively, DCs were incubated with His-hsp110 in the presence of BSA or autologous hsp110-purified from mouse spleen, followed by immunoblotting analysis of His-hsp110 association with immunoprecipitated SRA/CD204 (right). D and E . SRA/CD204 absence results in reduced hsp110 binding and internalization. WT and SRA −/− BMDCs were incubated with His-hsp110 for 30 min at either 4 °C ( D ) or 37 °C ( E ). Cells were washed and cultured at 37 °C for the times indicated. Total cell lysates were prepared and analyzed for the presence of His-hsp110 using His-tag antibodies. Immunoblots were quantified by densitometry analysis using ImageJ. The data are presented as fold of change in protein expression for each sample compared with WT DCs at 0 min. The ratio of His-hsp110 toβ-actin in WT DCs at 0 min is set to 1. * p
Figure Legend Snippet: SRA/CD204 absence reduces binding and internalization of hsp110 A. SRA/CD204 directly interacts with exogenous hsp110. WT BMDCs were incubated with His-hsp110 at 4 °C for 30 min, and then extensively washed with PBS. Cell lysates were immunoprecipitated with antibodies for SRA/CD204 (top) or His-tag (bottom). The immunocomplexes were subjected to immunoblotting analyses using antibodies for His-tag (1:4000, top) or SRA/CD204 (1:3000, bottom). IgG serves as a negative control. B. BMDCs were incubated with His-hsp110 or His-gp100 protein. SRA/CD204 was immunoprecipitated and analyzed for its association with hsp110 or gp100 using His-tag antibodies. C . Competition assays. DCs were incubated with biotinylated-His-hsp110 (40 μg/ml) in the presence of 3× or 9× excess of His-gp100 or His-Hsp110. Immunocomplexes pulled down by SRA/CD204 antibodies were analyzed by immunoblot using Avidin-HRP or anti-SRA antibodies (left). Alternatively, DCs were incubated with His-hsp110 in the presence of BSA or autologous hsp110-purified from mouse spleen, followed by immunoblotting analysis of His-hsp110 association with immunoprecipitated SRA/CD204 (right). D and E . SRA/CD204 absence results in reduced hsp110 binding and internalization. WT and SRA −/− BMDCs were incubated with His-hsp110 for 30 min at either 4 °C ( D ) or 37 °C ( E ). Cells were washed and cultured at 37 °C for the times indicated. Total cell lysates were prepared and analyzed for the presence of His-hsp110 using His-tag antibodies. Immunoblots were quantified by densitometry analysis using ImageJ. The data are presented as fold of change in protein expression for each sample compared with WT DCs at 0 min. The ratio of His-hsp110 toβ-actin in WT DCs at 0 min is set to 1. * p

Techniques Used: Binding Assay, Incubation, Immunoprecipitation, Negative Control, Avidin-Biotin Assay, Purification, Cell Culture, Western Blot, Expressing

14) Product Images from "Lysine pyrrolation is a naturally-occurring covalent modification involved in the production of DNA mimic proteins"

Article Title: Lysine pyrrolation is a naturally-occurring covalent modification involved in the production of DNA mimic proteins

Journal: Scientific Reports

doi: 10.1038/srep05343

Fluorescence spectra of SG free in solution and in complex with dsDNA or BDA-modified BSA. (a) Fluorescence spectra of SG in complex with dsDNA. dsDNA (1 mg/ml) was incubated with SG (100 nM) in TAE buffer for 30 min. (b) Fluorescence spectra of SG in complex with BDA-modified BSA. BSA or BDA-modified BSA (1 mg/ml) were incubated with SG (500 nM) in TAE buffer for 30 min. (c) A schematic illustration of the binding of DNA intercalators to the pyrrolated proteins.
Figure Legend Snippet: Fluorescence spectra of SG free in solution and in complex with dsDNA or BDA-modified BSA. (a) Fluorescence spectra of SG in complex with dsDNA. dsDNA (1 mg/ml) was incubated with SG (100 nM) in TAE buffer for 30 min. (b) Fluorescence spectra of SG in complex with BDA-modified BSA. BSA or BDA-modified BSA (1 mg/ml) were incubated with SG (500 nM) in TAE buffer for 30 min. (c) A schematic illustration of the binding of DNA intercalators to the pyrrolated proteins.

Techniques Used: Fluorescence, Modification, Incubation, Binding Assay

Pyrrolated proteins act as a DNA mimic. (a) Zeta potential of the BDA-treated proteins. BSA (1.0 mg/ml) was incubated with BDA (0–1 mM) in 10 mM sodium phosphate buffer (pH 7.4) for 24 at 37°C. (b) Temperature dependence of ionic conductivity for native and BDA-treated proteins. The BDA-modified protein was prepared by incubating 10 mg BSA with 10 mM BDA in 2.0 ml of distilled water at 37°C for 24 h. Native and BDA-treated proteins were dialyzed against distilled water, lyophilized, and subjected to the ionic conductivity measurement. (c) A schematic illustration of the electron transfer in pyrrolated proteins.
Figure Legend Snippet: Pyrrolated proteins act as a DNA mimic. (a) Zeta potential of the BDA-treated proteins. BSA (1.0 mg/ml) was incubated with BDA (0–1 mM) in 10 mM sodium phosphate buffer (pH 7.4) for 24 at 37°C. (b) Temperature dependence of ionic conductivity for native and BDA-treated proteins. The BDA-modified protein was prepared by incubating 10 mg BSA with 10 mM BDA in 2.0 ml of distilled water at 37°C for 24 h. Native and BDA-treated proteins were dialyzed against distilled water, lyophilized, and subjected to the ionic conductivity measurement. (c) A schematic illustration of the electron transfer in pyrrolated proteins.

Techniques Used: Activated Clotting Time Assay, Incubation, Modification

Formation of N ε -pyrrolelysine in the BDA-modified proteins. (a) HPLC detection of N ε -pyrrolelysine in the BDA-modified proteins. The BDA-modified protein was prepared by incubating BSA (1.0 mg/ml) with 1 mM BDA in PBS at 37°C for 24 h. The protein samples were hydrolyzed by 2 N NaOH under argon atmosphere for 18 h at 120°C. After the alkaline hydrolysis, the samples were neutralized with hydrochloric acid and analyzed by reverse-phase HPLC. Chromatogram: upper , BDA-treated BSA; lower , BSA. (b) Chemical structure of N ε -pyrrolelysine. (c) Measurement of N ε -pyrrolelysine generated in the BDA-modified proteins. The native and modified BSAs were analyzed by LC-ESI-MS in the selected ion monitoring (SIR) mode followed by alkaline hydrolysis. (d) Binding of SG to the BDA-tretaed BSA. BSA (1.0 mg/ml) was incubated with BDA (0–1 mM) in 1 ml of PBS at 37°C for 24 h. (e) Changes in electric properties on the molecular surface of both sides of native and BDA-modified BSA. The BDA-modified protein was prepared by incubating BSA (1.0 mg/ml) with 1 mM BDA in PBS at 37°C for 24 h. Panels : upper , native BSA; lower, BDA-treated BSA. The left and right panels represent the pair of images. The left panels shows molecules in the orientation with domain I on the left and further domains arranged counterclockwise, whereas in the right panels, domain I is on the right and the domains are arranged clockwise. Colored residues: red, negative amino acids; blue, positive amino acids; green, hydrophobic amino acids including pyrrolated lysine.
Figure Legend Snippet: Formation of N ε -pyrrolelysine in the BDA-modified proteins. (a) HPLC detection of N ε -pyrrolelysine in the BDA-modified proteins. The BDA-modified protein was prepared by incubating BSA (1.0 mg/ml) with 1 mM BDA in PBS at 37°C for 24 h. The protein samples were hydrolyzed by 2 N NaOH under argon atmosphere for 18 h at 120°C. After the alkaline hydrolysis, the samples were neutralized with hydrochloric acid and analyzed by reverse-phase HPLC. Chromatogram: upper , BDA-treated BSA; lower , BSA. (b) Chemical structure of N ε -pyrrolelysine. (c) Measurement of N ε -pyrrolelysine generated in the BDA-modified proteins. The native and modified BSAs were analyzed by LC-ESI-MS in the selected ion monitoring (SIR) mode followed by alkaline hydrolysis. (d) Binding of SG to the BDA-tretaed BSA. BSA (1.0 mg/ml) was incubated with BDA (0–1 mM) in 1 ml of PBS at 37°C for 24 h. (e) Changes in electric properties on the molecular surface of both sides of native and BDA-modified BSA. The BDA-modified protein was prepared by incubating BSA (1.0 mg/ml) with 1 mM BDA in PBS at 37°C for 24 h. Panels : upper , native BSA; lower, BDA-treated BSA. The left and right panels represent the pair of images. The left panels shows molecules in the orientation with domain I on the left and further domains arranged counterclockwise, whereas in the right panels, domain I is on the right and the domains are arranged clockwise. Colored residues: red, negative amino acids; blue, positive amino acids; green, hydrophobic amino acids including pyrrolated lysine.

Techniques Used: Modification, High Performance Liquid Chromatography, Generated, Mass Spectrometry, Binding Assay, Incubation

Pyrrolation transforms self-molecules into autoantigens. (a) Elevation of immune response to pyrrolated proteins and dsDNA in the balb/c mice immunized with the pyrrolated MSA. Female balb/c mice were immunized with complete Freund adjuvant and 50 μg of the BDA-modified MSA, and then boosted every 2 weeks with incomplete Freund adjuvant by emulsifying and intraperitoneal injection. The Ab titers were determined by ELISA using the BSA, BDA-modified BSA (pyrrolated BSA), and DNA as the absorbed antigens. Symbols: open circle , anti-BSA titer; closed circle , anti-pyrrolated BSA titer; closed triangle , anti-DNA titer. (b) Immunoreactivity of the anti-pyrrolated proteins mAb PSB established from the balb/c mice immunized with the pyrrolated MSA. The coating antigen was prepared by incubating BSA (1 mg/ml) with 1 mM aldehyde in 1 ml of PBS for 24 h at 37°C. Five microgram of antigen was coated per well on polystyrene plates and antibody binding detected. CRA, crotonaldehyde, ACR, acrolein; ONE, 4-oxo-2-nonenal; BDA, 1,4-butanedial.
Figure Legend Snippet: Pyrrolation transforms self-molecules into autoantigens. (a) Elevation of immune response to pyrrolated proteins and dsDNA in the balb/c mice immunized with the pyrrolated MSA. Female balb/c mice were immunized with complete Freund adjuvant and 50 μg of the BDA-modified MSA, and then boosted every 2 weeks with incomplete Freund adjuvant by emulsifying and intraperitoneal injection. The Ab titers were determined by ELISA using the BSA, BDA-modified BSA (pyrrolated BSA), and DNA as the absorbed antigens. Symbols: open circle , anti-BSA titer; closed circle , anti-pyrrolated BSA titer; closed triangle , anti-DNA titer. (b) Immunoreactivity of the anti-pyrrolated proteins mAb PSB established from the balb/c mice immunized with the pyrrolated MSA. The coating antigen was prepared by incubating BSA (1 mg/ml) with 1 mM aldehyde in 1 ml of PBS for 24 h at 37°C. Five microgram of antigen was coated per well on polystyrene plates and antibody binding detected. CRA, crotonaldehyde, ACR, acrolein; ONE, 4-oxo-2-nonenal; BDA, 1,4-butanedial.

Techniques Used: Mouse Assay, Modification, Injection, Enzyme-linked Immunosorbent Assay, Binding Assay

Pyrrolated proteins as a molecular target of autoimmunity. (a) Recognition of the pyrrolated proteins by anti-DNA autoAbs. Left , immunoblot analysis of the modified proteins using the sera from MRL- lpr mice. Right , immunoblot analysis of the modified proteins using the anti-DNA monoclonal IgG DSO established from female MRL- lpr mice. (b) Age-dependent elevation of antibody response to both DNA and pyrrolated proteins in SLE-prone MRL- lpr mice (n = 5) compared to those in the wild-type MRL-MpJ mice (n = 5). Left , IgG response. Right , IgM response. The Ab titers were determined by ELISA using BSA, BDA-treated BSA (pyrrolated BSA), and dsDNA as the absorbed antigens. Symbols: open circle , anti-BSA titer; closed circle , anti-pyrrolated BSA titer; closed triangle , anti-DNA titer. (c) Immunoreactivity of Abs eluted from the kidneys of the MRL-MpJ mice and MRL- lpr mice. Affinity of the Abs was determined by a direct antigen ELISA using BSA ( left ), pyrrolated BSA ( middle ), and dsDNA ( right ) as the absorbed antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between the MRL-MpJ and MRL- lpr mice are indicated by asterisks (*, P
Figure Legend Snippet: Pyrrolated proteins as a molecular target of autoimmunity. (a) Recognition of the pyrrolated proteins by anti-DNA autoAbs. Left , immunoblot analysis of the modified proteins using the sera from MRL- lpr mice. Right , immunoblot analysis of the modified proteins using the anti-DNA monoclonal IgG DSO established from female MRL- lpr mice. (b) Age-dependent elevation of antibody response to both DNA and pyrrolated proteins in SLE-prone MRL- lpr mice (n = 5) compared to those in the wild-type MRL-MpJ mice (n = 5). Left , IgG response. Right , IgM response. The Ab titers were determined by ELISA using BSA, BDA-treated BSA (pyrrolated BSA), and dsDNA as the absorbed antigens. Symbols: open circle , anti-BSA titer; closed circle , anti-pyrrolated BSA titer; closed triangle , anti-DNA titer. (c) Immunoreactivity of Abs eluted from the kidneys of the MRL-MpJ mice and MRL- lpr mice. Affinity of the Abs was determined by a direct antigen ELISA using BSA ( left ), pyrrolated BSA ( middle ), and dsDNA ( right ) as the absorbed antigens. The means were tested for statistical significance by Welch's test analysis. Statistically significant differences between the MRL-MpJ and MRL- lpr mice are indicated by asterisks (*, P

Techniques Used: Modification, Mouse Assay, Enzyme-linked Immunosorbent Assay

15) Product Images from "NanoLuc Luciferase – A Multifunctional Tool for High Throughput Antibody Screening"

Article Title: NanoLuc Luciferase – A Multifunctional Tool for High Throughput Antibody Screening

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2016.00027

Purified NanoLuc glow kinetic in different buffer conditions. Luminescence signal of purified Nanoluc (10 pmol/L) was measured at several time points in a 20 μL final volume of several buffer conditions : PBS (circle), PBS, 0.1% BSA (square); 50% Nano-Glo assay buffer (Promega, N112B), 50% PBS (triangle) or 50% Nano-Glo assay buffer 50% PBS, 0.1% BSA (diamond) with 1/100 (white) or 1/400 (black) furimazine final dilution. Graphs of a representative experiment, each experimental point was performed in triplicate, and bars correspond to standard deviation (SD).
Figure Legend Snippet: Purified NanoLuc glow kinetic in different buffer conditions. Luminescence signal of purified Nanoluc (10 pmol/L) was measured at several time points in a 20 μL final volume of several buffer conditions : PBS (circle), PBS, 0.1% BSA (square); 50% Nano-Glo assay buffer (Promega, N112B), 50% PBS (triangle) or 50% Nano-Glo assay buffer 50% PBS, 0.1% BSA (diamond) with 1/100 (white) or 1/400 (black) furimazine final dilution. Graphs of a representative experiment, each experimental point was performed in triplicate, and bars correspond to standard deviation (SD).

Techniques Used: Purification, Glo Assay, Standard Deviation

Western blot of targets 2 and 3 revealed with naked anti target 2 mAb and an anti-human kappa light chain-HRP secondary antibody (A), the Nluc fused to anti-target 2 antibody (B), the scFv-Nluc version of the anti-target 2 antibody (C) and an anti-target 3 scFv-Nluc (D). Membrane A labeling was revealed with ECL blotting substrate, while membranes (B–D) labeling was revealed with furimazine diluted 1000 fold in PBS, 0.1% BSA.
Figure Legend Snippet: Western blot of targets 2 and 3 revealed with naked anti target 2 mAb and an anti-human kappa light chain-HRP secondary antibody (A), the Nluc fused to anti-target 2 antibody (B), the scFv-Nluc version of the anti-target 2 antibody (C) and an anti-target 3 scFv-Nluc (D). Membrane A labeling was revealed with ECL blotting substrate, while membranes (B–D) labeling was revealed with furimazine diluted 1000 fold in PBS, 0.1% BSA.

Techniques Used: Western Blot, Labeling

Purified NanoLuc glow kinetic in different buffer conditions. Luminescence signal of purified Nanoluc (10 pmol/L) was measured at several time points in a 20 μL final volume of several buffer conditions : PBS (circle), PBS, 0.1% BSA (square); 50% Nano-Glo assay buffer (Promega, N112B), 50% PBS (triangle) or 50% Nano-Glo assay buffer 50% PBS, 0.1% BSA (diamond) with 1/100 (white) or 1/400 (black) furimazine final dilution. Graphs of a representative experiment, each experimental point was performed in triplicate, and bars correspond to standard deviation (SD).
Figure Legend Snippet: Purified NanoLuc glow kinetic in different buffer conditions. Luminescence signal of purified Nanoluc (10 pmol/L) was measured at several time points in a 20 μL final volume of several buffer conditions : PBS (circle), PBS, 0.1% BSA (square); 50% Nano-Glo assay buffer (Promega, N112B), 50% PBS (triangle) or 50% Nano-Glo assay buffer 50% PBS, 0.1% BSA (diamond) with 1/100 (white) or 1/400 (black) furimazine final dilution. Graphs of a representative experiment, each experimental point was performed in triplicate, and bars correspond to standard deviation (SD).

Techniques Used: Purification, Glo Assay, Standard Deviation

16) Product Images from "NanoLuc Luciferase – A Multifunctional Tool for High Throughput Antibody Screening"

Article Title: NanoLuc Luciferase – A Multifunctional Tool for High Throughput Antibody Screening

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2016.00027

Purified NanoLuc glow kinetic in different buffer conditions. Luminescence signal of purified Nanoluc (10 pmol/L) was measured at several time points in a 20 μL final volume of several buffer conditions : PBS (circle), PBS, 0.1% BSA (square); 50% Nano-Glo assay buffer (Promega, N112B), 50% PBS (triangle) or 50% Nano-Glo assay buffer 50% PBS, 0.1% BSA (diamond) with 1/100 (white) or 1/400 (black) furimazine final dilution. Graphs of a representative experiment, each experimental point was performed in triplicate, and bars correspond to standard deviation (SD).
Figure Legend Snippet: Purified NanoLuc glow kinetic in different buffer conditions. Luminescence signal of purified Nanoluc (10 pmol/L) was measured at several time points in a 20 μL final volume of several buffer conditions : PBS (circle), PBS, 0.1% BSA (square); 50% Nano-Glo assay buffer (Promega, N112B), 50% PBS (triangle) or 50% Nano-Glo assay buffer 50% PBS, 0.1% BSA (diamond) with 1/100 (white) or 1/400 (black) furimazine final dilution. Graphs of a representative experiment, each experimental point was performed in triplicate, and bars correspond to standard deviation (SD).

Techniques Used: Purification, Glo Assay, Standard Deviation

Western blot of targets 2 and 3 revealed with naked anti target 2 mAb and an anti-human kappa light chain-HRP secondary antibody (A), the Nluc fused to anti-target 2 antibody (B), the scFv-Nluc version of the anti-target 2 antibody (C) and an anti-target 3 scFv-Nluc (D). Membrane A labeling was revealed with ECL blotting substrate, while membranes (B–D) labeling was revealed with furimazine diluted 1000 fold in PBS, 0.1% BSA.
Figure Legend Snippet: Western blot of targets 2 and 3 revealed with naked anti target 2 mAb and an anti-human kappa light chain-HRP secondary antibody (A), the Nluc fused to anti-target 2 antibody (B), the scFv-Nluc version of the anti-target 2 antibody (C) and an anti-target 3 scFv-Nluc (D). Membrane A labeling was revealed with ECL blotting substrate, while membranes (B–D) labeling was revealed with furimazine diluted 1000 fold in PBS, 0.1% BSA.

Techniques Used: Western Blot, Labeling

Purified NanoLuc glow kinetic in different buffer conditions. Luminescence signal of purified Nanoluc (10 pmol/L) was measured at several time points in a 20 μL final volume of several buffer conditions : PBS (circle), PBS, 0.1% BSA (square); 50% Nano-Glo assay buffer (Promega, N112B), 50% PBS (triangle) or 50% Nano-Glo assay buffer 50% PBS, 0.1% BSA (diamond) with 1/100 (white) or 1/400 (black) furimazine final dilution. Graphs of a representative experiment, each experimental point was performed in triplicate, and bars correspond to standard deviation (SD).
Figure Legend Snippet: Purified NanoLuc glow kinetic in different buffer conditions. Luminescence signal of purified Nanoluc (10 pmol/L) was measured at several time points in a 20 μL final volume of several buffer conditions : PBS (circle), PBS, 0.1% BSA (square); 50% Nano-Glo assay buffer (Promega, N112B), 50% PBS (triangle) or 50% Nano-Glo assay buffer 50% PBS, 0.1% BSA (diamond) with 1/100 (white) or 1/400 (black) furimazine final dilution. Graphs of a representative experiment, each experimental point was performed in triplicate, and bars correspond to standard deviation (SD).

Techniques Used: Purification, Glo Assay, Standard Deviation

17) Product Images from "IL-4 impairs wound healing potential in the skin by repressing fibronectin expression"

Article Title: IL-4 impairs wound healing potential in the skin by repressing fibronectin expression

Journal: The Journal of allergy and clinical immunology

doi: 10.1016/j.jaci.2016.07.012

IL-4-stimulated keratinocytes show delayed wound healing ( A ) Human keratinocytes (HK) were differentiated for 5 days with calcium chloride in the presence or absence of IL-4 before analysis of FN1 expression by qRT-PCR. (B) Cell lysates were collected and the expression of fibronectin and β-actin was determined by western blot ( C ) Gene expression was analyzed 3h after scratching using qRT–PCR. ( D ) Percentage of scratch closed after 24h of culture, and ( E ) microscopy of human keratinocytes differentiated with calcium chloride alone or calcium chloride plus IL-4 for 5 days in control or plates covered with a substratum of BSA or fibronectin. Dashed red lines represent original wounds. Pictures are representative of 4 fields analyzed over at least three independent experiments. Data represents the mean of three independent experiments. *p
Figure Legend Snippet: IL-4-stimulated keratinocytes show delayed wound healing ( A ) Human keratinocytes (HK) were differentiated for 5 days with calcium chloride in the presence or absence of IL-4 before analysis of FN1 expression by qRT-PCR. (B) Cell lysates were collected and the expression of fibronectin and β-actin was determined by western blot ( C ) Gene expression was analyzed 3h after scratching using qRT–PCR. ( D ) Percentage of scratch closed after 24h of culture, and ( E ) microscopy of human keratinocytes differentiated with calcium chloride alone or calcium chloride plus IL-4 for 5 days in control or plates covered with a substratum of BSA or fibronectin. Dashed red lines represent original wounds. Pictures are representative of 4 fields analyzed over at least three independent experiments. Data represents the mean of three independent experiments. *p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Microscopy

18) Product Images from "Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics"

Article Title: Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics

Journal: Nature communications

doi: 10.1038/ncomms7632

Characterization of the UPF1 CP mRNP. Western blots of lysates of HEK293T cells transfected with either control (Ctrl) or UPF1 siRNA (100 nM) and 24 h later with plasmid encoding MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG either before (Input) or after anti(α)-FLAG immunoprecipitation (IP), the latter in the presence of BSA (−) or RNase ONE (+). EJC, exon-junction complex constituents. Representative of two independent experiments.
Figure Legend Snippet: Characterization of the UPF1 CP mRNP. Western blots of lysates of HEK293T cells transfected with either control (Ctrl) or UPF1 siRNA (100 nM) and 24 h later with plasmid encoding MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG either before (Input) or after anti(α)-FLAG immunoprecipitation (IP), the latter in the presence of BSA (−) or RNase ONE (+). EJC, exon-junction complex constituents. Representative of two independent experiments.

Techniques Used: Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation

19) Product Images from "Strong Antibody Responses Induced by Protein Antigens Conjugated onto the Surface of Lecithin-Based Nanoparticles"

Article Title: Strong Antibody Responses Induced by Protein Antigens Conjugated onto the Surface of Lecithin-Based Nanoparticles

Journal: Journal of controlled release : official journal of the Controlled Release Society

doi: 10.1016/j.jconrel.2009.08.023

The uptake of BSA-NPs by DC2.4 cells in culture A. The uptake of FITC-labeled BSA conjugated onto the nanoparticles by DC2.4 cells. Cells (1.0 × 10 6 ) were incubated with FITC-BSA-NPs or FITC-BSA for 6 h at 37°C or 4°C. Data reported are the ratio of the fluorescence intensity of cells treated with FITC-BSA-NPs over that with FITC-BSA. B. Microscopic graphs showing the uptake of the FITC-labeled, BSA-conjugated nanoparticles. Cells were incubated with BSA-NPs-FITC for 6 h at 37°C or 4°C and observed under a bright-field microscope (top panel) or a fluorescence microscope (bottom panel).
Figure Legend Snippet: The uptake of BSA-NPs by DC2.4 cells in culture A. The uptake of FITC-labeled BSA conjugated onto the nanoparticles by DC2.4 cells. Cells (1.0 × 10 6 ) were incubated with FITC-BSA-NPs or FITC-BSA for 6 h at 37°C or 4°C. Data reported are the ratio of the fluorescence intensity of cells treated with FITC-BSA-NPs over that with FITC-BSA. B. Microscopic graphs showing the uptake of the FITC-labeled, BSA-conjugated nanoparticles. Cells were incubated with BSA-NPs-FITC for 6 h at 37°C or 4°C and observed under a bright-field microscope (top panel) or a fluorescence microscope (bottom panel).

Techniques Used: Labeling, Incubation, Fluorescence, Microscopy

Related Articles

Mutagenesis:

Article Title: Out of Their Depth? Isolated Deep Populations of the Cosmopolitan Coral Desmophyllum dianthus May Be Highly Vulnerable to Environmental Change
Article Snippet: We targeted three different DNA regions, incorporating nuclear and mitochondrial markers with varying rates of mutation; the mitochondrial ribosomal subunit (16S) using the scleractinian-specific primers LP-16S-F and LP-16S-R , the Internal Transcribed Spacer region (ITS, spanning the ITS1, 5.8S and ITS2) using the universal primers ITS4 and ITS5 , and the Mitochondrial Control Region (MtC) using a mix of the scleractinian-specific primers Mt-Coral-Fwd-1, Mt-Coral-Fwd-2, Mt-Coral-Rev-1, Mt-Coral-Rev-2 . .. PCR reactions (50 µl) contained DNA template, 1× thermophilic DNA Polymerase buffer (Bioline), 1.5 mM MgCl2 (Promega), 3 U RedTaq DNA polymerase (Bioline), 0.2 µM of each primer (Sigma-Proligo), 80 µM of each dNTP and 0.5 µg Bovine Serum Albumen (Promega).

Size-exclusion Chromatography:

Article Title: Out of Their Depth? Isolated Deep Populations of the Cosmopolitan Coral Desmophyllum dianthus May Be Highly Vulnerable to Environmental Change
Article Snippet: PCR reactions (50 µl) contained DNA template, 1× thermophilic DNA Polymerase buffer (Bioline), 1.5 mM MgCl2 (Promega), 3 U RedTaq DNA polymerase (Bioline), 0.2 µM of each primer (Sigma-Proligo), 80 µM of each dNTP and 0.5 µg Bovine Serum Albumen (Promega). .. The thermal cycling profile consisted of an initial denaturation at 95°C for 5 min, then 35 cycles of a three-step program (95°C for 30 sec, 55°C for 30 sec and 72°C for 1 min), with a 10 min final extension at 72°C.

Incubation:

Article Title: Out of Their Depth? Isolated Deep Populations of the Cosmopolitan Coral Desmophyllum dianthus May Be Highly Vulnerable to Environmental Change
Article Snippet: Molecular protocols Genomic DNA was extracted from coral tissue with the Qiagen DNeasy Kit according to the manufacturer's specifications, but with an extended period of lysis (overnight incubation at 55°C). .. PCR reactions (50 µl) contained DNA template, 1× thermophilic DNA Polymerase buffer (Bioline), 1.5 mM MgCl2 (Promega), 3 U RedTaq DNA polymerase (Bioline), 0.2 µM of each primer (Sigma-Proligo), 80 µM of each dNTP and 0.5 µg Bovine Serum Albumen (Promega).

Purification:

Article Title: Out of Their Depth? Isolated Deep Populations of the Cosmopolitan Coral Desmophyllum dianthus May Be Highly Vulnerable to Environmental Change
Article Snippet: PCR reactions (50 µl) contained DNA template, 1× thermophilic DNA Polymerase buffer (Bioline), 1.5 mM MgCl2 (Promega), 3 U RedTaq DNA polymerase (Bioline), 0.2 µM of each primer (Sigma-Proligo), 80 µM of each dNTP and 0.5 µg Bovine Serum Albumen (Promega). .. PCR products were purified using the QIAquick PCR Purification kit (Qiagen) prior to sequencing on an ABI3730XL automated sequencer.

Polymerase Chain Reaction:

Article Title: Out of Their Depth? Isolated Deep Populations of the Cosmopolitan Coral Desmophyllum dianthus May Be Highly Vulnerable to Environmental Change
Article Snippet: .. PCR reactions (50 µl) contained DNA template, 1× thermophilic DNA Polymerase buffer (Bioline), 1.5 mM MgCl2 (Promega), 3 U RedTaq DNA polymerase (Bioline), 0.2 µM of each primer (Sigma-Proligo), 80 µM of each dNTP and 0.5 µg Bovine Serum Albumen (Promega). .. The thermal cycling profile consisted of an initial denaturation at 95°C for 5 min, then 35 cycles of a three-step program (95°C for 30 sec, 55°C for 30 sec and 72°C for 1 min), with a 10 min final extension at 72°C.

Generated:

Article Title: Out of Their Depth? Isolated Deep Populations of the Cosmopolitan Coral Desmophyllum dianthus May Be Highly Vulnerable to Environmental Change
Article Snippet: PCR reactions (50 µl) contained DNA template, 1× thermophilic DNA Polymerase buffer (Bioline), 1.5 mM MgCl2 (Promega), 3 U RedTaq DNA polymerase (Bioline), 0.2 µM of each primer (Sigma-Proligo), 80 µM of each dNTP and 0.5 µg Bovine Serum Albumen (Promega). .. Sequences were analysed either with Sequencher 4.5 software or MEGA3.1 , and consensus sequences generated for each sample using forward (5′-3′) and reverse (3′-5′) primer sequences.

Sequencing:

Article Title: Out of Their Depth? Isolated Deep Populations of the Cosmopolitan Coral Desmophyllum dianthus May Be Highly Vulnerable to Environmental Change
Article Snippet: PCR reactions (50 µl) contained DNA template, 1× thermophilic DNA Polymerase buffer (Bioline), 1.5 mM MgCl2 (Promega), 3 U RedTaq DNA polymerase (Bioline), 0.2 µM of each primer (Sigma-Proligo), 80 µM of each dNTP and 0.5 µg Bovine Serum Albumen (Promega). .. PCR products were purified using the QIAquick PCR Purification kit (Qiagen) prior to sequencing on an ABI3730XL automated sequencer.

Lysis:

Article Title: Out of Their Depth? Isolated Deep Populations of the Cosmopolitan Coral Desmophyllum dianthus May Be Highly Vulnerable to Environmental Change
Article Snippet: Molecular protocols Genomic DNA was extracted from coral tissue with the Qiagen DNeasy Kit according to the manufacturer's specifications, but with an extended period of lysis (overnight incubation at 55°C). .. PCR reactions (50 µl) contained DNA template, 1× thermophilic DNA Polymerase buffer (Bioline), 1.5 mM MgCl2 (Promega), 3 U RedTaq DNA polymerase (Bioline), 0.2 µM of each primer (Sigma-Proligo), 80 µM of each dNTP and 0.5 µg Bovine Serum Albumen (Promega).

Software:

Article Title: Out of Their Depth? Isolated Deep Populations of the Cosmopolitan Coral Desmophyllum dianthus May Be Highly Vulnerable to Environmental Change
Article Snippet: PCR reactions (50 µl) contained DNA template, 1× thermophilic DNA Polymerase buffer (Bioline), 1.5 mM MgCl2 (Promega), 3 U RedTaq DNA polymerase (Bioline), 0.2 µM of each primer (Sigma-Proligo), 80 µM of each dNTP and 0.5 µg Bovine Serum Albumen (Promega). .. Sequences were analysed either with Sequencher 4.5 software or MEGA3.1 , and consensus sequences generated for each sample using forward (5′-3′) and reverse (3′-5′) primer sequences.

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  • bsa  (Promega)
    99
    Promega bsa
    ( a ) <t>p22</t> binding to the DIC column is blocked by p150 Glued . Two identical DIC affinity columns were constructed. One column was blocked with excess p150 Glued ( fifth through seventh lanes ), whereas the other was blocked with <t>BSA</t> as a control ( second through fourth lanes ). Rat brain cytosol (1 ml) was then loaded ( first lane ), and the 1 M NaCl eluates ( fourth and seventh lanes ) were analyzed by Western blotting. Amounts of sample loaded in load and flow through lanes are equivalent (4 μl each), as are wash and 1 M eluate lanes (20 μl each from a total of 50 μl TCA precipitate). The results show that, like other dynactin subunits, p22 does not bind to a DIC column that is preblocked with p150 Glued . ( b and c ) p22 antibody immunoprecipitates the dynactin complex. Immunoprecipitations were carried out using antibodies to p150 Glued (1.5 ml), p22 (0.7 ml), and beads only (control) on rat brain cytosol (1 ml each). After thorough washing with RIPA buffer, the precipitates were eluted with 100 μl 1× Laemmli sample buffer. 1 μl each of control and anti-p150 Glued precipitate and 2 μl of anti-p22 precipitate were loaded and analyzed by SDS-PAGE ( b ) followed by Western blotting ( c ). A panel of dynactin subunit was used. The results demonstrate that p22 antibody coprecipitates the same subunits coprecipitated by anti-p150 Glued antibody.
    Bsa, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 128 article reviews
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    80
    Promega bsa basal
    Effect of secretin receptor gene silencing on the basal proliferative activity (by MTS assays, following incubation for 6, 24, 48 and 72 hours with 0.2% <t>BSA)</t> of large <t>cholangiocytes.</t> Data are mean ± SEM of 4 experiments. * p
    Bsa Basal, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 80 stars, based on 1 article reviews
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    ( a ) p22 binding to the DIC column is blocked by p150 Glued . Two identical DIC affinity columns were constructed. One column was blocked with excess p150 Glued ( fifth through seventh lanes ), whereas the other was blocked with BSA as a control ( second through fourth lanes ). Rat brain cytosol (1 ml) was then loaded ( first lane ), and the 1 M NaCl eluates ( fourth and seventh lanes ) were analyzed by Western blotting. Amounts of sample loaded in load and flow through lanes are equivalent (4 μl each), as are wash and 1 M eluate lanes (20 μl each from a total of 50 μl TCA precipitate). The results show that, like other dynactin subunits, p22 does not bind to a DIC column that is preblocked with p150 Glued . ( b and c ) p22 antibody immunoprecipitates the dynactin complex. Immunoprecipitations were carried out using antibodies to p150 Glued (1.5 ml), p22 (0.7 ml), and beads only (control) on rat brain cytosol (1 ml each). After thorough washing with RIPA buffer, the precipitates were eluted with 100 μl 1× Laemmli sample buffer. 1 μl each of control and anti-p150 Glued precipitate and 2 μl of anti-p22 precipitate were loaded and analyzed by SDS-PAGE ( b ) followed by Western blotting ( c ). A panel of dynactin subunit was used. The results demonstrate that p22 antibody coprecipitates the same subunits coprecipitated by anti-p150 Glued antibody.

    Journal: The Journal of Cell Biology

    Article Title: Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells

    doi:

    Figure Lengend Snippet: ( a ) p22 binding to the DIC column is blocked by p150 Glued . Two identical DIC affinity columns were constructed. One column was blocked with excess p150 Glued ( fifth through seventh lanes ), whereas the other was blocked with BSA as a control ( second through fourth lanes ). Rat brain cytosol (1 ml) was then loaded ( first lane ), and the 1 M NaCl eluates ( fourth and seventh lanes ) were analyzed by Western blotting. Amounts of sample loaded in load and flow through lanes are equivalent (4 μl each), as are wash and 1 M eluate lanes (20 μl each from a total of 50 μl TCA precipitate). The results show that, like other dynactin subunits, p22 does not bind to a DIC column that is preblocked with p150 Glued . ( b and c ) p22 antibody immunoprecipitates the dynactin complex. Immunoprecipitations were carried out using antibodies to p150 Glued (1.5 ml), p22 (0.7 ml), and beads only (control) on rat brain cytosol (1 ml each). After thorough washing with RIPA buffer, the precipitates were eluted with 100 μl 1× Laemmli sample buffer. 1 μl each of control and anti-p150 Glued precipitate and 2 μl of anti-p22 precipitate were loaded and analyzed by SDS-PAGE ( b ) followed by Western blotting ( c ). A panel of dynactin subunit was used. The results demonstrate that p22 antibody coprecipitates the same subunits coprecipitated by anti-p150 Glued antibody.

    Article Snippet: The purified recombinant p150Glued was covalently linked to activated CH-Sepharose as described ( ); a control column was generated by coupling an equivalent amount of BSA to the matrix. p22 labeled with [35 S]methionine (in a total of 200 μl reaction volume) was produced in an in vitro transcription/translation assay ( Promega Corp. , Madison, WI), diluted fivefold (to 1 ml) with HEM buffer (50 mM Na-Hepes, 1 mM EDTA, 2 mM MgCl2 , pH 7.0), divided into two and loaded onto either the p150Glued column or the BSA column.

    Techniques: Binding Assay, Construct, Western Blot, Flow Cytometry, SDS Page

    p22 binds to the p150 Glued subunit of dynactin. ATP-extract (500 μl) prepared from five rat brains was incubated with PBS alone (control) or with recombinant p50 (dynamitin) and subjected to linear density sucrose gradient (5–20%) for 18 h at 4°C in a Beckman SW41.Ti rotor at 32K rpm. Approximately 0.9-ml fractions were collected, and the fractions were analyzed by SDS-PAGE followed by Western blotting using antibodies to p150 Glued , Arp1, p50, and p22. In a , all subunits probed peak exclusively at fraction 5, corresponding to the 20- S peak. However, incubation of ATP-extract with recombinant p50 partially disrupts the dynactin complex as indicated by the presence of p150 Glued at fractions 9–11 ( b ). Interestingly, p22 is also found at the second peak at fraction 10. Note that the heavy p50 staining in b is due to excess recombinant p50 used for dynactin disruption. ( c ) A p150 Glued affinity column and a BSA control column were constructed and loaded with in vitro–translated and radio-labeled recombinant p22. The columns were extensively washed and eluted with 1 M NaCl. The loaded material ( load ), flow-through ( F.T. ), wash, and the eluate samples were analyzed by SDS-PAGE followed by autoradiography.

    Journal: The Journal of Cell Biology

    Article Title: Characterization of the p22 Subunit of Dynactin Reveals the Localization of Cytoplasmic Dynein and Dynactin to the Midbody of Dividing Cells

    doi:

    Figure Lengend Snippet: p22 binds to the p150 Glued subunit of dynactin. ATP-extract (500 μl) prepared from five rat brains was incubated with PBS alone (control) or with recombinant p50 (dynamitin) and subjected to linear density sucrose gradient (5–20%) for 18 h at 4°C in a Beckman SW41.Ti rotor at 32K rpm. Approximately 0.9-ml fractions were collected, and the fractions were analyzed by SDS-PAGE followed by Western blotting using antibodies to p150 Glued , Arp1, p50, and p22. In a , all subunits probed peak exclusively at fraction 5, corresponding to the 20- S peak. However, incubation of ATP-extract with recombinant p50 partially disrupts the dynactin complex as indicated by the presence of p150 Glued at fractions 9–11 ( b ). Interestingly, p22 is also found at the second peak at fraction 10. Note that the heavy p50 staining in b is due to excess recombinant p50 used for dynactin disruption. ( c ) A p150 Glued affinity column and a BSA control column were constructed and loaded with in vitro–translated and radio-labeled recombinant p22. The columns were extensively washed and eluted with 1 M NaCl. The loaded material ( load ), flow-through ( F.T. ), wash, and the eluate samples were analyzed by SDS-PAGE followed by autoradiography.

    Article Snippet: The purified recombinant p150Glued was covalently linked to activated CH-Sepharose as described ( ); a control column was generated by coupling an equivalent amount of BSA to the matrix. p22 labeled with [35 S]methionine (in a total of 200 μl reaction volume) was produced in an in vitro transcription/translation assay ( Promega Corp. , Madison, WI), diluted fivefold (to 1 ml) with HEM buffer (50 mM Na-Hepes, 1 mM EDTA, 2 mM MgCl2 , pH 7.0), divided into two and loaded onto either the p150Glued column or the BSA column.

    Techniques: Incubation, Recombinant, SDS Page, Western Blot, Staining, Affinity Column, Construct, In Vitro, Labeling, Flow Cytometry, Autoradiography

    Mitogen-activated protein kinase phosphorylation in advanced glycation end product-BSA-stimulated osteoarthritis chondrocytes. (a) Effect of epigallocatechin-3-gallate (EGCG) on mitogen-activated protein kinase phosphorylation in advanced glycation end product (AGE)-BSA-stimulated osteoarthritis (OA) chondrocytes. After pretreatment with EGCG (25 to 150 μM) for 1 hour at 37°C, primary human chondrocytes (70 to 80% confluent) were incubated with AGE-BSA (400 μg/ml) for 45 minutes, and then the phosphorylation of p38, JNK, and ERK was determined by western blot analysis. (b) Band images were digitally captured and the band intensities (pixels/band) were obtained using the Un-Scan-It software and are expressed in arbitrary optical density units. Data shown are cumulative of two experiments. OD values presented as mean ± standard deviation; data without a common letter differ, P

    Journal: Arthritis Research & Therapy

    Article Title: Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-? and matrix metalloproteinase-13 in human chondrocytes

    doi: 10.1186/ar2700

    Figure Lengend Snippet: Mitogen-activated protein kinase phosphorylation in advanced glycation end product-BSA-stimulated osteoarthritis chondrocytes. (a) Effect of epigallocatechin-3-gallate (EGCG) on mitogen-activated protein kinase phosphorylation in advanced glycation end product (AGE)-BSA-stimulated osteoarthritis (OA) chondrocytes. After pretreatment with EGCG (25 to 150 μM) for 1 hour at 37°C, primary human chondrocytes (70 to 80% confluent) were incubated with AGE-BSA (400 μg/ml) for 45 minutes, and then the phosphorylation of p38, JNK, and ERK was determined by western blot analysis. (b) Band images were digitally captured and the band intensities (pixels/band) were obtained using the Un-Scan-It software and are expressed in arbitrary optical density units. Data shown are cumulative of two experiments. OD values presented as mean ± standard deviation; data without a common letter differ, P

    Article Snippet: Chondrocytes were treated with various doses of AGE-BSA (20 to 600 μg/ml) and EGCG (25 to 200 μM), and after 24 hours the incubation cytotoxicity of AGE-BSA and EGCG was examined using the CytoTox-Glo™ Cytotoxicity Assay Kit (Promega, Madison, WI, USA).

    Techniques: Incubation, Western Blot, Software, Standard Deviation

    Gene expression and production of TNFα in advanced glycation end product-BSA-stimulated osteoarthritis chondrocytes. (a) Effect of epigallocatechin-3-gallate (EGCG), specific inhibitors for mitogen-activated protein kinases and NF-κB on the gene expression of TNFα in advanced glycation end product (AGE)-BSA-stimulated osteoarthritis (OA) chondrocytes. Primary chondrocytes were pretreated with EGCG (25 to 150 μM) for 2 hours and were stimulated by AGE-BSA (600 μg/ml) for 8 hours. Folds of TNFα mRNA expression, as compared with control and normalized to GAPDH, were determined by quantitative RT-PCR. Concentrations of specific inhibitors of JNK (SP600125), ERK (PD98059), p38 (SB202190) and NF-κB (MG-132) used in these studies were 10 μM, 50 μM, 100 μM and 100 μM, respectively. Native BSA (600 μg/ml) was used as a negative control. (b) Effect of EGCG on the production of TNFα in AGE-BSA-stimulated OA chondrocytes. Primary chondrocytes were pretreated with EGCG (25 to 150 μM) for 2 hours and were stimulated by AGE-BSA (600 μg/ml) for 24 hours. The production level of TNFα was determined by sandwich ELISA. Results are representative (mean ± standard error of the mean) of duplicate experiments with OA chondrocytes obtained from five age-matched and sex-matched OA donors; data without a common letter differ, P

    Journal: Arthritis Research & Therapy

    Article Title: Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-? and matrix metalloproteinase-13 in human chondrocytes

    doi: 10.1186/ar2700

    Figure Lengend Snippet: Gene expression and production of TNFα in advanced glycation end product-BSA-stimulated osteoarthritis chondrocytes. (a) Effect of epigallocatechin-3-gallate (EGCG), specific inhibitors for mitogen-activated protein kinases and NF-κB on the gene expression of TNFα in advanced glycation end product (AGE)-BSA-stimulated osteoarthritis (OA) chondrocytes. Primary chondrocytes were pretreated with EGCG (25 to 150 μM) for 2 hours and were stimulated by AGE-BSA (600 μg/ml) for 8 hours. Folds of TNFα mRNA expression, as compared with control and normalized to GAPDH, were determined by quantitative RT-PCR. Concentrations of specific inhibitors of JNK (SP600125), ERK (PD98059), p38 (SB202190) and NF-κB (MG-132) used in these studies were 10 μM, 50 μM, 100 μM and 100 μM, respectively. Native BSA (600 μg/ml) was used as a negative control. (b) Effect of EGCG on the production of TNFα in AGE-BSA-stimulated OA chondrocytes. Primary chondrocytes were pretreated with EGCG (25 to 150 μM) for 2 hours and were stimulated by AGE-BSA (600 μg/ml) for 24 hours. The production level of TNFα was determined by sandwich ELISA. Results are representative (mean ± standard error of the mean) of duplicate experiments with OA chondrocytes obtained from five age-matched and sex-matched OA donors; data without a common letter differ, P

    Article Snippet: Chondrocytes were treated with various doses of AGE-BSA (20 to 600 μg/ml) and EGCG (25 to 200 μM), and after 24 hours the incubation cytotoxicity of AGE-BSA and EGCG was examined using the CytoTox-Glo™ Cytotoxicity Assay Kit (Promega, Madison, WI, USA).

    Techniques: Expressing, Quantitative RT-PCR, Negative Control, Sandwich ELISA

    Gene expression and production of matrix metalloproteinase-13 in advanced glycation end product-BSA-stimulated osteoarthritis chondrocytes. (a) Effect of epigallocatechin-3-gallate (EGCG), specific inhibitors for mitogen-activated protein kinases and NF-κB on the gene expression of matrix metalloproteinase (MMP)-13 in advanced glycation end product (AGE)-BSA-stimulated osteoarthritis (OA) chondrocytes. Primary human chondrocytes were pretreated with EGCG (25 to 150 μM) for 2 hours and were stimulated with AGE-BSA (600 μg/ml) for 8 hours. Expression of MMP-13 mRNA was normalized to GAPDH and compared with the levels present in control. Concentrations of specific inhibitors of JNK (SP600125), ERK (PD98059), p38 (SB202190) and NF-κB (MG-132) used in these studies were 10 μM, 50 μM, 100 μM and 100 μM, respectively. Native BSA (600 μg/ml) was used as negative control. Results are representative (mean ± standard error of the mean) of duplicate experiments with chondrocytes obtained from five age-matched and sex-matched OA donors; data without a common letter differ, P

    Journal: Arthritis Research & Therapy

    Article Title: Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-? and matrix metalloproteinase-13 in human chondrocytes

    doi: 10.1186/ar2700

    Figure Lengend Snippet: Gene expression and production of matrix metalloproteinase-13 in advanced glycation end product-BSA-stimulated osteoarthritis chondrocytes. (a) Effect of epigallocatechin-3-gallate (EGCG), specific inhibitors for mitogen-activated protein kinases and NF-κB on the gene expression of matrix metalloproteinase (MMP)-13 in advanced glycation end product (AGE)-BSA-stimulated osteoarthritis (OA) chondrocytes. Primary human chondrocytes were pretreated with EGCG (25 to 150 μM) for 2 hours and were stimulated with AGE-BSA (600 μg/ml) for 8 hours. Expression of MMP-13 mRNA was normalized to GAPDH and compared with the levels present in control. Concentrations of specific inhibitors of JNK (SP600125), ERK (PD98059), p38 (SB202190) and NF-κB (MG-132) used in these studies were 10 μM, 50 μM, 100 μM and 100 μM, respectively. Native BSA (600 μg/ml) was used as negative control. Results are representative (mean ± standard error of the mean) of duplicate experiments with chondrocytes obtained from five age-matched and sex-matched OA donors; data without a common letter differ, P

    Article Snippet: Chondrocytes were treated with various doses of AGE-BSA (20 to 600 μg/ml) and EGCG (25 to 200 μM), and after 24 hours the incubation cytotoxicity of AGE-BSA and EGCG was examined using the CytoTox-Glo™ Cytotoxicity Assay Kit (Promega, Madison, WI, USA).

    Techniques: Expressing, Negative Control

    Absorbance spectra and electrophoresis of native BSA and advanced glycation end product-BSA. (a) Absorbance spectra of native BSA and advanced glycation end product (AGE)-BSA. Samples were incubated with or without glycoaldehyde in PBS, pH 7.2, with equal protein concentrations. (b) Electrophoresis of native BSA and AGE-BSA. Samples were electrophoresed using 10% SDS-PAGE with 4% stacking gel. The gel was run for 1.5 hours at 125 V. The precision plus protein standard (Bio-Rad) served as the molecular size marker. AGE-BSA was derived from the reaction between endotoxin-free BSA (2 mg/ml) and glycoaldehyde (70 mM).

    Journal: Arthritis Research & Therapy

    Article Title: Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-? and matrix metalloproteinase-13 in human chondrocytes

    doi: 10.1186/ar2700

    Figure Lengend Snippet: Absorbance spectra and electrophoresis of native BSA and advanced glycation end product-BSA. (a) Absorbance spectra of native BSA and advanced glycation end product (AGE)-BSA. Samples were incubated with or without glycoaldehyde in PBS, pH 7.2, with equal protein concentrations. (b) Electrophoresis of native BSA and AGE-BSA. Samples were electrophoresed using 10% SDS-PAGE with 4% stacking gel. The gel was run for 1.5 hours at 125 V. The precision plus protein standard (Bio-Rad) served as the molecular size marker. AGE-BSA was derived from the reaction between endotoxin-free BSA (2 mg/ml) and glycoaldehyde (70 mM).

    Article Snippet: Chondrocytes were treated with various doses of AGE-BSA (20 to 600 μg/ml) and EGCG (25 to 200 μM), and after 24 hours the incubation cytotoxicity of AGE-BSA and EGCG was examined using the CytoTox-Glo™ Cytotoxicity Assay Kit (Promega, Madison, WI, USA).

    Techniques: Electrophoresis, Incubation, SDS Page, Marker, Derivative Assay

    RBM24 inhibits the translation of the core protein by binding to the 5′ TR of pgRNA and blocking 80S ribosome assembly on HBV pgRNA. a The HepG2.2.15 cell line was transfected with siNC or siRBM24 (left panels), HepG2 cells were co-transfected with pHY106 and pRBM24 or empty vector (right panels). Cell lysates were collected at 48 hpt, and the expression of core and RBM24 was detected by western blotting. b HepG2 cells were co-transfected with pHA-core and pRBM24 or empty vector, and the expression of core and RBM24 was detected by western blotting. c HepG2 cells were co-transfected with pTR-core and the indicated siRNA or plasmid and harvested at 48 hpt. The expression of core and RBM24 was detected by western blotting. d HepG2 cells were transfected with the indicated plasmids, and luciferase activity was determined with Steady-Glo ® . The relative luciferase activity (RLA) values were calculated and are shown in the bar graph on the left. The luciferase activity of 5′ TR-associated luciferase reporter plasmids in vitro was detected (bar graph on the right). e The 5′ TR-luciferase RNA together with rhRBM24 or BSA was incubated in rabbit reticulocyte lysate (RRL). The ribosome complexes were separated by sucrose gradient ultracentrifugation. The distribution of biotin-RNA was detected using a dot-blot assay

    Journal: Emerging Microbes & Infections

    Article Title: RBM24 stabilizes hepatitis B virus pregenomic RNA but inhibits core protein translation by targeting the terminal redundancy sequence

    doi: 10.1038/s41426-018-0091-4

    Figure Lengend Snippet: RBM24 inhibits the translation of the core protein by binding to the 5′ TR of pgRNA and blocking 80S ribosome assembly on HBV pgRNA. a The HepG2.2.15 cell line was transfected with siNC or siRBM24 (left panels), HepG2 cells were co-transfected with pHY106 and pRBM24 or empty vector (right panels). Cell lysates were collected at 48 hpt, and the expression of core and RBM24 was detected by western blotting. b HepG2 cells were co-transfected with pHA-core and pRBM24 or empty vector, and the expression of core and RBM24 was detected by western blotting. c HepG2 cells were co-transfected with pTR-core and the indicated siRNA or plasmid and harvested at 48 hpt. The expression of core and RBM24 was detected by western blotting. d HepG2 cells were transfected with the indicated plasmids, and luciferase activity was determined with Steady-Glo ® . The relative luciferase activity (RLA) values were calculated and are shown in the bar graph on the left. The luciferase activity of 5′ TR-associated luciferase reporter plasmids in vitro was detected (bar graph on the right). e The 5′ TR-luciferase RNA together with rhRBM24 or BSA was incubated in rabbit reticulocyte lysate (RRL). The ribosome complexes were separated by sucrose gradient ultracentrifugation. The distribution of biotin-RNA was detected using a dot-blot assay

    Article Snippet: In brief, template RNA and rhRBM24 or a nonspecific control protein, BSA, were incubated in an RRL (Promega, Madison, WI, USA, L4960)-based translation reaction system followed by a luciferase activity assay with Steady-Glo® (Promega, Madison, WI, USA, E2520) .

    Techniques: Protein Binding, Blocking Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Luciferase, Activity Assay, In Vitro, Incubation, Dot Blot

    Effect of secretin receptor gene silencing on the basal proliferative activity (by MTS assays, following incubation for 6, 24, 48 and 72 hours with 0.2% BSA) of large cholangiocytes. Data are mean ± SEM of 4 experiments. * p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Knockout of secretin receptor reduces large cholangiocyte hyperplasia in mice with extrahepatic cholestasis induced by bile duct ligation

    doi: 10.1002/hep.23657

    Figure Lengend Snippet: Effect of secretin receptor gene silencing on the basal proliferative activity (by MTS assays, following incubation for 6, 24, 48 and 72 hours with 0.2% BSA) of large cholangiocytes. Data are mean ± SEM of 4 experiments. * p

    Article Snippet: Our small (negative control) and large cholangiocytes ( ) were treated at 37°C with: (i) 0.2% BSA (basal) or secretin (100 nM) for 48 hours in the absence or presence of pre-incubation (1 hour) with H89 (PKA inhibitor, 30 μM) or PD98059 (MEK inhibitor, 10 nM) before evaluating proliferation by CellTiter 96 Cell Proliferation Assay ( ) (Promega Corp., Madison, WI).

    Techniques: Activity Assay, Incubation

    [ A ] Effect of 0.2% BSA (basal) or secretin (100 nM) for 48 hours at 37°C on the proliferation (by MTS assays) of small and large cholangiocytes. Data are mean ± SEM of 14 experiments. * p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Knockout of secretin receptor reduces large cholangiocyte hyperplasia in mice with extrahepatic cholestasis induced by bile duct ligation

    doi: 10.1002/hep.23657

    Figure Lengend Snippet: [ A ] Effect of 0.2% BSA (basal) or secretin (100 nM) for 48 hours at 37°C on the proliferation (by MTS assays) of small and large cholangiocytes. Data are mean ± SEM of 14 experiments. * p

    Article Snippet: Our small (negative control) and large cholangiocytes ( ) were treated at 37°C with: (i) 0.2% BSA (basal) or secretin (100 nM) for 48 hours in the absence or presence of pre-incubation (1 hour) with H89 (PKA inhibitor, 30 μM) or PD98059 (MEK inhibitor, 10 nM) before evaluating proliferation by CellTiter 96 Cell Proliferation Assay ( ) (Promega Corp., Madison, WI).

    Techniques: