Structured Review

Millipore bsa
The kinetics of fluorescence changes of PED6 incorporated in DOPC:PED6 8:2 LUV (data from Figure S1 ). The time 0 min corresponds to the addition of the following samples: buffer (light blue), urea (dark blue), <t>BSA</t> (green), <t>Crotoxin</t> (black), CyaA IP (orange), and CyaA UBC (red). ( A ) Increase of fluorescence emission intensity at 515 nm. ( B ) Ratio of fluorescence intensity at 515 nm, F 515 , at time t normalized by initial fluorescence at 515 nm. Inset shows same data with an expanded Y -axis (from 0.95 and 1.05). ( C ) Ratio of fluorescence intensity at 515 nm over fluorescence intensity at 575 nm: F 515 /F 575 . ( D ) The F 515 /F 575 values are normalized to the initial F 515 /F 575 value at 0 min (see methods for details).
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Images

1) Product Images from "The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis Has No Detectable Phospholipase A (PLA) Activity In Vitro"

Article Title: The Adenylate Cyclase (CyaA) Toxin from Bordetella pertussis Has No Detectable Phospholipase A (PLA) Activity In Vitro

Journal: Toxins

doi: 10.3390/toxins11020111

The kinetics of fluorescence changes of PED6 incorporated in DOPC:PED6 8:2 LUV (data from Figure S1 ). The time 0 min corresponds to the addition of the following samples: buffer (light blue), urea (dark blue), BSA (green), Crotoxin (black), CyaA IP (orange), and CyaA UBC (red). ( A ) Increase of fluorescence emission intensity at 515 nm. ( B ) Ratio of fluorescence intensity at 515 nm, F 515 , at time t normalized by initial fluorescence at 515 nm. Inset shows same data with an expanded Y -axis (from 0.95 and 1.05). ( C ) Ratio of fluorescence intensity at 515 nm over fluorescence intensity at 575 nm: F 515 /F 575 . ( D ) The F 515 /F 575 values are normalized to the initial F 515 /F 575 value at 0 min (see methods for details).
Figure Legend Snippet: The kinetics of fluorescence changes of PED6 incorporated in DOPC:PED6 8:2 LUV (data from Figure S1 ). The time 0 min corresponds to the addition of the following samples: buffer (light blue), urea (dark blue), BSA (green), Crotoxin (black), CyaA IP (orange), and CyaA UBC (red). ( A ) Increase of fluorescence emission intensity at 515 nm. ( B ) Ratio of fluorescence intensity at 515 nm, F 515 , at time t normalized by initial fluorescence at 515 nm. Inset shows same data with an expanded Y -axis (from 0.95 and 1.05). ( C ) Ratio of fluorescence intensity at 515 nm over fluorescence intensity at 575 nm: F 515 /F 575 . ( D ) The F 515 /F 575 values are normalized to the initial F 515 /F 575 value at 0 min (see methods for details).

Techniques Used: Fluorescence

2) Product Images from "Identification and characterization of photomedins: novel olfactomedin-domain-containing proteins with chondroitin sulphate-E-binding activity"

Article Title: Identification and characterization of photomedins: novel olfactomedin-domain-containing proteins with chondroitin sulphate-E-binding activity

Journal: Biochemical Journal

doi: 10.1042/BJ20050120

Binding of recombinant photomedins to ECM proteins and glycosaminoglycans Microtitre plates (96-well) were coated with the following ECM proteins or glycosaminoglycans: 20 μg/ml collagens type I, II, III, IV, V and VI (col1, col2, col3, col4, col5, and col6), laminin-1, laminin-2, laminin-10 purified from human placenta (h laminin-10), recombinant laminin-10 (r laminin-10), fibronectin, sternal proteoglycan and decorin, as well as 100 μg/ml gelatin and 500 μg/ml matrigel, and 20 μg/ml CS-C, CS-D, CS-E, dermatan sulphate (DS), hyaluronan (HA) heparan sulphate (HS) and heparin (Hep). Glycosaminoglycans used had been conjugated to dipalmitoyl phosphatidylethanolamine to facilitate their adsorption on to hydrophobic surfaces. Plates were blocked with BSA and incubated with 5 μg/ml of recombinant photomedins with a FLAG tag for 2 h at room temperature, followed by detection of bound photomedins with horse-radish-peroxidase-conjugated anti-FLAG antibody. Bars represent the absorbance at 490 nm. Error bars indicate S.D. ( n =4).
Figure Legend Snippet: Binding of recombinant photomedins to ECM proteins and glycosaminoglycans Microtitre plates (96-well) were coated with the following ECM proteins or glycosaminoglycans: 20 μg/ml collagens type I, II, III, IV, V and VI (col1, col2, col3, col4, col5, and col6), laminin-1, laminin-2, laminin-10 purified from human placenta (h laminin-10), recombinant laminin-10 (r laminin-10), fibronectin, sternal proteoglycan and decorin, as well as 100 μg/ml gelatin and 500 μg/ml matrigel, and 20 μg/ml CS-C, CS-D, CS-E, dermatan sulphate (DS), hyaluronan (HA) heparan sulphate (HS) and heparin (Hep). Glycosaminoglycans used had been conjugated to dipalmitoyl phosphatidylethanolamine to facilitate their adsorption on to hydrophobic surfaces. Plates were blocked with BSA and incubated with 5 μg/ml of recombinant photomedins with a FLAG tag for 2 h at room temperature, followed by detection of bound photomedins with horse-radish-peroxidase-conjugated anti-FLAG antibody. Bars represent the absorbance at 490 nm. Error bars indicate S.D. ( n =4).

Techniques Used: Binding Assay, Recombinant, Purification, Adsorption, Incubation, FLAG-tag

3) Product Images from "Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities"

Article Title: Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2016.89

HeLa cell attachment assay for Fc-CD97ECD WT and its GPS site mutant and truncated forms. (A) FACS profile of attachment of Fc-CD97ECD WT with several cell lines. Biotin-RA/RB/OVA/BSA and Fc fragment marginally attached to HeLa cells (bottom right panel). (B) FACS profile of HeLa cell attachment with four Fc-CD97ECD proteins. (C) Mean fluorescence intense (MFI) data of HeLa cell attachment with four Fc-CD97ECD proteins. Quantification from three independent experiments: mean±SD. ** P
Figure Legend Snippet: HeLa cell attachment assay for Fc-CD97ECD WT and its GPS site mutant and truncated forms. (A) FACS profile of attachment of Fc-CD97ECD WT with several cell lines. Biotin-RA/RB/OVA/BSA and Fc fragment marginally attached to HeLa cells (bottom right panel). (B) FACS profile of HeLa cell attachment with four Fc-CD97ECD proteins. (C) Mean fluorescence intense (MFI) data of HeLa cell attachment with four Fc-CD97ECD proteins. Quantification from three independent experiments: mean±SD. ** P

Techniques Used: Cell Attachment Assay, Mutagenesis, FACS, Fluorescence

4) Product Images from "Anti-Inflammatory and Vasoprotective Activity of a Retroviral-Derived Peptide, Homologous to Human Endogenous Retroviruses: Endothelial Cell Effects"

Article Title: Anti-Inflammatory and Vasoprotective Activity of a Retroviral-Derived Peptide, Homologous to Human Endogenous Retroviruses: Endothelial Cell Effects

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052693

MN10021 inhibits dermal vascular leak in a reverse passive Arthus reaction. Groups of 5 Hartley strain guinea pigs (male; ∼250 g) were injected s.c. with 1.0 ml of either saline or the indicated dose of MN10021. Eighteen hours later the flanks of the animals were shaved and both 6.25 and 12.5 µl of rabbit anti-BSA antiserum injected i.d. at each of two sites, one on each flank. The animals were then injected i.v. with 1.0 ml of PBS containing BSA (1.0 mg/ml) and Evans Blue dye (2.0 mg/ml). Six hours later the animals were euthanized by barbiturate overdose, the skin on the back removed, and the diameter of the extravasation of the Evans Blue dye measured for each site on the ventral surface of the skin. The values represent the mean ± SE of the 10 measurements obtained for each of the treatment conditions. ** p
Figure Legend Snippet: MN10021 inhibits dermal vascular leak in a reverse passive Arthus reaction. Groups of 5 Hartley strain guinea pigs (male; ∼250 g) were injected s.c. with 1.0 ml of either saline or the indicated dose of MN10021. Eighteen hours later the flanks of the animals were shaved and both 6.25 and 12.5 µl of rabbit anti-BSA antiserum injected i.d. at each of two sites, one on each flank. The animals were then injected i.v. with 1.0 ml of PBS containing BSA (1.0 mg/ml) and Evans Blue dye (2.0 mg/ml). Six hours later the animals were euthanized by barbiturate overdose, the skin on the back removed, and the diameter of the extravasation of the Evans Blue dye measured for each site on the ventral surface of the skin. The values represent the mean ± SE of the 10 measurements obtained for each of the treatment conditions. ** p

Techniques Used: Injection

Binding of [ 125 I] – MN20054 to Fibroblasts. HUVEC and WI-38 fibroblasts (CCL-75™; ATCC) were grown to confluence in 12-well tissue culture plates. To each of triplicate wells was added 20 pmol of [ 125 I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, or a 100-fold molar excess of MN10021. The plates were incubated for 60 min at 37°C with gentle rocking, the wells aspirated and quickly washed 3X with 2.0 ml each of ice-cold PBS/BSA and 1.0 ml of 1.0 N NaOH added to each well to solubilize the cells. Nine-tenths ml of solubilized cells was then removed from each well and cell-associated radioactivity determined by gamma scintillation spectrophotometry.
Figure Legend Snippet: Binding of [ 125 I] – MN20054 to Fibroblasts. HUVEC and WI-38 fibroblasts (CCL-75™; ATCC) were grown to confluence in 12-well tissue culture plates. To each of triplicate wells was added 20 pmol of [ 125 I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, or a 100-fold molar excess of MN10021. The plates were incubated for 60 min at 37°C with gentle rocking, the wells aspirated and quickly washed 3X with 2.0 ml each of ice-cold PBS/BSA and 1.0 ml of 1.0 N NaOH added to each well to solubilize the cells. Nine-tenths ml of solubilized cells was then removed from each well and cell-associated radioactivity determined by gamma scintillation spectrophotometry.

Techniques Used: Binding Assay, Incubation, Radioactivity, Spectrophotometry

Monocyte and endothelial cell binding of MN10021 and MN20054. A. Human blood monocytes, isolated as described in Methods, were adjusted to 5×10 6 /ml in PBS (0.5% BSA, pH 7.5). Two-tenths ml of cell suspension was added to each of triplicate 12×75 mm polypropylene tubes and the tubes were incubated for 60 min at 37°C with 20 pmol of [ 125 I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, a 100-fold molar excess of MN10021, or a 100-fold molar excess of MN20050. The contents of each tube were then layered onto 0.8 ml of 10% sucrose in Eppendorf tubes, centrifuged, and the cell-associated radioactivity determined by gamma scintillation spectrophotometry of the cell pellet. B. Binding/uptake of [ 125 I]-MN20054 by human monocytes was performed as described for panel A with the following exceptions: (1) half the cells were pretreated for 30 min at 37°C with cytochalasin B to inhibit endocytosis and (2) competition was performed only with a 100-fold molar excess of MN10021. C. HUVEC were grown to confluence in 12-well tissue culture plates. To each of triplicate wells was added 20 pmol of [ 125 I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, a 100-fold molar excess of MN10021, or a 100-fold molar excess of MN20050. The plates were incubated for 60 min at 37°C with gentle rocking, the wells aspirated and quickly washed 3X with 2.0 ml each of ice-cold PBS/BSA and 1.0 ml of 1.0 N NaOH added to each well to solubilize the cells. Nine-tenths ml of solubilized cells was then removed from each well and cell-associated radioactivity determined by gamma scintillation spectrophotometry.
Figure Legend Snippet: Monocyte and endothelial cell binding of MN10021 and MN20054. A. Human blood monocytes, isolated as described in Methods, were adjusted to 5×10 6 /ml in PBS (0.5% BSA, pH 7.5). Two-tenths ml of cell suspension was added to each of triplicate 12×75 mm polypropylene tubes and the tubes were incubated for 60 min at 37°C with 20 pmol of [ 125 I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, a 100-fold molar excess of MN10021, or a 100-fold molar excess of MN20050. The contents of each tube were then layered onto 0.8 ml of 10% sucrose in Eppendorf tubes, centrifuged, and the cell-associated radioactivity determined by gamma scintillation spectrophotometry of the cell pellet. B. Binding/uptake of [ 125 I]-MN20054 by human monocytes was performed as described for panel A with the following exceptions: (1) half the cells were pretreated for 30 min at 37°C with cytochalasin B to inhibit endocytosis and (2) competition was performed only with a 100-fold molar excess of MN10021. C. HUVEC were grown to confluence in 12-well tissue culture plates. To each of triplicate wells was added 20 pmol of [ 125 I]-MN20054 in the presence of either buffer alone, a 100-fold molar excess of MN20054, a 100-fold molar excess of MN10021, or a 100-fold molar excess of MN20050. The plates were incubated for 60 min at 37°C with gentle rocking, the wells aspirated and quickly washed 3X with 2.0 ml each of ice-cold PBS/BSA and 1.0 ml of 1.0 N NaOH added to each well to solubilize the cells. Nine-tenths ml of solubilized cells was then removed from each well and cell-associated radioactivity determined by gamma scintillation spectrophotometry.

Techniques Used: Binding Assay, Isolation, Incubation, Radioactivity, Spectrophotometry

5) Product Images from "Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion"

Article Title: Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005780

Penetration of C. neoformans through reconstituted ECM. The ECM invasion chambers are composed of matrigel (basement membrane) layered on membranes with 8 µm pores. Strain JEC21 was incubated with plasminogen in phosphate-buffered saline with BSA in the presence or absence of tissue-derived plasminogen activator (tPA), incubated in the upper chamber of the transwell for 24 hours at 37°C prior to analysis of colony counts from the lower well (* (p = 0.0093); ** (p = 0.0084)).
Figure Legend Snippet: Penetration of C. neoformans through reconstituted ECM. The ECM invasion chambers are composed of matrigel (basement membrane) layered on membranes with 8 µm pores. Strain JEC21 was incubated with plasminogen in phosphate-buffered saline with BSA in the presence or absence of tissue-derived plasminogen activator (tPA), incubated in the upper chamber of the transwell for 24 hours at 37°C prior to analysis of colony counts from the lower well (* (p = 0.0093); ** (p = 0.0084)).

Techniques Used: Incubation, Derivative Assay

Plasminogen binds selectively and specifically to the cell-surface of intact C. neoformans strains. (A–B) Conversion of plasminogen (Plg) into plasmin heavy chain (Pla H ) and light chain (Pla L ) on the surface of intact C. neoformans serotype D and A strains. (A) Serotype D strain JEC21 was incubated in the presence or absence of plasminogen, tissue plasminogen activator (tPA), and/or the plasmin inhibitor aprotinin in phosphate-buffered saline with BSA. Cell wall proteins were released by boiling labeled cells in SDS-extraction buffer and fractionated by SDS-PAGE, transferred to PVDF, and Western blotted with polyclonal anti-plasminogen antibody. Lane descriptions as follow: cells (JEC21) only (1), 100 µg plasminogen (2), plasminogen and 100 ng tPA (3), plasminogen, tPA, and 1 unit aprotinin (4). (B) Serotype A strains C23 and A1 38-2 were incubated in the presence or absence of plasminogen and/or tPA for 4 hrs at 37°C prior to Western blot analysis as described above. Lanes: cells (C23) only (1), C23 with 15 µg plasminogen (2), C23 with plasminogen and 100 ng tPA (3), cells (A1 38-2) only (4), A1 38-2 with 15 µg plasminogen (5), and A1 38-2 with plasminogen and tPA (6). (C) Plasminogen associates with the cell wall of intact cells. Cells (1×10 10 ) from log phase cultures (JEC21) were incubated 4 hr at 37°C in the presence (lane 1) or absence (lane 3) of 50 µg plasminogen and separated into cell wall and cytosol components, as described in Methods . Membranes (lane 2, 4) from cell walls were extracted and each fraction examined for the presence of plasminogen by Western blot analysis. Sample loading was uniform at 5 µg per well. (D) Sulfo-NHS-biotin and plasminogen compete for cell-surface binding sites. Log phase cells (JEC21) were initially labeled with sulfo-NHS-biotin in 0-, 1-, 10-, 100-fold molar equivalents of plasminogen then labeled 1 hr at 37°C with 50 µg plasminogen (lanes 1–4, respectively).
Figure Legend Snippet: Plasminogen binds selectively and specifically to the cell-surface of intact C. neoformans strains. (A–B) Conversion of plasminogen (Plg) into plasmin heavy chain (Pla H ) and light chain (Pla L ) on the surface of intact C. neoformans serotype D and A strains. (A) Serotype D strain JEC21 was incubated in the presence or absence of plasminogen, tissue plasminogen activator (tPA), and/or the plasmin inhibitor aprotinin in phosphate-buffered saline with BSA. Cell wall proteins were released by boiling labeled cells in SDS-extraction buffer and fractionated by SDS-PAGE, transferred to PVDF, and Western blotted with polyclonal anti-plasminogen antibody. Lane descriptions as follow: cells (JEC21) only (1), 100 µg plasminogen (2), plasminogen and 100 ng tPA (3), plasminogen, tPA, and 1 unit aprotinin (4). (B) Serotype A strains C23 and A1 38-2 were incubated in the presence or absence of plasminogen and/or tPA for 4 hrs at 37°C prior to Western blot analysis as described above. Lanes: cells (C23) only (1), C23 with 15 µg plasminogen (2), C23 with plasminogen and 100 ng tPA (3), cells (A1 38-2) only (4), A1 38-2 with 15 µg plasminogen (5), and A1 38-2 with plasminogen and tPA (6). (C) Plasminogen associates with the cell wall of intact cells. Cells (1×10 10 ) from log phase cultures (JEC21) were incubated 4 hr at 37°C in the presence (lane 1) or absence (lane 3) of 50 µg plasminogen and separated into cell wall and cytosol components, as described in Methods . Membranes (lane 2, 4) from cell walls were extracted and each fraction examined for the presence of plasminogen by Western blot analysis. Sample loading was uniform at 5 µg per well. (D) Sulfo-NHS-biotin and plasminogen compete for cell-surface binding sites. Log phase cells (JEC21) were initially labeled with sulfo-NHS-biotin in 0-, 1-, 10-, 100-fold molar equivalents of plasminogen then labeled 1 hr at 37°C with 50 µg plasminogen (lanes 1–4, respectively).

Techniques Used: Proximity Ligation Assay, Incubation, Labeling, SDS Page, Western Blot, Binding Assay

6) Product Images from "Fetuin-A/Albumin-Mineral Complexes Resembling Serum Calcium Granules and Putative Nanobacteria: Demonstration of a Dual Inhibition-Seeding Concept"

Article Title: Fetuin-A/Albumin-Mineral Complexes Resembling Serum Calcium Granules and Putative Nanobacteria: Demonstration of a Dual Inhibition-Seeding Concept

Journal: PLoS ONE

doi: 10.1371/journal.pone.0008058

Seeding of NB-like particles by boiled fetuin-A and albumin in metastable versus supersaturated medium. (A) Metastable medium: BSF and bovine serum albumin (BSA) were boiled for the time indicated on the left side. The boiled protein solutions were then diluted into DMEM, separately or together, to the final concentrations indicated on the top. A 650 was then monitored following inoculation (“Day 1”) and after incubation in cell culture conditions for 1 month, as indicated on the right. Inoculation of either untreated BSF or BSF that had been boiled for either 10, 30, or 120 min did not result in any turbidity changes after 1 month of incubation. In contrast, pre-boiled BSA produced either bell-shaped or linear turbidity changes depending on the amount of boiling time. When added together, pre-boiled BSF and BSA produced additive effects. (B) Supersaturated medium: BSF and BSA were added to medium as in (A) that was also inoculated with 1 mM each of CaCl 2 and NaH 2 PO 4 . Notice the various patterns of turbidity changes associated with the three protein combinations. See the text for further details.
Figure Legend Snippet: Seeding of NB-like particles by boiled fetuin-A and albumin in metastable versus supersaturated medium. (A) Metastable medium: BSF and bovine serum albumin (BSA) were boiled for the time indicated on the left side. The boiled protein solutions were then diluted into DMEM, separately or together, to the final concentrations indicated on the top. A 650 was then monitored following inoculation (“Day 1”) and after incubation in cell culture conditions for 1 month, as indicated on the right. Inoculation of either untreated BSF or BSF that had been boiled for either 10, 30, or 120 min did not result in any turbidity changes after 1 month of incubation. In contrast, pre-boiled BSA produced either bell-shaped or linear turbidity changes depending on the amount of boiling time. When added together, pre-boiled BSF and BSA produced additive effects. (B) Supersaturated medium: BSF and BSA were added to medium as in (A) that was also inoculated with 1 mM each of CaCl 2 and NaH 2 PO 4 . Notice the various patterns of turbidity changes associated with the three protein combinations. See the text for further details.

Techniques Used: Incubation, Cell Culture, Produced

7) Product Images from "Identification of EBP50: A PDZ-containing Phosphoprotein that Associates with Members of the Ezrin-Radixin-Moesin Family "

Article Title: Identification of EBP50: A PDZ-containing Phosphoprotein that Associates with Members of the Ezrin-Radixin-Moesin Family

Journal: The Journal of Cell Biology

doi:

Identification of N-ERMAD binding proteins. ( A ) Lysis buffer, or placental extracts, or brain extracts were mixed with BSA–agarose ( BSA ), ezrin–N-ERMAD-agarose ( E-N ), or moesin– N-ERMAD-agarose ( M-N ), and then were washed extensively in 0.5 M NaCl and bound proteins eluted and resolved on a 6–20% silver stained gradient SDS gel. For some reactions, a fourfold molar excess of competitor ezrin N-ERMAD (+ C ) or mock competitor BSA (+ mock ) was added to the extract before mixing with moesin–N-ERMAD-agarose. ( B ) One quarter the amount of the same samples shown in A were resolved on a 10% gel, transferred to PVDF, and probed with biotinylated ezrin– N-ERMAD. Brackets indicate the N-ERMAD binding proteins, and arrowheads indicate ezrin. The mobilities of standard proteins are indicated in kD. DF , dye front.
Figure Legend Snippet: Identification of N-ERMAD binding proteins. ( A ) Lysis buffer, or placental extracts, or brain extracts were mixed with BSA–agarose ( BSA ), ezrin–N-ERMAD-agarose ( E-N ), or moesin– N-ERMAD-agarose ( M-N ), and then were washed extensively in 0.5 M NaCl and bound proteins eluted and resolved on a 6–20% silver stained gradient SDS gel. For some reactions, a fourfold molar excess of competitor ezrin N-ERMAD (+ C ) or mock competitor BSA (+ mock ) was added to the extract before mixing with moesin–N-ERMAD-agarose. ( B ) One quarter the amount of the same samples shown in A were resolved on a 10% gel, transferred to PVDF, and probed with biotinylated ezrin– N-ERMAD. Brackets indicate the N-ERMAD binding proteins, and arrowheads indicate ezrin. The mobilities of standard proteins are indicated in kD. DF , dye front.

Techniques Used: Binding Assay, Lysis, Staining, SDS-Gel

8) Product Images from "Subnuclear Trafficking of Glucocorticoid Receptors In Vitro: Chromatin Recycling and Nuclear Export"

Article Title: Subnuclear Trafficking of Glucocorticoid Receptors In Vitro: Chromatin Recycling and Nuclear Export

Journal: The Journal of Cell Biology

doi:

In vitro nuclear export of GR is an ATP-driven process. Hormone-withdrawn GrH2 cells were permeabilized and then incubated with 10 mg/ml of BSA in transport buffer containing 20 mM sodium molybdate at 30°C with the following additions: ( A ) 4 mM ATP; ( B ) 4 mM GTP; ( C ) 4 mM each of ATP and GTPγS; ( D ) 4 mM each of GTP and AMP-PNP. GR in fixed cells was detected by IIF.
Figure Legend Snippet: In vitro nuclear export of GR is an ATP-driven process. Hormone-withdrawn GrH2 cells were permeabilized and then incubated with 10 mg/ml of BSA in transport buffer containing 20 mM sodium molybdate at 30°C with the following additions: ( A ) 4 mM ATP; ( B ) 4 mM GTP; ( C ) 4 mM each of ATP and GTPγS; ( D ) 4 mM each of GTP and AMP-PNP. GR in fixed cells was detected by IIF.

Techniques Used: In Vitro, Incubation

In vitro nuclear export of GR stimulated by tyrosine phosphatase inhibitors is blocked by the tyrosine kinase inhibitors. Permeabilized, hormone-withdrawn GrH2 cells were incubated with BSA in transport buffer containing 4 mM ATP for 20 min at 30°C, in the absence ( A , C , E , G , and H ) or presence ( B , D , and F ) of 0.2 mM genistein. 20 mM sodium molybdate was included in A , B , and H , 7.5 mM sodium vanadate in C and D , 50 μg/ml of heparin in E and F , 1 μM microcystin in G , and 1 mM tyrphostin AG126 ( AG 126 ) in H. GR was detected in fixed cells by IIF.
Figure Legend Snippet: In vitro nuclear export of GR stimulated by tyrosine phosphatase inhibitors is blocked by the tyrosine kinase inhibitors. Permeabilized, hormone-withdrawn GrH2 cells were incubated with BSA in transport buffer containing 4 mM ATP for 20 min at 30°C, in the absence ( A , C , E , G , and H ) or presence ( B , D , and F ) of 0.2 mM genistein. 20 mM sodium molybdate was included in A , B , and H , 7.5 mM sodium vanadate in C and D , 50 μg/ml of heparin in E and F , 1 μM microcystin in G , and 1 mM tyrphostin AG126 ( AG 126 ) in H. GR was detected in fixed cells by IIF.

Techniques Used: In Vitro, Incubation

( a ) Western blot analysis of molybdate-stimulated in vitro GR nuclear export. (Lanes 1–8 ) GrH2 cells treated with corticosterone ( Cort ) for 1 h and then withdrawn from hormone for 20 min. (Lanes 9–12 ) GrH2 cells treated with corticosterone for 80 min. Harvested cells were permeabilized in suspension, and aliquots of intact nuclei were incubated with 50 μl of reaction mixture containing 10 mg/ml BSA in transport buffer, 4 mM ATP (lanes 1 , 2 , 5 , 6 , 9 , and 10 ), or 4 mM GTP (lanes 3 , 7 , and 11 ) with an energy-regenerating system, and 20 mM sodium molybdate where indicated (lanes 2 , 3 , 4 , 6 , 7 , 8 , 10 , 11 , and 12 ). After a 20min incubation at 30°C, each nuclear suspension was split into two identical samples. One sample was subjected to SDS-PAGE directly (lanes 1–4 , Whole ). The other identical sample was washed, and nuclei were recovered and subjected to SDS-PAGE for detection of the remaining nuclear GR (lanes 5–12 , Nuc Prep ). ( b ) Quantification of GR levels observed in Western blots by densitometry. GR levels were normalized to the internal control NuMA protein. (Bars 1–8 ) Average of four experiments; (bars 9–12 ) average of two experiments. Whole , whole reaction mix; Nuc Prep , nuclear pellets.
Figure Legend Snippet: ( a ) Western blot analysis of molybdate-stimulated in vitro GR nuclear export. (Lanes 1–8 ) GrH2 cells treated with corticosterone ( Cort ) for 1 h and then withdrawn from hormone for 20 min. (Lanes 9–12 ) GrH2 cells treated with corticosterone for 80 min. Harvested cells were permeabilized in suspension, and aliquots of intact nuclei were incubated with 50 μl of reaction mixture containing 10 mg/ml BSA in transport buffer, 4 mM ATP (lanes 1 , 2 , 5 , 6 , 9 , and 10 ), or 4 mM GTP (lanes 3 , 7 , and 11 ) with an energy-regenerating system, and 20 mM sodium molybdate where indicated (lanes 2 , 3 , 4 , 6 , 7 , 8 , 10 , 11 , and 12 ). After a 20min incubation at 30°C, each nuclear suspension was split into two identical samples. One sample was subjected to SDS-PAGE directly (lanes 1–4 , Whole ). The other identical sample was washed, and nuclei were recovered and subjected to SDS-PAGE for detection of the remaining nuclear GR (lanes 5–12 , Nuc Prep ). ( b ) Quantification of GR levels observed in Western blots by densitometry. GR levels were normalized to the internal control NuMA protein. (Bars 1–8 ) Average of four experiments; (bars 9–12 ) average of two experiments. Whole , whole reaction mix; Nuc Prep , nuclear pellets.

Techniques Used: Western Blot, In Vitro, Incubation, SDS Page

Molybdate-stimulated in vitro nuclear export of VAN556 GR and hsp90. CHO cells stably transfected with VAN556 GR were permeabilized and then incubated with 50 μl of 10 mg/ml BSA in transport buffer for 20 min at 30°C with the following additions: ( A and B ) 4 mM ATP; ( C and D ) 4 mM ATP and 20 mM sodium molybdate; ( E and F ) 20 mM sodium molybdate. GR ( A , C , and E ) and hsp90 ( B , D , and F ) were detected in fixed cells by costaining with the BuGR2 mAb and TSTA rabbit serum.
Figure Legend Snippet: Molybdate-stimulated in vitro nuclear export of VAN556 GR and hsp90. CHO cells stably transfected with VAN556 GR were permeabilized and then incubated with 50 μl of 10 mg/ml BSA in transport buffer for 20 min at 30°C with the following additions: ( A and B ) 4 mM ATP; ( C and D ) 4 mM ATP and 20 mM sodium molybdate; ( E and F ) 20 mM sodium molybdate. GR ( A , C , and E ) and hsp90 ( B , D , and F ) were detected in fixed cells by costaining with the BuGR2 mAb and TSTA rabbit serum.

Techniques Used: In Vitro, Stable Transfection, Transfection, Incubation

Stimulation of in vitro GR nuclear export by sodium molybdate. Hormone-withdrawn GrH2 cells ( A–E , G , and H ) were permeabilized and then incubated with 10 mg/ml of BSA in transport buffer at 30°C ( A , B , and D–H ) or 0°C ( C ) with the following additions: ( A ) ATP; ( B and C ) ATP and 20 mM sodium molybdate; ( D ) 20 mM sodium molybdate; ( E ) 0.2 mg/ml WGA, ATP, and 20 mM sodium molybdate; ( F ) corticosterone-treated GrH2 cells were permeabilized and then incubated for 20 min at 30°C with ATP and 20 mM sodium molybdate; ( G ) ATP, 20 mM sodium molybdate, and the Ab-1 anti-NuMA antibody; and ( H ) ATP and 20 mM sodium molybdate. In A–F , cells were fixed with methanol and subjected to IIF to detect GR. In G , permeabilized cells were fixed after the incubation described above and treated with a FITC-coupled secondary antibody to detect the Ab-1 anti-NuMA primary antibody. In H , ATP- and molybdate-treated permeabilized cells were fixed and then subjected to IIF with the Ab-1 antibody to detect nuclear NuMA.
Figure Legend Snippet: Stimulation of in vitro GR nuclear export by sodium molybdate. Hormone-withdrawn GrH2 cells ( A–E , G , and H ) were permeabilized and then incubated with 10 mg/ml of BSA in transport buffer at 30°C ( A , B , and D–H ) or 0°C ( C ) with the following additions: ( A ) ATP; ( B and C ) ATP and 20 mM sodium molybdate; ( D ) 20 mM sodium molybdate; ( E ) 0.2 mg/ml WGA, ATP, and 20 mM sodium molybdate; ( F ) corticosterone-treated GrH2 cells were permeabilized and then incubated for 20 min at 30°C with ATP and 20 mM sodium molybdate; ( G ) ATP, 20 mM sodium molybdate, and the Ab-1 anti-NuMA antibody; and ( H ) ATP and 20 mM sodium molybdate. In A–F , cells were fixed with methanol and subjected to IIF to detect GR. In G , permeabilized cells were fixed after the incubation described above and treated with a FITC-coupled secondary antibody to detect the Ab-1 anti-NuMA primary antibody. In H , ATP- and molybdate-treated permeabilized cells were fixed and then subjected to IIF with the Ab-1 antibody to detect nuclear NuMA.

Techniques Used: In Vitro, Incubation, Whole Genome Amplification

Selective effects of tungstate on in vitro nuclear protein export. Permeabilized HeLa cells were incubated for 20 min at 30°C with BSA in transport buffer containing 20 mM sodium tungstate, in the absence ( A , C , E , and G ) or presence of 4 mM ATP ( B , D , F , and H ). Fixed cells were subjected to costaining to detect either hnRNP A1 ( A and B ) and hsp56 ( C and D ), or hnRNP C ( G and H ) and hsp90 ( E and F ) in the same fields of cells.
Figure Legend Snippet: Selective effects of tungstate on in vitro nuclear protein export. Permeabilized HeLa cells were incubated for 20 min at 30°C with BSA in transport buffer containing 20 mM sodium tungstate, in the absence ( A , C , E , and G ) or presence of 4 mM ATP ( B , D , F , and H ). Fixed cells were subjected to costaining to detect either hnRNP A1 ( A and B ) and hsp56 ( C and D ), or hnRNP C ( G and H ) and hsp90 ( E and F ) in the same fields of cells.

Techniques Used: In Vitro, Incubation

9) Product Images from "Efficient Conjugation of Aflatoxin M1 With Bovine Serum Albumin through Aflatoxin M1-(O-carboxymethyl) Oxime and Production of Anti-aflatoxin M1 Antibodies"

Article Title: Efficient Conjugation of Aflatoxin M1 With Bovine Serum Albumin through Aflatoxin M1-(O-carboxymethyl) Oxime and Production of Anti-aflatoxin M1 Antibodies

Journal: Jundishapur Journal of Microbiology

doi: 10.5812/jjm.8(4)2015.16850

UV-VIS Spectrum Obtained From Coupling AFM1-oxime to BSA; Using UV-VIS Spectrophotometry The peaks at wave lengths of 280 nm and 365 nm correspond to BSA and BSA-AFM1 conjugate, respectively.
Figure Legend Snippet: UV-VIS Spectrum Obtained From Coupling AFM1-oxime to BSA; Using UV-VIS Spectrophotometry The peaks at wave lengths of 280 nm and 365 nm correspond to BSA and BSA-AFM1 conjugate, respectively.

Techniques Used: Spectrophotometry

10) Product Images from "Albumin-induced podocyte injury and protection are associated with regulation of COX-2."

Article Title: Albumin-induced podocyte injury and protection are associated with regulation of COX-2.

Journal: Kidney international

doi: 10.1038/ki.2014.196

SA-associated factors contribute to COX-2 induction in podocytes. Serum-starved podocytes were exposed to 40 mg/ml of BSA, charcoal-treated FA/endotoxin-free BSA, FA/globulin-free BSA, endotoxin-free BSA, HSA and recombinant HSA made in yeast (rHSA). (A) Cells were harvested after 4 h, processed for SDS-PAGE and western blotting and analyzed for COX-2 and GAPDH. (B) Total RNA was extracted and COX-2 mRNA was measured by RT-PCR and normalized to the β-actin mRNA (***P
Figure Legend Snippet: SA-associated factors contribute to COX-2 induction in podocytes. Serum-starved podocytes were exposed to 40 mg/ml of BSA, charcoal-treated FA/endotoxin-free BSA, FA/globulin-free BSA, endotoxin-free BSA, HSA and recombinant HSA made in yeast (rHSA). (A) Cells were harvested after 4 h, processed for SDS-PAGE and western blotting and analyzed for COX-2 and GAPDH. (B) Total RNA was extracted and COX-2 mRNA was measured by RT-PCR and normalized to the β-actin mRNA (***P

Techniques Used: Recombinant, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction

11) Product Images from "Binding of dihydromyricetin and its metal ion complexes with bovine serum albumin"

Article Title: Binding of dihydromyricetin and its metal ion complexes with bovine serum albumin

Journal: Biotechnology, Biotechnological Equipment

doi: 10.1080/13102818.2014.915077

Energy transfer between BSA and DMY
Figure Legend Snippet: Energy transfer between BSA and DMY

Techniques Used:

Energy transfer between BSA and DMY
Figure Legend Snippet: Energy transfer between BSA and DMY

Techniques Used:

Quenching effect of DMY and its complexes on BSA fluorescence intensity. Different concentrations of DMY (A), DMY–Cu (II) (B), DMY–Mn (II) (C) and DMY-Zn (II) (D): a–h: 0.0 × 10 −5 mol L −1 (a), 1.0 × 10 −5 mol L −1 (b), 2.0 × 10 −5 mol L −1 (c), 3.0 × 10 −5 mol L −1 (d), 4.0 × 10 −5 mol L −1 (e), 5.0 × 10 −5 mol L −1 (f), 6.0 × 10 −5 mol L −1 (g), 7.0 × 10 −5 mol L −1 (h); λ ex = 280 nm; T = 300 K.
Figure Legend Snippet: Quenching effect of DMY and its complexes on BSA fluorescence intensity. Different concentrations of DMY (A), DMY–Cu (II) (B), DMY–Mn (II) (C) and DMY-Zn (II) (D): a–h: 0.0 × 10 −5 mol L −1 (a), 1.0 × 10 −5 mol L −1 (b), 2.0 × 10 −5 mol L −1 (c), 3.0 × 10 −5 mol L −1 (d), 4.0 × 10 −5 mol L −1 (e), 5.0 × 10 −5 mol L −1 (f), 6.0 × 10 −5 mol L −1 (g), 7.0 × 10 −5 mol L −1 (h); λ ex = 280 nm; T = 300 K.

Techniques Used: Fluorescence

Energy transfer between BSA and DMY
Figure Legend Snippet: Energy transfer between BSA and DMY

Techniques Used:

UV-vis spectra of DMY (A) or its complexes (B–D) in the presence (a–f) or absence of BSA (g). Different concentrations of DMY (A), DMY-Cu (II) (B), DMY-Mn (II) (C) and DMY-Zn (II) (D): 0.0 × 10 −5 mol L −1 (a), 1.0 ×1 0 −5 mol L −1 (b), 2.0 × 10 −5 mol L −1 (c), 3.0 × 10 −5 mol L −1 (d), 4.0 × 10 −5 mol L −1 (e), 5.0 × 10 −5 mol L −1 (f); C BSA = 5.0 × 10 −6 mol L −1 . In the absence of BSA (g), C DMY = C DMY-Cu (II) = C DMY-Mn (II) = C DMY-Zn(II) = 2.0 × 10 −5 mol L −1 .
Figure Legend Snippet: UV-vis spectra of DMY (A) or its complexes (B–D) in the presence (a–f) or absence of BSA (g). Different concentrations of DMY (A), DMY-Cu (II) (B), DMY-Mn (II) (C) and DMY-Zn (II) (D): 0.0 × 10 −5 mol L −1 (a), 1.0 ×1 0 −5 mol L −1 (b), 2.0 × 10 −5 mol L −1 (c), 3.0 × 10 −5 mol L −1 (d), 4.0 × 10 −5 mol L −1 (e), 5.0 × 10 −5 mol L −1 (f); C BSA = 5.0 × 10 −6 mol L −1 . In the absence of BSA (g), C DMY = C DMY-Cu (II) = C DMY-Mn (II) = C DMY-Zn(II) = 2.0 × 10 −5 mol L −1 .

Techniques Used:

Overlap of the fluorescence emission spectrum of BSA (a) and the UV–vis absorption spectra (b) of DMY (A), DMY-Cu (II) (B), DMY-Mn (II) (C) and DMY-Zn (II) (D). C BSA = 1.00 × 10 −6 mol L −1 , C DMY = C DMY-Cu (II) = C DMY-Mn (II) = C DMY-Zn(II) = 3.0 × 10 −5 mol L −1 .
Figure Legend Snippet: Overlap of the fluorescence emission spectrum of BSA (a) and the UV–vis absorption spectra (b) of DMY (A), DMY-Cu (II) (B), DMY-Mn (II) (C) and DMY-Zn (II) (D). C BSA = 1.00 × 10 −6 mol L −1 , C DMY = C DMY-Cu (II) = C DMY-Mn (II) = C DMY-Zn(II) = 3.0 × 10 −5 mol L −1 .

Techniques Used: Fluorescence

Stern-Volmer plots for BSA fluorescence quenching by DMY, DMY-Cu (II), DMY-Mn (II) and DMY-Zn (II) complexes at 300 and 310 K.
Figure Legend Snippet: Stern-Volmer plots for BSA fluorescence quenching by DMY, DMY-Cu (II), DMY-Mn (II) and DMY-Zn (II) complexes at 300 and 310 K.

Techniques Used: Fluorescence

12) Product Images from "Direct Oxidation and Covalent Binding of Isoniazid to Rodent Liver and Human Hepatic Microsomes: Humans Are More Like Mice than Rats"

Article Title: Direct Oxidation and Covalent Binding of Isoniazid to Rodent Liver and Human Hepatic Microsomes: Humans Are More Like Mice than Rats

Journal: Chemical Research in Toxicology

doi: 10.1021/tx300341r

Specificity of the anti-INH antibody. (A) Antibody specificity was tested by ELISA. The plate was either coated with BSA modified with INA (BSA-INA) or BSA alone. Preimmune serum (S Pre ) or serum after immunization with Blue Carrier Protein modified with INA (S Aft ) diluted at 1:100,000 was used as the primary antibody. In the third and fifth column, the primary antibody was preincubated with INH or NAL, respectively, at a concentration of 200 μM for 30 min at room temperature. (B) Antibody was tested for cross-reactivity with binding due to AcHz. Female C57BL/6 mice were treated with either INH or AcHz (Fisher Scientific) by gavage for 7 days at 50 mg/kg/day ( n = 2 for each group). There was no binding to hepatic proteins from untreated control and AcHz-treated mice, whereas hepatic proteins from INH-treated mice showed a large number of bands modified with INH. (C) Antiserum was tested for specificity on Western blots by preincubation with INH at 200 μM or 2 mM for 1 h at 4 °C, which prevented binding to the INH-modified hepatic proteins. (D) Binding of the anti-INH serum to INH-modified liver proteins on Western blots was compared to that of the preimmune serum from the same animal.
Figure Legend Snippet: Specificity of the anti-INH antibody. (A) Antibody specificity was tested by ELISA. The plate was either coated with BSA modified with INA (BSA-INA) or BSA alone. Preimmune serum (S Pre ) or serum after immunization with Blue Carrier Protein modified with INA (S Aft ) diluted at 1:100,000 was used as the primary antibody. In the third and fifth column, the primary antibody was preincubated with INH or NAL, respectively, at a concentration of 200 μM for 30 min at room temperature. (B) Antibody was tested for cross-reactivity with binding due to AcHz. Female C57BL/6 mice were treated with either INH or AcHz (Fisher Scientific) by gavage for 7 days at 50 mg/kg/day ( n = 2 for each group). There was no binding to hepatic proteins from untreated control and AcHz-treated mice, whereas hepatic proteins from INH-treated mice showed a large number of bands modified with INH. (C) Antiserum was tested for specificity on Western blots by preincubation with INH at 200 μM or 2 mM for 1 h at 4 °C, which prevented binding to the INH-modified hepatic proteins. (D) Binding of the anti-INH serum to INH-modified liver proteins on Western blots was compared to that of the preimmune serum from the same animal.

Techniques Used: Enzyme-linked Immunosorbent Assay, Modification, Concentration Assay, Binding Assay, Mouse Assay, Western Blot

13) Product Images from "First Total Synthesis of a Naturally Occurring Iodinated 5?-Deoxyxylofuranosyl Marine Nucleoside"

Article Title: First Total Synthesis of a Naturally Occurring Iodinated 5?-Deoxyxylofuranosyl Marine Nucleoside

Journal: Marine Drugs

doi: 10.3390/md10040881

Total synthesis of pyrrolo[2,3- d ]pyrimidine nucleoside 1 . Reagents and conditions: ( a ) (a1) Conc. H 2 SO 4 , acetone, 0 °C, 3 h; (a2) 5% Na 2 CO 3 aq., 30 min, 87%; ( b ) TsCl (1.1 eq.), THF, Et 3 N, 0 °C, overnight, 92%; ( c ) LiAlH 4 (0.5 eq.), THF, reflux, 6 h, 95%; ( d ) BzCl (1.1 eq), CH 2 Cl 2 , Et 3 N, 2 h, 98%; ( e ) Conc. H 2 SO 4 , Ac 2 O, 26 h, 78%; ( f ) POCl 3 (excess), reflux, 3 h, 98%; ( g ) NIS (1.01 eq.), DMF, 0 °C, 2 h, 98%; ( h ) 4 (1 eq.), BSA (1.2 eq.), TMSOTf (1 eq.), CH 3 CN, rt-80 °C, 12 h 56%; ( i ) Sat. NH 3 in MeOH (excess), 130 °C, 12 h, 82%.
Figure Legend Snippet: Total synthesis of pyrrolo[2,3- d ]pyrimidine nucleoside 1 . Reagents and conditions: ( a ) (a1) Conc. H 2 SO 4 , acetone, 0 °C, 3 h; (a2) 5% Na 2 CO 3 aq., 30 min, 87%; ( b ) TsCl (1.1 eq.), THF, Et 3 N, 0 °C, overnight, 92%; ( c ) LiAlH 4 (0.5 eq.), THF, reflux, 6 h, 95%; ( d ) BzCl (1.1 eq), CH 2 Cl 2 , Et 3 N, 2 h, 98%; ( e ) Conc. H 2 SO 4 , Ac 2 O, 26 h, 78%; ( f ) POCl 3 (excess), reflux, 3 h, 98%; ( g ) NIS (1.01 eq.), DMF, 0 °C, 2 h, 98%; ( h ) 4 (1 eq.), BSA (1.2 eq.), TMSOTf (1 eq.), CH 3 CN, rt-80 °C, 12 h 56%; ( i ) Sat. NH 3 in MeOH (excess), 130 °C, 12 h, 82%.

Techniques Used: Reflux

14) Product Images from "Role of Transcription Factor Acetylation in Diabetic Kidney Disease"

Article Title: Role of Transcription Factor Acetylation in Diabetic Kidney Disease

Journal: Diabetes

doi: 10.2337/db13-1810

Effects of bromodomain inhibitor in DN. MS417, a bromodomain inhibitor, suppressed acetylation of Stat3 and NF-κB in podocytes treated with AGE. Podocytes were incubated with either BSA or AGE-BSA together with DMSO or MS417 (1.0 μmol/L) for 24 h. Western blot analysis was performed in these cells for acetyl, phosphor- and total p65 ( A ) and STAT3 ( C ). The representative blots of three independent experiments are shown. The densitometry analyses of these Western blots are shown for p65 ( B ) and STAT3 ( D ). The ratios of acetyl-protein or phosphor-protein to total protein were calculated for p65 and STAT3. The fold changes relative to cells treated with BSA + DMSO are shown. * P
Figure Legend Snippet: Effects of bromodomain inhibitor in DN. MS417, a bromodomain inhibitor, suppressed acetylation of Stat3 and NF-κB in podocytes treated with AGE. Podocytes were incubated with either BSA or AGE-BSA together with DMSO or MS417 (1.0 μmol/L) for 24 h. Western blot analysis was performed in these cells for acetyl, phosphor- and total p65 ( A ) and STAT3 ( C ). The representative blots of three independent experiments are shown. The densitometry analyses of these Western blots are shown for p65 ( B ) and STAT3 ( D ). The ratios of acetyl-protein or phosphor-protein to total protein were calculated for p65 and STAT3. The fold changes relative to cells treated with BSA + DMSO are shown. * P

Techniques Used: Incubation, Western Blot

AGE induced acetylation of p65 and STAT3, which is required for their transcriptional activation. A : Human podocytes were cultured with AGE or BSA at the indicated doses for 24 h. Cell lysates were subject to Western blot analysis for acetyl-, phosphor-, and total p65 and STAT3. The representative blots of three independent experiments are shown. B : The Western blots from all experiments were quantified by densitometry analysis. The ratios of acetyl-protein or phosphor-protein to total protein were calculated for p65 and STAT3. The fold changes relative to BSA-treated cells are shown. * P
Figure Legend Snippet: AGE induced acetylation of p65 and STAT3, which is required for their transcriptional activation. A : Human podocytes were cultured with AGE or BSA at the indicated doses for 24 h. Cell lysates were subject to Western blot analysis for acetyl-, phosphor-, and total p65 and STAT3. The representative blots of three independent experiments are shown. B : The Western blots from all experiments were quantified by densitometry analysis. The ratios of acetyl-protein or phosphor-protein to total protein were calculated for p65 and STAT3. The fold changes relative to BSA-treated cells are shown. * P

Techniques Used: Activation Assay, Cell Culture, Western Blot

Acetylation of p65 and STAT3 is required for AGE-induced transcriptional activation of their target genes. Podocytes were infected with p65 or STAT3 constructs with mutated acetyl residues for 3 days then stimulated with AGE or BSA at the indicated doses for an additional 24 h. Total RNA was isolated from these cells for real-time PCR analysis of NF-κB target genes ( CXCL5 [ A ] and CCL17 [ B ]) and STAT3 target genes ( SOC3 [ C ] and Casp9 [ D ]). For chromatin immunoprecipitation (ChiP) assay for NF-κB and STAT3 target genes, podocytes were infected with p65 or STAT3 constructs with mutated acetyl residues for 3 days then stimulated with AGE or BSA at the indicated doses for an additional 24 h. ChiP assay also was performed in these cells to determine the binding of p65 and STAT3 on the promoter sites of their target genes ( CXCL5 [ E ], CCL17 [ F ], SOC3 [ G ], and Casp9 [ H ]). Immunoprecipitated DNA samples were subjected to PCR analysis and are expressed as a percentage of input. * P
Figure Legend Snippet: Acetylation of p65 and STAT3 is required for AGE-induced transcriptional activation of their target genes. Podocytes were infected with p65 or STAT3 constructs with mutated acetyl residues for 3 days then stimulated with AGE or BSA at the indicated doses for an additional 24 h. Total RNA was isolated from these cells for real-time PCR analysis of NF-κB target genes ( CXCL5 [ A ] and CCL17 [ B ]) and STAT3 target genes ( SOC3 [ C ] and Casp9 [ D ]). For chromatin immunoprecipitation (ChiP) assay for NF-κB and STAT3 target genes, podocytes were infected with p65 or STAT3 constructs with mutated acetyl residues for 3 days then stimulated with AGE or BSA at the indicated doses for an additional 24 h. ChiP assay also was performed in these cells to determine the binding of p65 and STAT3 on the promoter sites of their target genes ( CXCL5 [ E ], CCL17 [ F ], SOC3 [ G ], and Casp9 [ H ]). Immunoprecipitated DNA samples were subjected to PCR analysis and are expressed as a percentage of input. * P

Techniques Used: Activation Assay, Infection, Construct, Isolation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

15) Product Images from "Adiponectin, chemerin, cytokines, and dipeptidyl peptidase 4 are released from human adipose tissue in a depot-dependent manner: an in vitro system including human serum albumin"

Article Title: Adiponectin, chemerin, cytokines, and dipeptidyl peptidase 4 are released from human adipose tissue in a depot-dependent manner: an in vitro system including human serum albumin

Journal: BMC Endocrine Disorders

doi: 10.1186/1472-6823-14-7

Effects of BSA and HSA on cytokine release from human adipose tissue. Adipose tissue was incubated for 2, 4, 6, 8, and 24 h in albumin-free medium (blue) and medium supplemented with 1% HSA (red), 1% BSA Fraction V (purple), or 1% BSA Essentially Fatty Acid Free (black). Concentrations of nine cytokines in the conditioned media were analyzed with a multiplex immunoassay; (A) granulocyte-macrophage colony-stimulating factor (B) interferon-γ (C) IL-1β (D) IL-2 (E) IL-6 (F) IL-8 (G) IL-10 (H) IL-12p70 (I) TNF-α. Insets show detailed plots of cytokine release in medium without albumin and in medium with HSA. Concentrations are ng/mL/g adipose tissue for IL-6 and IL-8 and pg/mL/g adipose tissue for the remaining cytokines. Values mean ± SEM (n = 3). Both media containing BSA markedly induced the release of all nine cytokines vs albumin-free medium or medium containing HSA over 24 h (p
Figure Legend Snippet: Effects of BSA and HSA on cytokine release from human adipose tissue. Adipose tissue was incubated for 2, 4, 6, 8, and 24 h in albumin-free medium (blue) and medium supplemented with 1% HSA (red), 1% BSA Fraction V (purple), or 1% BSA Essentially Fatty Acid Free (black). Concentrations of nine cytokines in the conditioned media were analyzed with a multiplex immunoassay; (A) granulocyte-macrophage colony-stimulating factor (B) interferon-γ (C) IL-1β (D) IL-2 (E) IL-6 (F) IL-8 (G) IL-10 (H) IL-12p70 (I) TNF-α. Insets show detailed plots of cytokine release in medium without albumin and in medium with HSA. Concentrations are ng/mL/g adipose tissue for IL-6 and IL-8 and pg/mL/g adipose tissue for the remaining cytokines. Values mean ± SEM (n = 3). Both media containing BSA markedly induced the release of all nine cytokines vs albumin-free medium or medium containing HSA over 24 h (p

Techniques Used: Incubation, Multiplex Assay

16) Product Images from "Stem Cell Antigen-1 (Sca-1) Regulates Mammary Tumor Development and Cell Migration"

Article Title: Stem Cell Antigen-1 (Sca-1) Regulates Mammary Tumor Development and Cell Migration

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027841

Sca-1 repression leads to increased adhesion to collagen I, collagen IV and fibronectin. Cell adhesion of 100,000 cells/well coated with fibronectin, vitronectin, laminin, collagen I or collagen IV comparing shLuc (open bars), shSca-1 (grey bars) and shSca-1+Sca-1 (black bars). (A). Relative adhesion was normalized to a BSA control, (* p
Figure Legend Snippet: Sca-1 repression leads to increased adhesion to collagen I, collagen IV and fibronectin. Cell adhesion of 100,000 cells/well coated with fibronectin, vitronectin, laminin, collagen I or collagen IV comparing shLuc (open bars), shSca-1 (grey bars) and shSca-1+Sca-1 (black bars). (A). Relative adhesion was normalized to a BSA control, (* p

Techniques Used:

17) Product Images from "Resolubilization of Protein from Water-Insoluble Phlorotannin–Protein Complexes upon Acidification"

Article Title: Resolubilization of Protein from Water-Insoluble Phlorotannin–Protein Complexes upon Acidification

Journal: Journal of Agricultural and Food Chemistry

doi: 10.1021/acs.jafc.7b03779

Fluorescence emission spectra (λ ex = 280 nm) of (A) β-casein and (B) BSA quenched by an increasing tannin concentration. Stern–Volmer plots (λ em = 350 nm) for binding of PhT (0–25 μM assuming an average MW = 2000 Da) (●) and PGG (0–50 μM) (□) to (C) β-casein and (D) BSA at pH 8.0.
Figure Legend Snippet: Fluorescence emission spectra (λ ex = 280 nm) of (A) β-casein and (B) BSA quenched by an increasing tannin concentration. Stern–Volmer plots (λ em = 350 nm) for binding of PhT (0–25 μM assuming an average MW = 2000 Da) (●) and PGG (0–50 μM) (□) to (C) β-casein and (D) BSA at pH 8.0.

Techniques Used: Fluorescence, Concentration Assay, Binding Assay

Proportions (%) of (A and C) precipitated protein and (B and D) precipitated PhT (×) after precipitation of protein (□) or mixtures of PhT/protein (○) for (A and B) β-casein and (C and D) BSA as a function of pH. (- - -) pI of the proteins.
Figure Legend Snippet: Proportions (%) of (A and C) precipitated protein and (B and D) precipitated PhT (×) after precipitation of protein (□) or mixtures of PhT/protein (○) for (A and B) β-casein and (C and D) BSA as a function of pH. (- - -) pI of the proteins.

Techniques Used:

(A) Fluorescence spectra of BSA (λ ex = 280 nm) at pH 8 (−), pH 6.0 (- - -), and pH 3.0 (···). Stern–Volmer plots (λ em = 350 nm) of (B) 5 μM β-casein and (C) 5 μM BSA quenched by an increasing PhT concentration (0–25 μM, assuming an average MW = 2000 Da) at pH 8 (●), pH 6 (□), and pH 3 (×).
Figure Legend Snippet: (A) Fluorescence spectra of BSA (λ ex = 280 nm) at pH 8 (−), pH 6.0 (- - -), and pH 3.0 (···). Stern–Volmer plots (λ em = 350 nm) of (B) 5 μM β-casein and (C) 5 μM BSA quenched by an increasing PhT concentration (0–25 μM, assuming an average MW = 2000 Da) at pH 8 (●), pH 6 (□), and pH 3 (×).

Techniques Used: Fluorescence, Concentration Assay

Protein (%) remaining in precipitation after resolubilization from precipitated β-casein (□) from PhT/protein combinations (○) and PhT/protein/PEG combinations (◇) for (A) β-casein and (B) BSA as a function of pH. In absence of tannins, BSA did not precipitate.
Figure Legend Snippet: Protein (%) remaining in precipitation after resolubilization from precipitated β-casein (□) from PhT/protein combinations (○) and PhT/protein/PEG combinations (◇) for (A) β-casein and (B) BSA as a function of pH. In absence of tannins, BSA did not precipitate.

Techniques Used:

18) Product Images from "Gradients of substrate-bound laminin orient axonal specification of neurons"

Article Title: Gradients of substrate-bound laminin orient axonal specification of neurons

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

doi: 10.1073/pnas.192457199

( A ) Schematic drawing of the design of a typical microfluidic network in PDMS that we used for the fabrication of immobilized gradients. Solutions of laminin and BSA were injected into the microfluidic network (inlets). Several streams, each carrying different concentrations of laminin and BSA, were generated in the gradient mixer and combined in a single channel to form a gradient perpendicular to the direction of the flow. The initial step profile created at the junction blurs slightly due to diffusion as the fluid travels downstream. The right-hand side of the diagram shows cross-sectional images of the concentration profile (using pure buffer and fluorescein, D = 5.0 × 10 −6 cm 2 ⋅s −1 ) of the flow visualized by using a confocal microscope. The shadows in the corners of the confocal images are due to the distortion of the light at the channel walls. ( B ) The diagram summarizes the steps from the fabrication of the substrate-bound gradient to the cultivation of neurons on the gradient immobilized on the substrate. A gradient was deposited on the floor of the channels by adsorption from the flowing stream. The network of microfluidic channels was removed, inverted, and placed in a culture dish. Neurons were plated on the floor of the channel in the region where the gradient had formed. ( C ) An idealized schematic diagram of the cross section of the surface composition of a substrate-bound gradient composed of laminin and BSA on PLL.
Figure Legend Snippet: ( A ) Schematic drawing of the design of a typical microfluidic network in PDMS that we used for the fabrication of immobilized gradients. Solutions of laminin and BSA were injected into the microfluidic network (inlets). Several streams, each carrying different concentrations of laminin and BSA, were generated in the gradient mixer and combined in a single channel to form a gradient perpendicular to the direction of the flow. The initial step profile created at the junction blurs slightly due to diffusion as the fluid travels downstream. The right-hand side of the diagram shows cross-sectional images of the concentration profile (using pure buffer and fluorescein, D = 5.0 × 10 −6 cm 2 ⋅s −1 ) of the flow visualized by using a confocal microscope. The shadows in the corners of the confocal images are due to the distortion of the light at the channel walls. ( B ) The diagram summarizes the steps from the fabrication of the substrate-bound gradient to the cultivation of neurons on the gradient immobilized on the substrate. A gradient was deposited on the floor of the channels by adsorption from the flowing stream. The network of microfluidic channels was removed, inverted, and placed in a culture dish. Neurons were plated on the floor of the channel in the region where the gradient had formed. ( C ) An idealized schematic diagram of the cross section of the surface composition of a substrate-bound gradient composed of laminin and BSA on PLL.

Techniques Used: Injection, Generated, Flow Cytometry, Diffusion-based Assay, Concentration Assay, Microscopy, Adsorption

( A ) Superimposed drawings of all processes of neurons grown on gradients of laminin on PLL. Neurons were grouped and superimposed according to the orientation of the tip of the axon (the longest process) after 24 h in culture. This analysis was done for each gradient slope separately (columns). The single axon of a neuron is drawn in black; the remaining short processes (dendrites) are drawn in gray. Neurons with axons extending in the direction of increasing laminin concentration are shown in the first row. Neurons with axons extending parallel to the gradient or in the direction of decreasing laminin concentration are shown in the second and third row, respectively. The shapes of the gradients in laminin and BSA in solution that were used to fabricate the substrate-bound gradient are given in the graphs below. The gradients in laminin and BSA were always formed on a homogeneous layer of PLL. In each drawing, the number of neurons in each classification (+, 0, or −) is listed with the total number of neurons grown on a particular gradient on the upper left corner. ( B ) Definition of the angle α of axonal process grown on a gradient in laminin. The sectors that formed the basis for the classification in (+)-, (0)-, and (−)-responding neurons are indicated. ( C ) Summary of the percentage of (+)-, (0)-, and (−)-responding neurons cultivated on gradients with different slopes in laminin.
Figure Legend Snippet: ( A ) Superimposed drawings of all processes of neurons grown on gradients of laminin on PLL. Neurons were grouped and superimposed according to the orientation of the tip of the axon (the longest process) after 24 h in culture. This analysis was done for each gradient slope separately (columns). The single axon of a neuron is drawn in black; the remaining short processes (dendrites) are drawn in gray. Neurons with axons extending in the direction of increasing laminin concentration are shown in the first row. Neurons with axons extending parallel to the gradient or in the direction of decreasing laminin concentration are shown in the second and third row, respectively. The shapes of the gradients in laminin and BSA in solution that were used to fabricate the substrate-bound gradient are given in the graphs below. The gradients in laminin and BSA were always formed on a homogeneous layer of PLL. In each drawing, the number of neurons in each classification (+, 0, or −) is listed with the total number of neurons grown on a particular gradient on the upper left corner. ( B ) Definition of the angle α of axonal process grown on a gradient in laminin. The sectors that formed the basis for the classification in (+)-, (0)-, and (−)-responding neurons are indicated. ( C ) Summary of the percentage of (+)-, (0)-, and (−)-responding neurons cultivated on gradients with different slopes in laminin.

Techniques Used: Concentration Assay

19) Product Images from "Lewisx and α2,3-Sialyl Glycans and Their Receptors TAG-1, Contactin, and L1 Mediate CD24-Dependent Neurite Outgrowth"

Article Title: Lewisx and α2,3-Sialyl Glycans and Their Receptors TAG-1, Contactin, and L1 Mediate CD24-Dependent Neurite Outgrowth

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.4361-08.2009

TAG-1 and Contactin bind to CD24 and are receptors for Lewis x . A , Lysates from CHO cells transfected with TAX-1 (CHO/TAX-1), Contactin (CHO/Contactin), or NCAM120 (CHO/NCAM120) were incubated with epoxy beads coated with Lewis x –BSA (Le x -BSA), N -acetyllactosamine–BSA (LN-BSA), and BSA. After pull-down, beads were analyzed by Western blotting using antibodies to TAG-1 (lanes 1–4), Contactin (lanes 5–8), or NCAM (lanes 9–12). B , Immunoprecipitates from brain homogenate using CD24 antibody (α-CD24) or control rat IgG (rIgG) were analyzed by Western blotting using antibodies against L1, TAG-1, Contactin, NCAM, or CD24. C , CD24-coupled epoxy beads were incubated with TAX-1-Fc, Contactin-Fc, CHL1-Fc, or Fc alone. After pull-down, beads (PD CD24) were analyzed by Western blotting using HRP-coupled anti-human antibody. D , E , Cerebellar tissue (input) was used for immunoprecipitations with CD24 with antibody (α-CD24), with control rat IgG (rIgG) or without antibody (−) or for immunoprecipitations with TAG-1 antibody (α-TAG-1), Contactin antibody (α-Contactin), or control goat IgG (gIgG) or without antibody (−). Immunoprecipitates were analyzed by Western blotting using antibodies against CD24, TAG-1, Contactin, or CHL1.
Figure Legend Snippet: TAG-1 and Contactin bind to CD24 and are receptors for Lewis x . A , Lysates from CHO cells transfected with TAX-1 (CHO/TAX-1), Contactin (CHO/Contactin), or NCAM120 (CHO/NCAM120) were incubated with epoxy beads coated with Lewis x –BSA (Le x -BSA), N -acetyllactosamine–BSA (LN-BSA), and BSA. After pull-down, beads were analyzed by Western blotting using antibodies to TAG-1 (lanes 1–4), Contactin (lanes 5–8), or NCAM (lanes 9–12). B , Immunoprecipitates from brain homogenate using CD24 antibody (α-CD24) or control rat IgG (rIgG) were analyzed by Western blotting using antibodies against L1, TAG-1, Contactin, NCAM, or CD24. C , CD24-coupled epoxy beads were incubated with TAX-1-Fc, Contactin-Fc, CHL1-Fc, or Fc alone. After pull-down, beads (PD CD24) were analyzed by Western blotting using HRP-coupled anti-human antibody. D , E , Cerebellar tissue (input) was used for immunoprecipitations with CD24 with antibody (α-CD24), with control rat IgG (rIgG) or without antibody (−) or for immunoprecipitations with TAG-1 antibody (α-TAG-1), Contactin antibody (α-Contactin), or control goat IgG (gIgG) or without antibody (−). Immunoprecipitates were analyzed by Western blotting using antibodies against CD24, TAG-1, Contactin, or CHL1.

Techniques Used: Transfection, Incubation, Western Blot

20) Product Images from "Enzyme-Linked Immunosorbent Assay (ELISA) and Blocking with Bovine Serum Albumin (BSA) - Not all BSAs are alike"

Article Title: Enzyme-Linked Immunosorbent Assay (ELISA) and Blocking with Bovine Serum Albumin (BSA) - Not all BSAs are alike

Journal: Journal of immunological methods

doi: 10.1016/j.jim.2012.06.009

Specific vs. non-specific binding of VCP to C3b or various BSA preparations
Figure Legend Snippet: Specific vs. non-specific binding of VCP to C3b or various BSA preparations

Techniques Used: Binding Assay

21) Product Images from "Directly Coupled High-Performance Liquid Chromatography-Accelerator Mass Spectrometry Measurement of Chemically Modified Protein and Peptides"

Article Title: Directly Coupled High-Performance Liquid Chromatography-Accelerator Mass Spectrometry Measurement of Chemically Modified Protein and Peptides

Journal: Analytical chemistry

doi: 10.1021/ac303609n

Absolute quantitation of an albumin 14 C-iodoacetamide modification competitive binding experiment. Treated and untreated BSA samples were exposed to 1 pCi 14 C-iodoacetamide and described as follows: 14 C-iodoacetamide-modified native BSA ( 14 C-IAC-native):native
Figure Legend Snippet: Absolute quantitation of an albumin 14 C-iodoacetamide modification competitive binding experiment. Treated and untreated BSA samples were exposed to 1 pCi 14 C-iodoacetamide and described as follows: 14 C-iodoacetamide-modified native BSA ( 14 C-IAC-native):native

Techniques Used: Quantitation Assay, Modification, Binding Assay

22) Product Images from "Nitric Oxide as an Endogenous Mutagen for Sendai Virus without Antiviral Activity"

Article Title: Nitric Oxide as an Endogenous Mutagen for Sendai Virus without Antiviral Activity

Journal: Journal of Virology

doi: 10.1128/JVI.78.16.8709-8719.2004

Effects of NO formed from GS-NO and SNAP on propagation of SeV and influenza virus. (A to D) After MDCK monolayers were inoculated with influenza virus or SeV at a multiplicity of infection of 3.0 PFU per cell, infected cells were incubated in DMEM containing 0.2% BSA and various concentrations of the NO donors GS-NO and SNAP. (E and F) MDCK cells were infected with SeV or influenza virus at a multiplicity of infection (MOI) of 0.1 or 0.01 PFU per cell, followed by culture with 0.1 mM SNAP as just described. At different time points after infection, the yield of virus in culture supernatants was assessed by use of the plaque-forming assay. Data are means ± standard error ( n = 3). See text for details.
Figure Legend Snippet: Effects of NO formed from GS-NO and SNAP on propagation of SeV and influenza virus. (A to D) After MDCK monolayers were inoculated with influenza virus or SeV at a multiplicity of infection of 3.0 PFU per cell, infected cells were incubated in DMEM containing 0.2% BSA and various concentrations of the NO donors GS-NO and SNAP. (E and F) MDCK cells were infected with SeV or influenza virus at a multiplicity of infection (MOI) of 0.1 or 0.01 PFU per cell, followed by culture with 0.1 mM SNAP as just described. At different time points after infection, the yield of virus in culture supernatants was assessed by use of the plaque-forming assay. Data are means ± standard error ( n = 3). See text for details.

Techniques Used: Infection, Incubation

23) Product Images from "TRIVALENT LIGANDS WITH RIGID DNA SPACERS REVEAL STRUCTURAL REQUIREMENTS FOR IgE RECEPTOR SIGNALING IN RBL MAST CELLS"

Article Title: TRIVALENT LIGANDS WITH RIGID DNA SPACERS REVEAL STRUCTURAL REQUIREMENTS FOR IgE RECEPTOR SIGNALING IN RBL MAST CELLS

Journal: ACS chemical biology

doi: 10.1021/cb7001472

Ca 2+ mobilization stimulated by Yn-DNP 3 ligands a) Representative Ca 2+ responses of RBL mast cells as stimulated by DNP-BSA (1 μg/ml) or the Yn-DNP 3 ligands (30 nM) as specified on individual plots. The baseline was set to 0.0, and ligand addition at 100s is indicated by the arrow. Cells were suspended in BSS including 1.8 mM Ca 2+ except the designated plot for DNP-BSA (top panel) for which Ca 2+ was omitted from the buffer. b) Ca 2+ mobilization integrated for 600 sec after ligand addition, and averaged for each Yn-DNP 3 from 5−7 independent experiments. and expressed relative to integrated response to 1μg/μl DNP-BSA. Error bars represent standard errors.
Figure Legend Snippet: Ca 2+ mobilization stimulated by Yn-DNP 3 ligands a) Representative Ca 2+ responses of RBL mast cells as stimulated by DNP-BSA (1 μg/ml) or the Yn-DNP 3 ligands (30 nM) as specified on individual plots. The baseline was set to 0.0, and ligand addition at 100s is indicated by the arrow. Cells were suspended in BSS including 1.8 mM Ca 2+ except the designated plot for DNP-BSA (top panel) for which Ca 2+ was omitted from the buffer. b) Ca 2+ mobilization integrated for 600 sec after ligand addition, and averaged for each Yn-DNP 3 from 5−7 independent experiments. and expressed relative to integrated response to 1μg/μl DNP-BSA. Error bars represent standard errors.

Techniques Used: Size-exclusion Chromatography

24) Product Images from "Role of the receptor for advanced glycation end products in hepatic fibrosis"

Article Title: Role of the receptor for advanced glycation end products in hepatic fibrosis

Journal: World Journal of Gastroenterology : WJG

doi: 10.3748/wjg.15.5789

AGE-BSA does not activate p44/42 (A) and p38 (B) MAPK signaling in hepatic stellate cells. Kinase phosphorylation was determined in CFSC-2G and HSC-T6 HSC using quantitative western blotting with phosphospecific antibodies relative to total kinase antibodies
Figure Legend Snippet: AGE-BSA does not activate p44/42 (A) and p38 (B) MAPK signaling in hepatic stellate cells. Kinase phosphorylation was determined in CFSC-2G and HSC-T6 HSC using quantitative western blotting with phosphospecific antibodies relative to total kinase antibodies

Techniques Used: Western Blot

AGE do not stimulate DNA synthesis in hepatic stellate cells. DNA synthesis as a surrogate of proliferation was determined in CFSC-2G (A) and HSC-T6 (B) HSC after a 24 h incubation with increasing concentrations of FCS, AGE-BSA or CML-BSA in 0% FCS. Data
Figure Legend Snippet: AGE do not stimulate DNA synthesis in hepatic stellate cells. DNA synthesis as a surrogate of proliferation was determined in CFSC-2G (A) and HSC-T6 (B) HSC after a 24 h incubation with increasing concentrations of FCS, AGE-BSA or CML-BSA in 0% FCS. Data

Techniques Used: DNA Synthesis, Incubation

25) Product Images from "A General Strategy for Generating Gradients of Bioactive Proteins on Electrospun Nanofiber Mats by Masking with Bovine Serum Albumin"

Article Title: A General Strategy for Generating Gradients of Bioactive Proteins on Electrospun Nanofiber Mats by Masking with Bovine Serum Albumin

Journal: Journal of materials chemistry. B, Materials for biology and medicine

doi: 10.1039/C7TB00974G

SEM images of random PCL nanofibers (A) before and (B–;D) after plasma treatment for 2 min, followed by soaking in (B) PBS, (C) 0.1% BSA solution, and (D) 0.5% BSA solution, respectively, for 1 h. The morphology and texture of the nanofibers are essentially preserved during the treatments.
Figure Legend Snippet: SEM images of random PCL nanofibers (A) before and (B–;D) after plasma treatment for 2 min, followed by soaking in (B) PBS, (C) 0.1% BSA solution, and (D) 0.5% BSA solution, respectively, for 1 h. The morphology and texture of the nanofibers are essentially preserved during the treatments.

Techniques Used:

(A) Plots of the relative fluorescence intensities for the BSA-FITC adsorbed on the nanofibers as a function of soaking time in the 0.1% and 0.5% BSA solutions, respectively. The corresponding representative fluorescence micrographs of the nanofibers are also included (upper row: 0.1% BSA, and lower row: 0.5%). The extent of BSA adsorbed on PCL nanofibers is dependent on the duration of exposure to BSA solution (n=3 for each time point). The BSA concentration also affects the rate at which BSA adsorbs onto the nanofibers. (B) Representative fluorescence micrographs and their corresponding relative fluorescence intensities at different positions of nanofiber strips by varying the duration of contact time with a 0.1% BSA solution over a distance of 35 mm (n=9 at each position). A gradient in BSA can be clearly seen across the strip of nanofibers.
Figure Legend Snippet: (A) Plots of the relative fluorescence intensities for the BSA-FITC adsorbed on the nanofibers as a function of soaking time in the 0.1% and 0.5% BSA solutions, respectively. The corresponding representative fluorescence micrographs of the nanofibers are also included (upper row: 0.1% BSA, and lower row: 0.5%). The extent of BSA adsorbed on PCL nanofibers is dependent on the duration of exposure to BSA solution (n=3 for each time point). The BSA concentration also affects the rate at which BSA adsorbs onto the nanofibers. (B) Representative fluorescence micrographs and their corresponding relative fluorescence intensities at different positions of nanofiber strips by varying the duration of contact time with a 0.1% BSA solution over a distance of 35 mm (n=9 at each position). A gradient in BSA can be clearly seen across the strip of nanofibers.

Techniques Used: Fluorescence, Concentration Assay, Stripping Membranes

26) Product Images from "Spectral-domain optical coherence phase microscopy for label-free multiplexed protein microarray assay"

Article Title: Spectral-domain optical coherence phase microscopy for label-free multiplexed protein microarray assay

Journal: Biosensors & bioelectronics

doi: 10.1016/j.bios.2009.06.028

Calculated kinetic association and dissociation parameters for BSA and HSA spots with αHSA. (a) Sensorgrams for each spot. (b) and (c) present k a and k d averaged over 5 spots for each species. The error bar denotes standard deviation.
Figure Legend Snippet: Calculated kinetic association and dissociation parameters for BSA and HSA spots with αHSA. (a) Sensorgrams for each spot. (b) and (c) present k a and k d averaged over 5 spots for each species. The error bar denotes standard deviation.

Techniques Used: Standard Deviation

27) Product Images from "Comparison of the Sensitivities of WaterLOGSY and Saturation Transfer Difference NMR Experiments"

Article Title: Comparison of the Sensitivities of WaterLOGSY and Saturation Transfer Difference NMR Experiments

Journal: Journal of biomolecular NMR

doi: 10.1007/s10858-014-9848-9

Examples of WaterLOGSY and STD spectra used to compare sensitivities for (a) KET-BSA, (b) TBHQ-HA and (c) CAM-Ribosome 70S. In all cases the relaxation delays, saturation/mixing times, receiver gain and total experimental times were identical.
Figure Legend Snippet: Examples of WaterLOGSY and STD spectra used to compare sensitivities for (a) KET-BSA, (b) TBHQ-HA and (c) CAM-Ribosome 70S. In all cases the relaxation delays, saturation/mixing times, receiver gain and total experimental times were identical.

Techniques Used: Chick Chorioallantoic Membrane Assay

28) Product Images from "Neutrophil lactoferrin release induced by IgA immune complexes differed from that induced by cross-linking of Fc? receptors (Fc?R) with a monoclonal antibody, MIP8a"

Article Title: Neutrophil lactoferrin release induced by IgA immune complexes differed from that induced by cross-linking of Fc? receptors (Fc?R) with a monoclonal antibody, MIP8a

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.2000.01254.x

Inhibition of neutrophil lactoferrin release by MIP8a and MIP10c. Neutrophils (100 μl) (4 × 10 6 cells/ml) were incubated with MIP8a F(ab′) 2 fragments or MIP10c F(ab′) 2 fragments in 96-well plates at room temperature for 10 min. Then 50 μl of IgA/NIP-BSA IC (60 μg/ml) were added to each well. After incubation at 37°C for 2 h, lactoferrin released from neutrophils was measured. ▪, MIP10c; ▴, MIP8a. Results are representative of three separate experiments.
Figure Legend Snippet: Inhibition of neutrophil lactoferrin release by MIP8a and MIP10c. Neutrophils (100 μl) (4 × 10 6 cells/ml) were incubated with MIP8a F(ab′) 2 fragments or MIP10c F(ab′) 2 fragments in 96-well plates at room temperature for 10 min. Then 50 μl of IgA/NIP-BSA IC (60 μg/ml) were added to each well. After incubation at 37°C for 2 h, lactoferrin released from neutrophils was measured. ▪, MIP10c; ▴, MIP8a. Results are representative of three separate experiments.

Techniques Used: Inhibition, Incubation

Lactoferrin release induced by MIP8a and IgA immune complexes (IC). (a) Dose–response of MIP8a-induced lactoferrin release. Neutrophils (100 μl) (4 × 10 6 cells/ml) were incubated at room temperature for 10 min with 25 μl MIP8a F(ab′) 2 fragments as indicated. Then 25 μl of F(ab′) 2 fragments of rabbit anti-mouse IgG (120 μg/ml) were added to each well. After incubation at 37°C for 2 h, lactoferrin released from neutrophils was measured. (b) Time course of MIP8a- and IgA IC-induced lactoferrin release. For MIP8a-induced lactoferrin release, 100 μl of neutrophils (4 × 10 6 cells/ml) were incubated at room temperature for 10 min with 25 μl of MIP8a F(ab′) 2 fragments (40 μg/ml). Then 25 μl of F(ab′) 2 fragments of rabbit anti-mouse IgG (120 μg/ml) were added to each well. For IgA IC-induced lactoferrin release, 100 μ l of neutrophils (4 × 10 6 cells/ml) were incubated with 50 μl of IgA/NIP-BSA IC (60 μg IgA/ml). After incubation at 37°C for the times indicated, lactoferrin released from neutrophils was measured. ▪, IgA IC; ▴, MIP8a. Results are representative of three separate experiments.
Figure Legend Snippet: Lactoferrin release induced by MIP8a and IgA immune complexes (IC). (a) Dose–response of MIP8a-induced lactoferrin release. Neutrophils (100 μl) (4 × 10 6 cells/ml) were incubated at room temperature for 10 min with 25 μl MIP8a F(ab′) 2 fragments as indicated. Then 25 μl of F(ab′) 2 fragments of rabbit anti-mouse IgG (120 μg/ml) were added to each well. After incubation at 37°C for 2 h, lactoferrin released from neutrophils was measured. (b) Time course of MIP8a- and IgA IC-induced lactoferrin release. For MIP8a-induced lactoferrin release, 100 μl of neutrophils (4 × 10 6 cells/ml) were incubated at room temperature for 10 min with 25 μl of MIP8a F(ab′) 2 fragments (40 μg/ml). Then 25 μl of F(ab′) 2 fragments of rabbit anti-mouse IgG (120 μg/ml) were added to each well. For IgA IC-induced lactoferrin release, 100 μ l of neutrophils (4 × 10 6 cells/ml) were incubated with 50 μl of IgA/NIP-BSA IC (60 μg IgA/ml). After incubation at 37°C for the times indicated, lactoferrin released from neutrophils was measured. ▪, IgA IC; ▴, MIP8a. Results are representative of three separate experiments.

Techniques Used: Incubation

29) Product Images from "Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase"

Article Title: Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg610

Fig. 1. Site-specific GreB–RNAP photocrosslinking. ( A ) Autoradiogram of 8% Tris–glycine SDS–PAGE after UV-irradiation of nine [ 35 S]ASDPC-derivatized GreB-Cys mutants in the presence of RNAP core and BSA as a carrier protein. Numbers on top of the gel indicate the position of the Cys substitution in GreB. GreB-68 is a wt GreB used as a negative control. Positions of free β, β′, BSA and GreB are indicated by arrows. ( B ), shown as ribbons and a schematic representation of their crosslinking targets.
Figure Legend Snippet: Fig. 1. Site-specific GreB–RNAP photocrosslinking. ( A ) Autoradiogram of 8% Tris–glycine SDS–PAGE after UV-irradiation of nine [ 35 S]ASDPC-derivatized GreB-Cys mutants in the presence of RNAP core and BSA as a carrier protein. Numbers on top of the gel indicate the position of the Cys substitution in GreB. GreB-68 is a wt GreB used as a negative control. Positions of free β, β′, BSA and GreB are indicated by arrows. ( B ), shown as ribbons and a schematic representation of their crosslinking targets.

Techniques Used: SDS Page, Irradiation, Negative Control

30) Product Images from "ToF-SIMS and XPS Characterization of Protein Films Adsorbed onto Bare and Sodium Styrene Sulfonate Grafted Gold Substrates"

Article Title: ToF-SIMS and XPS Characterization of Protein Films Adsorbed onto Bare and Sodium Styrene Sulfonate Grafted Gold Substrates

Journal: Langmuir : the ACS journal of surfaces and colloids

doi: 10.1021/acs.langmuir.5b04743

Amino acid peak list PCA results. Panels (A) and (B) are the scores and loadings plots for BSA. Panels (C) and (D) are the scores and loadings plots for Fgn. Panels (E) and (F) and the scores and loadings plots for IgG. In panels (A), (C), and (E),
Figure Legend Snippet: Amino acid peak list PCA results. Panels (A) and (B) are the scores and loadings plots for BSA. Panels (C) and (D) are the scores and loadings plots for Fgn. Panels (E) and (F) and the scores and loadings plots for IgG. In panels (A), (C), and (E),

Techniques Used:

XPS isotherms for (A) BSA, (B) Fgn, (C) IgG, and (D) plasma adsorption onto gold and NaSS surfaces. Panels (E) through (H) are isotherms of the PC1 scores obtained from PCA using peak lists containing all peaks that are at least 3x the background intensity.
Figure Legend Snippet: XPS isotherms for (A) BSA, (B) Fgn, (C) IgG, and (D) plasma adsorption onto gold and NaSS surfaces. Panels (E) through (H) are isotherms of the PC1 scores obtained from PCA using peak lists containing all peaks that are at least 3x the background intensity.

Techniques Used: Adsorption

ToF-SIMS peak intensity ratios reveal changes in adsorbed protein structure for (A) BSA and (B) IgG adsorbed onto gold and NaSS surfaces. Insets show the crystal structure of each protein. In (A) Asn and Tyr are highlighted in yellow while His is highlighted
Figure Legend Snippet: ToF-SIMS peak intensity ratios reveal changes in adsorbed protein structure for (A) BSA and (B) IgG adsorbed onto gold and NaSS surfaces. Insets show the crystal structure of each protein. In (A) Asn and Tyr are highlighted in yellow while His is highlighted

Techniques Used:

ToF-SIMS peak intensity ratios suggest levels of denaturation of (A) BSA, (B) Fgn, (C) and IgG adsorbed onto gold and NaSS surfaces. Insets show the crystal structure of each protein. Bars represent mean amino acid peak ratios for proteins adsorbed from
Figure Legend Snippet: ToF-SIMS peak intensity ratios suggest levels of denaturation of (A) BSA, (B) Fgn, (C) and IgG adsorbed onto gold and NaSS surfaces. Insets show the crystal structure of each protein. Bars represent mean amino acid peak ratios for proteins adsorbed from

Techniques Used:

PC models for gold (A and B) and NaSS (C and D) surfaces. The models were constructed using the amino acids peaks from 100 μg/ml plasma adsorption ToF-SIMS data. Single-component BSA ( ), Fgn ( ), and IgG ( ) spectra were projected into the models.
Figure Legend Snippet: PC models for gold (A and B) and NaSS (C and D) surfaces. The models were constructed using the amino acids peaks from 100 μg/ml plasma adsorption ToF-SIMS data. Single-component BSA ( ), Fgn ( ), and IgG ( ) spectra were projected into the models.

Techniques Used: Construct, Adsorption

31) Product Images from "Mass spectrometry-based proteomics of oxidative stress: Identification of 4-hydroxy-2-nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins"

Article Title: Mass spectrometry-based proteomics of oxidative stress: Identification of 4-hydroxy-2-nonenal (HNE) adducts of amino acids using lysozyme and bovine serum albumin as model proteins

Journal: Electrophoresis

doi: 10.1002/elps.201600134

Example of pyrrole-type His modification by HNE in BSA, at the peptide and amino acid levels. (A) XIC of peak with m/z 713.26 (2+) in BSA sample treated with 0, 2, 5 and 10 mM HNE. (B) Summed spectra from the XIC of the peak with m/z 713.26 (2+). (C) Product ions (MS/MS fragmentation) of the precursor ion from HNE-treated BSA, with m/z 713.26 (2+).
Figure Legend Snippet: Example of pyrrole-type His modification by HNE in BSA, at the peptide and amino acid levels. (A) XIC of peak with m/z 713.26 (2+) in BSA sample treated with 0, 2, 5 and 10 mM HNE. (B) Summed spectra from the XIC of the peak with m/z 713.26 (2+). (C) Product ions (MS/MS fragmentation) of the precursor ion from HNE-treated BSA, with m/z 713.26 (2+).

Techniques Used: Modification, Mass Spectrometry

Example of HNE modification of Arg residue in BSA, at the peptide and amino acid levels. (A) Extracted ion chromatograms (XIC) of the peak with m/z 651.31 (2+) in BSA sample treated with 0, 2, 5 and 10 mM HNE. (B) Summed spectra from the XIC of the peak with m/z 651.31 (2+). (C) Product ions (MS/MS fragmentation) of the precursor ion from HNE-treated BSA, with m/z 651.31 (2+).
Figure Legend Snippet: Example of HNE modification of Arg residue in BSA, at the peptide and amino acid levels. (A) Extracted ion chromatograms (XIC) of the peak with m/z 651.31 (2+) in BSA sample treated with 0, 2, 5 and 10 mM HNE. (B) Summed spectra from the XIC of the peak with m/z 651.31 (2+). (C) Product ions (MS/MS fragmentation) of the precursor ion from HNE-treated BSA, with m/z 651.31 (2+).

Techniques Used: Modification, Mass Spectrometry

Example of HNE modification of Thr residue in BSA, at the peptide and amino acid levels. (A) XIC of the peak with m/z 990.45 (2+) in BSA sample treated with 0, 2, 5 and 10 mM HNE. (B) Summed spectra from the XIC of the peak with m/z 990.45 (2+). (C) Product ions (MS/MS fragmentation) of the precursor ion from HNE-treated BSA, with m/z 990.45 (2+).
Figure Legend Snippet: Example of HNE modification of Thr residue in BSA, at the peptide and amino acid levels. (A) XIC of the peak with m/z 990.45 (2+) in BSA sample treated with 0, 2, 5 and 10 mM HNE. (B) Summed spectra from the XIC of the peak with m/z 990.45 (2+). (C) Product ions (MS/MS fragmentation) of the precursor ion from HNE-treated BSA, with m/z 990.45 (2+).

Techniques Used: Modification, Mass Spectrometry

32) Product Images from "Btk-dependent Rac activation and actin rearrangement following Fc?RI aggregation promotes enhanced chemotactic responses of mast cells"

Article Title: Btk-dependent Rac activation and actin rearrangement following Fc?RI aggregation promotes enhanced chemotactic responses of mast cells

Journal: Journal of Cell Science

doi: 10.1242/jcs.071043

Synergistic cell migration is primarily dependent on chemotaxis. ( A ) IgE-sensitized BMMCs were washed three times with HEPES buffer containing 0.5% BSA, and then cells were stimulated with indicated agonists [antigen, Ag (10 ng/ml), SCF (10 ng/ml), adenosine (1 μM), PGE 2 (100 nM)]. After 3 hours, cell-free supernatants (Sup) from antigen and/or other agonist-stimulated BMMCs were applied to the lower chamber. Sensitized or unsensitized BMMCs were placed in the upper chambers. After incubation for 4 hours, BMMCs migrating to the lower chambers were collected and counted. ( B ) Sensitized BMMCs were preincubated with or without actinomycin D (5 μg/ml) for 30 minutes, and cell migration was measured. ( C ) To test whether the cell migration was chemotaxis or chemokinesis, indicated agonists were placed in the lower chamber or both upper and lower chambers. Sensitized BMMCs were placed in upper chambers. After 4 hours, BMMCs migrating to the lower chambers were collected and counted. Results are means ± s.e. of three separate experiments. * P
Figure Legend Snippet: Synergistic cell migration is primarily dependent on chemotaxis. ( A ) IgE-sensitized BMMCs were washed three times with HEPES buffer containing 0.5% BSA, and then cells were stimulated with indicated agonists [antigen, Ag (10 ng/ml), SCF (10 ng/ml), adenosine (1 μM), PGE 2 (100 nM)]. After 3 hours, cell-free supernatants (Sup) from antigen and/or other agonist-stimulated BMMCs were applied to the lower chamber. Sensitized or unsensitized BMMCs were placed in the upper chambers. After incubation for 4 hours, BMMCs migrating to the lower chambers were collected and counted. ( B ) Sensitized BMMCs were preincubated with or without actinomycin D (5 μg/ml) for 30 minutes, and cell migration was measured. ( C ) To test whether the cell migration was chemotaxis or chemokinesis, indicated agonists were placed in the lower chamber or both upper and lower chambers. Sensitized BMMCs were placed in upper chambers. After 4 hours, BMMCs migrating to the lower chambers were collected and counted. Results are means ± s.e. of three separate experiments. * P

Techniques Used: Migration, Chemotaxis Assay, Incubation

The role of PI3K in synergistic chemotactic responses. ( A ) Sensitized BMMCs were preincubated with or without wortmannin (100 nM), in the upper chamber and placed in 600 μl HEPES buffer containing 0.5% BSA and wortmannin for 30 minutes. The upper chambers were then replaced in the lower chambers containing the indicated agonists. ( B ) Sensitized BMMCs were preincubated with indicated inhibitors for 20 minutes and then stimulated with the indicated agonists for 5 minutes. Following electrophoresis and membrane transfer, proteins were probed using anti-phospho-AKT (Ser473 -P ). To normalize protein loading, membranes were stripped and probed for β-actin, or alternatively identically loaded samples were probed for β-actin. The data shown are from three separate experiments, each repeated at least three times, with identical results, on separate cell preparations. ( C ) Sensitized BMMCs were preincubated with or without AS 252424 (3 μM) or IC 87114 (3 μM) in the upper chambers and placed in 600 μl HEPES buffer containing 0.5% BSA and indicated inhibitors for 30 minutes, and then upper chambers were replaced in the lower chamber containing the indicated agonists. Results in A and C are means ± s.e. of three separate experiments. * P
Figure Legend Snippet: The role of PI3K in synergistic chemotactic responses. ( A ) Sensitized BMMCs were preincubated with or without wortmannin (100 nM), in the upper chamber and placed in 600 μl HEPES buffer containing 0.5% BSA and wortmannin for 30 minutes. The upper chambers were then replaced in the lower chambers containing the indicated agonists. ( B ) Sensitized BMMCs were preincubated with indicated inhibitors for 20 minutes and then stimulated with the indicated agonists for 5 minutes. Following electrophoresis and membrane transfer, proteins were probed using anti-phospho-AKT (Ser473 -P ). To normalize protein loading, membranes were stripped and probed for β-actin, or alternatively identically loaded samples were probed for β-actin. The data shown are from three separate experiments, each repeated at least three times, with identical results, on separate cell preparations. ( C ) Sensitized BMMCs were preincubated with or without AS 252424 (3 μM) or IC 87114 (3 μM) in the upper chambers and placed in 600 μl HEPES buffer containing 0.5% BSA and indicated inhibitors for 30 minutes, and then upper chambers were replaced in the lower chamber containing the indicated agonists. Results in A and C are means ± s.e. of three separate experiments. * P

Techniques Used: Electrophoresis

33) Product Images from "Increasing Dietary Medium-Chain Fatty Acid Ratio Mitigates High-fat Diet-Induced Non-Alcoholic Steatohepatitis by Regulating Autophagy"

Article Title: Increasing Dietary Medium-Chain Fatty Acid Ratio Mitigates High-fat Diet-Induced Non-Alcoholic Steatohepatitis by Regulating Autophagy

Journal: Scientific Reports

doi: 10.1038/s41598-017-14376-y

Increasing MCFA ratio attenuated LCFAs-induced fat accumulation, insulin resistance, autophagy impairment, ER stress, and apoptosis in HepG2 cells. To investigate whether MCFAs can directly exert their protective effects in hepatic cells, the fat accumulation, insulin resistance, autophagy impairment, ER stress, and apoptosis were evaluated in fat-loaded HepG2 cells. For intracellular lipid quantification, 48-hour fat-loaded cells were fixed then stained with Nile Red and Hoechst 33342 ( A ). For the insulin sensitivity test, cells were incubated with 100 nM insulin for an additional 10 min after a 48-hour fatty acid treatment. Representative immunoblots and densitometric quantification of phospho-Akt (Ser473) ( B ), and the calculated insulin-stimulated Akt phosphorylation fold change results ( C ) are presented. Representative SQSTM1/p62 and LC3 immunoblots and densitometric results show that increasing MCFA/LCFA ratio protected HepG2 cells against LCFA-induced autophagy impairment ( D ). Autophagic flux changes were also confirmed by analyzing Bafilomycin A1 (50 nM, 3 hours)-induced LC3 II protein accumulation in BSA or fatty acid-treated HepG2 cells ( E ). Western blotting results of CHOP and cleaved caspase 3 show that MCFAs also attenuated LCFAs-induced ER stress and apoptosis in HepG2 cells ( F ). For densitometric analyses of Western blotting data, β-actin or total Akt (for Akt phosphorylation analysis) were used as the loading control. Values are mean ± SEM (n = 3). *, # (vs. BSA treated cells without insulin stimulation), and † (vs. SDF-treated cells without insulin stimulation) indicate statistical significance, P
Figure Legend Snippet: Increasing MCFA ratio attenuated LCFAs-induced fat accumulation, insulin resistance, autophagy impairment, ER stress, and apoptosis in HepG2 cells. To investigate whether MCFAs can directly exert their protective effects in hepatic cells, the fat accumulation, insulin resistance, autophagy impairment, ER stress, and apoptosis were evaluated in fat-loaded HepG2 cells. For intracellular lipid quantification, 48-hour fat-loaded cells were fixed then stained with Nile Red and Hoechst 33342 ( A ). For the insulin sensitivity test, cells were incubated with 100 nM insulin for an additional 10 min after a 48-hour fatty acid treatment. Representative immunoblots and densitometric quantification of phospho-Akt (Ser473) ( B ), and the calculated insulin-stimulated Akt phosphorylation fold change results ( C ) are presented. Representative SQSTM1/p62 and LC3 immunoblots and densitometric results show that increasing MCFA/LCFA ratio protected HepG2 cells against LCFA-induced autophagy impairment ( D ). Autophagic flux changes were also confirmed by analyzing Bafilomycin A1 (50 nM, 3 hours)-induced LC3 II protein accumulation in BSA or fatty acid-treated HepG2 cells ( E ). Western blotting results of CHOP and cleaved caspase 3 show that MCFAs also attenuated LCFAs-induced ER stress and apoptosis in HepG2 cells ( F ). For densitometric analyses of Western blotting data, β-actin or total Akt (for Akt phosphorylation analysis) were used as the loading control. Values are mean ± SEM (n = 3). *, # (vs. BSA treated cells without insulin stimulation), and † (vs. SDF-treated cells without insulin stimulation) indicate statistical significance, P

Techniques Used: Staining, Incubation, Western Blot

34) Product Images from "The TRPM8 Protein Is a Testosterone Receptor"

Article Title: The TRPM8 Protein Is a Testosterone Receptor

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.610873

Testosterone-induced TRPM8 activity in intracellular Ca 2+ measurements in different cell types. Fluorescence measurements of the intracellular Ca 2+ concentration were performed on prostate PC3 cells and DRG neurons transiently expressing TRPM8. a–d , fluorescence measurements obtained from PC3 cells that were transiently transfected with the TRPM8 (0.7 μg) and GFP (0.2 μg) constructs. a , representative recording of the menthol-induced activation of TRPM8 channels ( n = 6). b , representative recording of the testosterone-induced responses of TRPM8 channels ( n = 8). c , TRPM8 channels were also induced by an impermeable variant of testosterone that was conjugated to BSA ( BSA-Testosterone ), indicating that at least one of the androgen-interacting sites is located on the extracellular side of the TRPM8 protein ( n = 6). d , summary of the menthol- and testosterone-induced TRPM8 responses of PC3 cells ( error bars , S.E.). e–h , fluorescence measurements obtained from DRG neurons transiently transfected with the TRPM8 (3 μg) and GFP (0.5 μg) constructs. e , representative recording of the menthol-induced activation of TRPM8 channels ( n = 4). f , representative recording of the testosterone-induced responses of TRPM8 channels ( n = 6). g , the control recording of DRG neurons that were not transfected with TRPM8 shows no response to either testosterone or menthol ( n = 3). h , summary of the menthol- and testosterone-induced TRPM8 responses of DRG neurons ( error bars , S.E.).
Figure Legend Snippet: Testosterone-induced TRPM8 activity in intracellular Ca 2+ measurements in different cell types. Fluorescence measurements of the intracellular Ca 2+ concentration were performed on prostate PC3 cells and DRG neurons transiently expressing TRPM8. a–d , fluorescence measurements obtained from PC3 cells that were transiently transfected with the TRPM8 (0.7 μg) and GFP (0.2 μg) constructs. a , representative recording of the menthol-induced activation of TRPM8 channels ( n = 6). b , representative recording of the testosterone-induced responses of TRPM8 channels ( n = 8). c , TRPM8 channels were also induced by an impermeable variant of testosterone that was conjugated to BSA ( BSA-Testosterone ), indicating that at least one of the androgen-interacting sites is located on the extracellular side of the TRPM8 protein ( n = 6). d , summary of the menthol- and testosterone-induced TRPM8 responses of PC3 cells ( error bars , S.E.). e–h , fluorescence measurements obtained from DRG neurons transiently transfected with the TRPM8 (3 μg) and GFP (0.5 μg) constructs. e , representative recording of the menthol-induced activation of TRPM8 channels ( n = 4). f , representative recording of the testosterone-induced responses of TRPM8 channels ( n = 6). g , the control recording of DRG neurons that were not transfected with TRPM8 shows no response to either testosterone or menthol ( n = 3). h , summary of the menthol- and testosterone-induced TRPM8 responses of DRG neurons ( error bars , S.E.).

Techniques Used: Activity Assay, Fluorescence, Concentration Assay, Expressing, Transfection, Construct, Activation Assay, Variant Assay

Testosterone-induced Ca 2+ uptake in HEK-293 TRPM8 stable cells cultured under the steroid-deprived conditions. Fluorescence measurements of intracellular Ca 2+ concentration were performed on HEK-293 cells stably expressing the TRPM8 protein. The cells were cultured in the steroid-deprived medium using 0.25% charcoal and 0.0025% dextran for up to 38 h. Menthol-induced ( a ), testosterone-induced ( b ), and BSA-testosterone-induced (c) TRPM8 activity is shown. A summary is presented in d. Error bars , S.E.
Figure Legend Snippet: Testosterone-induced Ca 2+ uptake in HEK-293 TRPM8 stable cells cultured under the steroid-deprived conditions. Fluorescence measurements of intracellular Ca 2+ concentration were performed on HEK-293 cells stably expressing the TRPM8 protein. The cells were cultured in the steroid-deprived medium using 0.25% charcoal and 0.0025% dextran for up to 38 h. Menthol-induced ( a ), testosterone-induced ( b ), and BSA-testosterone-induced (c) TRPM8 activity is shown. A summary is presented in d. Error bars , S.E.

Techniques Used: Cell Culture, Fluorescence, Concentration Assay, Stable Transfection, Expressing, Activity Assay

Testosterone-induced Ca 2+ signals observed on HEK-293 cells stably expressing TRPM8. Fluorescence measurements of intracellular Ca 2+ concentration were performed on HEK-293 cells expressing the TRPM8 protein. Shown are the menthol-induced Ca 2+ response ( a ) and testosterone-induced ( b ) and impermeable analog BSA-testosterone-induced ( c ) TRPM8 activities. A summary is shown in d. Error bars , S.E.
Figure Legend Snippet: Testosterone-induced Ca 2+ signals observed on HEK-293 cells stably expressing TRPM8. Fluorescence measurements of intracellular Ca 2+ concentration were performed on HEK-293 cells expressing the TRPM8 protein. Shown are the menthol-induced Ca 2+ response ( a ) and testosterone-induced ( b ) and impermeable analog BSA-testosterone-induced ( c ) TRPM8 activities. A summary is shown in d. Error bars , S.E.

Techniques Used: Stable Transfection, Expressing, Fluorescence, Concentration Assay

35) Product Images from "Divergent expression patterns of pituitary gonadotropin subunit and GnRH receptor genes to continuous GnRH in vitro and in vivo"

Article Title: Divergent expression patterns of pituitary gonadotropin subunit and GnRH receptor genes to continuous GnRH in vitro and in vivo

Journal: Scientific Reports

doi: 10.1038/s41598-019-56480-1

Time-course of GnRH receptor agonists on gonadotropin subunit genes, Gnrhr , and Dmp1 expression in cultured pituitary cells. ( A ) Effects of continuous GnRH application on gene expression. Note the transient nature of Fshb , Gnrhr , and Dmp1 expression, a slow and continuous upregulation in Cga expression, and a transient and minor inhibition of Lhb expression during GnRH treatment. Data shown are mean ± SEM values, normalized to the zero time point controls, from three to nine independent experiments per time point, each performed in quadruplicate. Asterisks indicate significant differences vs. zero time point. ( B ) A gene-specific patterns of expression in response to application of D-Ala6. Top panel indicates duration of first (blue) and second (red) 10 nM D-Ala6 application and washout periods (green). The data shown are mean ± SEM values from a representative experiment; when not visible, SEM values were within circles. Asterisks indicate a significant difference between pairs. In both panels, horizontal gray lines indicate basal gene expression and vertical dotted lines indicate times needed to reach the peak in expression. If not otherwise specified, in this and following figures experiments were performed with cells from postpubertal female rats. Immediately after dispersion, cells (1.5 million per well) were seeded in 24-well plates and cultured overnight in horse serum-containing GnRH-free medium. After 20 h incubation, medium was replaced with fresh 0.1% BSA-containing medium with GnRH or D-Ala6.
Figure Legend Snippet: Time-course of GnRH receptor agonists on gonadotropin subunit genes, Gnrhr , and Dmp1 expression in cultured pituitary cells. ( A ) Effects of continuous GnRH application on gene expression. Note the transient nature of Fshb , Gnrhr , and Dmp1 expression, a slow and continuous upregulation in Cga expression, and a transient and minor inhibition of Lhb expression during GnRH treatment. Data shown are mean ± SEM values, normalized to the zero time point controls, from three to nine independent experiments per time point, each performed in quadruplicate. Asterisks indicate significant differences vs. zero time point. ( B ) A gene-specific patterns of expression in response to application of D-Ala6. Top panel indicates duration of first (blue) and second (red) 10 nM D-Ala6 application and washout periods (green). The data shown are mean ± SEM values from a representative experiment; when not visible, SEM values were within circles. Asterisks indicate a significant difference between pairs. In both panels, horizontal gray lines indicate basal gene expression and vertical dotted lines indicate times needed to reach the peak in expression. If not otherwise specified, in this and following figures experiments were performed with cells from postpubertal female rats. Immediately after dispersion, cells (1.5 million per well) were seeded in 24-well plates and cultured overnight in horse serum-containing GnRH-free medium. After 20 h incubation, medium was replaced with fresh 0.1% BSA-containing medium with GnRH or D-Ala6.

Techniques Used: Expressing, Cell Culture, Inhibition, Incubation

36) Product Images from "Electrochemical Studies of Camptothecin and Its Interaction with Human Serum Albumin"

Article Title: Electrochemical Studies of Camptothecin and Its Interaction with Human Serum Albumin

Journal: International Journal of Molecular Sciences

doi:

Plot of the decrease of the camptothecin peak current versus the addition of different volume of HSA/BSA in the solutions. HSA/BSA concentration: 10 mg/ml (1.52×10 −4 M). Others same as in Figure 1 .
Figure Legend Snippet: Plot of the decrease of the camptothecin peak current versus the addition of different volume of HSA/BSA in the solutions. HSA/BSA concentration: 10 mg/ml (1.52×10 −4 M). Others same as in Figure 1 .

Techniques Used: Concentration Assay

37) Product Images from "Human Lung Mast Cell Products Regulate Airway Smooth Muscle CXCL10 Levels"

Article Title: Human Lung Mast Cell Products Regulate Airway Smooth Muscle CXCL10 Levels

Journal: Journal of Allergy

doi: 10.1155/2014/875105

The proteolytic effects of tryptase on rhCXCL10 in a cell free system. The rhCXCL10 made up in serum-free DMEM supplemented with 0.1% BSA was placed in 96-well culture plates and incubated with tryptase ± leupeptin, heat-inactivated tryptase (HI), or its vehicle for 24 h under the same conditions as the ASM cells. CXCL10 was detected using ELISA. * P
Figure Legend Snippet: The proteolytic effects of tryptase on rhCXCL10 in a cell free system. The rhCXCL10 made up in serum-free DMEM supplemented with 0.1% BSA was placed in 96-well culture plates and incubated with tryptase ± leupeptin, heat-inactivated tryptase (HI), or its vehicle for 24 h under the same conditions as the ASM cells. CXCL10 was detected using ELISA. * P

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

38) Product Images from "Human alveolar epithelial type II cells in primary culture"

Article Title: Human alveolar epithelial type II cells in primary culture

Journal: Physiological Reports

doi: 10.14814/phy2.12288

Expression of SPC and absence of expression of AQP-5 in AEII cell. Cultured AEII cells or Hela cells (5 × 10 4 cells/mL) were seeded for 12 h, fixed in 4% paraformaldehyde, and then were permeabilized using 0.1% Triton X-100 followed by blockade using 2% BSA. The cells were immunostained by using rabbit anti-human pro-SPC polyclonal antibody and goat anti-human AQP-5 polyclonal antibody over night at 4°C. After washing, the cells were incubated with appropriate secondary antibodies for 1 h at 37°C in dark. The coverslips were mounted after washing, and analyzed under a confocal microscope.
Figure Legend Snippet: Expression of SPC and absence of expression of AQP-5 in AEII cell. Cultured AEII cells or Hela cells (5 × 10 4 cells/mL) were seeded for 12 h, fixed in 4% paraformaldehyde, and then were permeabilized using 0.1% Triton X-100 followed by blockade using 2% BSA. The cells were immunostained by using rabbit anti-human pro-SPC polyclonal antibody and goat anti-human AQP-5 polyclonal antibody over night at 4°C. After washing, the cells were incubated with appropriate secondary antibodies for 1 h at 37°C in dark. The coverslips were mounted after washing, and analyzed under a confocal microscope.

Techniques Used: Expressing, Cell Culture, Incubation, Microscopy

39) Product Images from "Integrin αIIbβ3 Transmembrane Domain Separation Mediates Bi-Directional Signaling across the Plasma Membrane"

Article Title: Integrin αIIbβ3 Transmembrane Domain Separation Mediates Bi-Directional Signaling across the Plasma Membrane

Journal: PLoS ONE

doi: 10.1371/journal.pone.0116208

TM separation promotes activation of FAK whereas PI3K regulates Akt and Erk1/2 but not FAK in outside-in signaling. Cells were seeded on Fbg (20μg/mL)-coated dishes with or without 1mM DTT or with 1.5μM/mL Wortmannin at 37°C for 1h and then lysed and subjected to western blot. Cells seeded on 1% BSA coated dishes were used as control. (A)FAK activation induced by immobilized ligand Fbg is TM domain separation dependent. TM-clasped mutants expressed reduced phosphorylation on Y397. (B) Treatment with Wortmannin ablated Akt and Erk1/2 activation. Removing Wortmannin before cell spreading restored Erk1/2 activation without inducing Akt activation. This may implicate that PI3K but not Akt activity is required for cell survival and proliferation mediated by Erk1/2 in outside-in signaling. (C) Wortmannin treatment did not alter activation of FAK, implicating parallel pathways in outside-in signaling.
Figure Legend Snippet: TM separation promotes activation of FAK whereas PI3K regulates Akt and Erk1/2 but not FAK in outside-in signaling. Cells were seeded on Fbg (20μg/mL)-coated dishes with or without 1mM DTT or with 1.5μM/mL Wortmannin at 37°C for 1h and then lysed and subjected to western blot. Cells seeded on 1% BSA coated dishes were used as control. (A)FAK activation induced by immobilized ligand Fbg is TM domain separation dependent. TM-clasped mutants expressed reduced phosphorylation on Y397. (B) Treatment with Wortmannin ablated Akt and Erk1/2 activation. Removing Wortmannin before cell spreading restored Erk1/2 activation without inducing Akt activation. This may implicate that PI3K but not Akt activity is required for cell survival and proliferation mediated by Erk1/2 in outside-in signaling. (C) Wortmannin treatment did not alter activation of FAK, implicating parallel pathways in outside-in signaling.

Techniques Used: Activation Assay, Western Blot, Activity Assay

40) Product Images from "Lysosomal trafficking functions of mucolipin-1 in murine macrophages"

Article Title: Lysosomal trafficking functions of mucolipin-1 in murine macrophages

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-8-54

MCOLN1 RNAi clones . A) Northern blot done on 15 μg of total RNA isolated from RAW264.7, LS9, or LS10 cells. The same filter was probed for MCOLN1 , stripped, and re-probed for GAPDH , stripped again and re-probed for MCOLN2 . B) Confocal images of RAW264.7, LS9, and LS10 cells stained with Acridine Orange (AO). Bar is 6 μm. C) Quantitation of the intensity of staining of AO-stained compartments and of dextran-Oregon Green (OG)-stained compartments. Bars represent standard deviations. D) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with Dextran-Cascade Blue (DEX-CB, red). BSA-Bodipy LacCer (B-LACCER, green) was added for 30 minutes and chased for the indicated times before fixation. Arrows indicate co-localization of the two markers. Bar is 5 μm.
Figure Legend Snippet: MCOLN1 RNAi clones . A) Northern blot done on 15 μg of total RNA isolated from RAW264.7, LS9, or LS10 cells. The same filter was probed for MCOLN1 , stripped, and re-probed for GAPDH , stripped again and re-probed for MCOLN2 . B) Confocal images of RAW264.7, LS9, and LS10 cells stained with Acridine Orange (AO). Bar is 6 μm. C) Quantitation of the intensity of staining of AO-stained compartments and of dextran-Oregon Green (OG)-stained compartments. Bars represent standard deviations. D) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with Dextran-Cascade Blue (DEX-CB, red). BSA-Bodipy LacCer (B-LACCER, green) was added for 30 minutes and chased for the indicated times before fixation. Arrows indicate co-localization of the two markers. Bar is 5 μm.

Techniques Used: Northern Blot, Isolation, Staining, Quantitation Assay

Trafficking and endocytosis defect of proteins in MCOLN1 RNAi clones . A) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with BSA-AlexaFluor 594 (BSA-AF 594, red). BSA-AlexaFluor 488 (BSA-AF 488, green) was added for 10 minutes to the cells and the cells were chased for the indicated times before fixation. Bar is 5 μm. B) Western blots of HEL that was endocytosed for 5 minutes and chased for the indicated times. C) Quantitation of the HEL that remains in cells relative to the 0 time point. Bars represent standard deviations from two independent assays.
Figure Legend Snippet: Trafficking and endocytosis defect of proteins in MCOLN1 RNAi clones . A) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with BSA-AlexaFluor 594 (BSA-AF 594, red). BSA-AlexaFluor 488 (BSA-AF 488, green) was added for 10 minutes to the cells and the cells were chased for the indicated times before fixation. Bar is 5 μm. B) Western blots of HEL that was endocytosed for 5 minutes and chased for the indicated times. C) Quantitation of the HEL that remains in cells relative to the 0 time point. Bars represent standard deviations from two independent assays.

Techniques Used: Western Blot, Quantitation Assay

41) Product Images from "Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction"

Article Title: Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddq087

Brn-2 forms SDS-insoluble complex with mutant Nhtt in neuro2a cells and in vitro . ( A ) Co-aggregation of Brn-2 with mutant Nhtt in transfected neuro2a cells. Neuro2a cells were transfected with expression vector for Brn-1, Brn-2, Oct-1, RPF-1, PQBP-1 or LacZ tagged with V5 together with expression vector for Nhtt18Q-EGFP-NLS (left) or Nhtt150Q-EGFP-NLS (right). After 24 h, cells were subjected to SDS–PAGE and western blot analysis using anti-V5 (upper) or anti-GFP (lower) antibody. Bands for Nhtt18Q-EGFP-NLS are indicated by arrowhead and positions at the top of the gel are indicated by arrows. Bands for soluble Nhtt150Q-EGFP-NLS were not observed in the gel, possibly due to its efficient insolubilization, but they were detected at the top of the gel. In the case of Brn-1 and Brn-2, 1/2.5 the amount of plasmid DNA was used to make their expression levels similar to those of other proteins. ( B ) Co-aggregation of Brn-2 with mutant Nhtt in vitro . HRV-3C-treated Nhtt18Q, Nhtt62Q or BSA (0.2 mg/ml) was co-incubated with different concentrations of HRV-3C-treated His-TF-Brn-2 (0, 0.2, 0.5 or 1 mg/ml) at 37°C as indicated to the left of panels. After 20 h, the samples were subjected to filter trap assay and the aggregated proteins were detected with anti-huntingtin or anti-Brn-2 (C-2AP).
Figure Legend Snippet: Brn-2 forms SDS-insoluble complex with mutant Nhtt in neuro2a cells and in vitro . ( A ) Co-aggregation of Brn-2 with mutant Nhtt in transfected neuro2a cells. Neuro2a cells were transfected with expression vector for Brn-1, Brn-2, Oct-1, RPF-1, PQBP-1 or LacZ tagged with V5 together with expression vector for Nhtt18Q-EGFP-NLS (left) or Nhtt150Q-EGFP-NLS (right). After 24 h, cells were subjected to SDS–PAGE and western blot analysis using anti-V5 (upper) or anti-GFP (lower) antibody. Bands for Nhtt18Q-EGFP-NLS are indicated by arrowhead and positions at the top of the gel are indicated by arrows. Bands for soluble Nhtt150Q-EGFP-NLS were not observed in the gel, possibly due to its efficient insolubilization, but they were detected at the top of the gel. In the case of Brn-1 and Brn-2, 1/2.5 the amount of plasmid DNA was used to make their expression levels similar to those of other proteins. ( B ) Co-aggregation of Brn-2 with mutant Nhtt in vitro . HRV-3C-treated Nhtt18Q, Nhtt62Q or BSA (0.2 mg/ml) was co-incubated with different concentrations of HRV-3C-treated His-TF-Brn-2 (0, 0.2, 0.5 or 1 mg/ml) at 37°C as indicated to the left of panels. After 20 h, the samples were subjected to filter trap assay and the aggregated proteins were detected with anti-huntingtin or anti-Brn-2 (C-2AP).

Techniques Used: Mutagenesis, In Vitro, Transfection, Expressing, Plasmid Preparation, SDS Page, Western Blot, Incubation, TRAP Assay

42) Product Images from "Understanding the Impact of Extracellular Polymeric Substances on Lead Release in Drinking Water Systems"

Article Title: Understanding the Impact of Extracellular Polymeric Substances on Lead Release in Drinking Water Systems

Journal: ACS Omega

doi: 10.1021/acsomega.8b02363

Representative chromatograms and UV–vis absorption spectra showing the complexation of metal ions and reduction of cytochrome c. Panel (A) shows the complexation of Pb by sEPS1 (alginate and BSA) on the anode (orange), the complexation of Fe by sEPS1 on the cathode (blue), and the complexation of Pb when Cyto. c (alginate, BSA, and cytochrome c) was on the anode (red). Panel (B) shows the contrast of UV–vis absorption spectra from cytochrome c before and after corrosion tests on the anode and cathode in which anaerobic or aerobic states the condition on the cathode side and those after the colon indicate the location of Cyto. c.
Figure Legend Snippet: Representative chromatograms and UV–vis absorption spectra showing the complexation of metal ions and reduction of cytochrome c. Panel (A) shows the complexation of Pb by sEPS1 (alginate and BSA) on the anode (orange), the complexation of Fe by sEPS1 on the cathode (blue), and the complexation of Pb when Cyto. c (alginate, BSA, and cytochrome c) was on the anode (red). Panel (B) shows the contrast of UV–vis absorption spectra from cytochrome c before and after corrosion tests on the anode and cathode in which anaerobic or aerobic states the condition on the cathode side and those after the colon indicate the location of Cyto. c.

Techniques Used:

43) Product Images from "Effects of pentoxifylline on the histological and ultra-structural features of vitrified mouse ovarian tissue: An experimental study"

Article Title: Effects of pentoxifylline on the histological and ultra-structural features of vitrified mouse ovarian tissue: An experimental study

Journal: International Journal of Reproductive Biomedicine

doi:

Experimental design, phosphate buffer solution (PBS); bovine serum albumin (BSA); pentoxifylline (PTX).
Figure Legend Snippet: Experimental design, phosphate buffer solution (PBS); bovine serum albumin (BSA); pentoxifylline (PTX).

Techniques Used:

44) Product Images from "Quantitative Detection of Digoxin in Plasma Using Small‐Molecule Immunoassay in a Recyclable Gravity‐Driven Microfluidic Chip"

Article Title: Quantitative Detection of Digoxin in Plasma Using Small‐Molecule Immunoassay in a Recyclable Gravity‐Driven Microfluidic Chip

Journal: Advanced Science

doi: 10.1002/advs.201802051

Digoxin detection in plasma diluted 1:2. Plots of normalized fluorescence intensity of Atto 488‐labeled anti‐Dig antibody (1.11 n m ) and Atto 680‐labeled streptavidin (20.20 n m ) (1:2 diluted probe system), and the corresponding signal ratio in digoxin detection under different conditions: a) detection buffer; b) diluted plasma (1:2) with detection buffer (about 60 µL of plasma is needed in each sample). c) Comparison of plotted concentration regression curves from (a) and (b). The concentrations given in all graphs are before dilution. We used recycled PS–BSA–Digg beads. Plotted values are mean values with standard deviations compared to the mean value ( N = 3).
Figure Legend Snippet: Digoxin detection in plasma diluted 1:2. Plots of normalized fluorescence intensity of Atto 488‐labeled anti‐Dig antibody (1.11 n m ) and Atto 680‐labeled streptavidin (20.20 n m ) (1:2 diluted probe system), and the corresponding signal ratio in digoxin detection under different conditions: a) detection buffer; b) diluted plasma (1:2) with detection buffer (about 60 µL of plasma is needed in each sample). c) Comparison of plotted concentration regression curves from (a) and (b). The concentrations given in all graphs are before dilution. We used recycled PS–BSA–Digg beads. Plotted values are mean values with standard deviations compared to the mean value ( N = 3).

Techniques Used: Fluorescence, Labeling, Concentration Assay

Illustration of the method and responses of the assay to digoxin at different concentrations. a) The principle of digoxin detection. b) Fluorescence emission spectra of Atto 488‐labeled anti‐Dig antibody (3.33 n m ) from the supernatant after being incubated with different concentrations of digoxin, subsequent incubation with PS–BSA–Digg beads (10 µL), and followed by removal of the beads. c) Normalized fluorescence intensities of Atto 488 and Atto 680. The signal ratio of Atto 488/Atto 680 is calculated accordingly. d) Investigation of the advantages of the two signal‐mode strategy over single signal‐mode measurements by comparing day‐to‐day variation of the detection system. Box and whisker plots of the normalized fluorescence intensity and corresponding signal ratio. Horizontal lines are medians, boxes show the interquartile range (IQR), and error bars show the full range excluding outliers (crosses) defined as being more than ±1.5 IQR outside the box. Asterisks indicate statistically significant differences ( P
Figure Legend Snippet: Illustration of the method and responses of the assay to digoxin at different concentrations. a) The principle of digoxin detection. b) Fluorescence emission spectra of Atto 488‐labeled anti‐Dig antibody (3.33 n m ) from the supernatant after being incubated with different concentrations of digoxin, subsequent incubation with PS–BSA–Digg beads (10 µL), and followed by removal of the beads. c) Normalized fluorescence intensities of Atto 488 and Atto 680. The signal ratio of Atto 488/Atto 680 is calculated accordingly. d) Investigation of the advantages of the two signal‐mode strategy over single signal‐mode measurements by comparing day‐to‐day variation of the detection system. Box and whisker plots of the normalized fluorescence intensity and corresponding signal ratio. Horizontal lines are medians, boxes show the interquartile range (IQR), and error bars show the full range excluding outliers (crosses) defined as being more than ±1.5 IQR outside the box. Asterisks indicate statistically significant differences ( P

Techniques Used: Fluorescence, Labeling, Incubation, Whisker Assay

Conceptual view of the G‐Chip and digoxin detection in plasma using the G‐Chip with only 10 µL plasma. a) Schematic view of the designed G‐Chip. b) An optical image of a fabricated chip loaded with red dye. c) Conceptual view of pillar arrays inside the fluid channel. d) Conceptual view and dimensions of the integrated size‐exclusion filters (inset shows the dimensions of the pillars that compose the filters). e) The merged image of bright‐field and fluorescence image of the filters loaded with 20 µm PS–BSA–Digg beads with adsorbed Atto 488‐labeled anti‐Dig antibody. f) Normalized fluorescence intensity of 0.42 n m Atto 488‐labeled anti‐Dig antibody (black dots) and 7.58 n m Atto 680‐labeled streptavidin (red dots) in diluted plasma, and the corresponding signal ratio (purple dots) at various digoxin concentrations. The sample consisting of plasma mixed with labeled antibodies is diluted 1:7 before entering the G‐Chip. However, the concentrations given in (f) are of digoxin in plasma before dilution. All used PS–BSA–Digg beads are recycled beads. Plotted values are mean values with standard deviations ( N = 3).
Figure Legend Snippet: Conceptual view of the G‐Chip and digoxin detection in plasma using the G‐Chip with only 10 µL plasma. a) Schematic view of the designed G‐Chip. b) An optical image of a fabricated chip loaded with red dye. c) Conceptual view of pillar arrays inside the fluid channel. d) Conceptual view and dimensions of the integrated size‐exclusion filters (inset shows the dimensions of the pillars that compose the filters). e) The merged image of bright‐field and fluorescence image of the filters loaded with 20 µm PS–BSA–Digg beads with adsorbed Atto 488‐labeled anti‐Dig antibody. f) Normalized fluorescence intensity of 0.42 n m Atto 488‐labeled anti‐Dig antibody (black dots) and 7.58 n m Atto 680‐labeled streptavidin (red dots) in diluted plasma, and the corresponding signal ratio (purple dots) at various digoxin concentrations. The sample consisting of plasma mixed with labeled antibodies is diluted 1:7 before entering the G‐Chip. However, the concentrations given in (f) are of digoxin in plasma before dilution. All used PS–BSA–Digg beads are recycled beads. Plotted values are mean values with standard deviations ( N = 3).

Techniques Used: Chromatin Immunoprecipitation, Fluorescence, Labeling

45) Product Images from "Functions of Armigeres subalbatus C-type lectins in innate immunity"

Article Title: Functions of Armigeres subalbatus C-type lectins in innate immunity

Journal: Insect biochemistry and molecular biology

doi: 10.1016/j.ibmb.2014.06.010

Binding of recombinant As CTLs to LPS in the presence of free microbial components or saccharides Purified recombinant As CTLs were diluted in the binding buffer (50 mM Tris-HCl, 50 mM NaCl, pH8.0) containing 5 mM CaCl 2 and 0.1 mg/ml BSA to 10μg/ml and pre-incubated with increasing concentrations of free microbial components (A–E) or sacc harides (F), and the mixtures were then added to the 96-well plates coated with LPS from E. coli 026:B6. Binding of recombinant proteins to LPS was determined by ELISA assay using anti-polyhistidine antibody as described in the Materials and Methods. The figure showed total binding of recombinant proteins to LPS. Each point (or bar) represents the mean of four individual measurements ± SEM.“**” indicates significant difference (p
Figure Legend Snippet: Binding of recombinant As CTLs to LPS in the presence of free microbial components or saccharides Purified recombinant As CTLs were diluted in the binding buffer (50 mM Tris-HCl, 50 mM NaCl, pH8.0) containing 5 mM CaCl 2 and 0.1 mg/ml BSA to 10μg/ml and pre-incubated with increasing concentrations of free microbial components (A–E) or sacc harides (F), and the mixtures were then added to the 96-well plates coated with LPS from E. coli 026:B6. Binding of recombinant proteins to LPS was determined by ELISA assay using anti-polyhistidine antibody as described in the Materials and Methods. The figure showed total binding of recombinant proteins to LPS. Each point (or bar) represents the mean of four individual measurements ± SEM.“**” indicates significant difference (p

Techniques Used: Binding Assay, Recombinant, Purification, Incubation, Enzyme-linked Immunosorbent Assay

46) Product Images from "Identification and characterization of photomedins: novel olfactomedin-domain-containing proteins with chondroitin sulphate-E-binding activity"

Article Title: Identification and characterization of photomedins: novel olfactomedin-domain-containing proteins with chondroitin sulphate-E-binding activity

Journal: Biochemical Journal

doi: 10.1042/BJ20050120

Binding of recombinant photomedins to ECM proteins and glycosaminoglycans Microtitre plates (96-well) were coated with the following ECM proteins or glycosaminoglycans: 20 μg/ml collagens type I, II, III, IV, V and VI (col1, col2, col3, col4, col5, and col6), laminin-1, laminin-2, laminin-10 purified from human placenta (h laminin-10), recombinant laminin-10 (r laminin-10), fibronectin, sternal proteoglycan and decorin, as well as 100 μg/ml gelatin and 500 μg/ml matrigel, and 20 μg/ml CS-C, CS-D, CS-E, dermatan sulphate (DS), hyaluronan (HA) heparan sulphate (HS) and heparin (Hep). Glycosaminoglycans used had been conjugated to dipalmitoyl phosphatidylethanolamine to facilitate their adsorption on to hydrophobic surfaces. Plates were blocked with BSA and incubated with 5 μg/ml of recombinant photomedins with a FLAG tag for 2 h at room temperature, followed by detection of bound photomedins with horse-radish-peroxidase-conjugated anti-FLAG antibody. Bars represent the absorbance at 490 nm. Error bars indicate S.D. ( n =4).
Figure Legend Snippet: Binding of recombinant photomedins to ECM proteins and glycosaminoglycans Microtitre plates (96-well) were coated with the following ECM proteins or glycosaminoglycans: 20 μg/ml collagens type I, II, III, IV, V and VI (col1, col2, col3, col4, col5, and col6), laminin-1, laminin-2, laminin-10 purified from human placenta (h laminin-10), recombinant laminin-10 (r laminin-10), fibronectin, sternal proteoglycan and decorin, as well as 100 μg/ml gelatin and 500 μg/ml matrigel, and 20 μg/ml CS-C, CS-D, CS-E, dermatan sulphate (DS), hyaluronan (HA) heparan sulphate (HS) and heparin (Hep). Glycosaminoglycans used had been conjugated to dipalmitoyl phosphatidylethanolamine to facilitate their adsorption on to hydrophobic surfaces. Plates were blocked with BSA and incubated with 5 μg/ml of recombinant photomedins with a FLAG tag for 2 h at room temperature, followed by detection of bound photomedins with horse-radish-peroxidase-conjugated anti-FLAG antibody. Bars represent the absorbance at 490 nm. Error bars indicate S.D. ( n =4).

Techniques Used: Binding Assay, Recombinant, Purification, Adsorption, Incubation, FLAG-tag

47) Product Images from "Developmental Pathways Pervade Stem Cell Responses to Evolving Extracellular Matrices of 3D Bioprinted Microenvironments"

Article Title: Developmental Pathways Pervade Stem Cell Responses to Evolving Extracellular Matrices of 3D Bioprinted Microenvironments

Journal: Stem Cells International

doi: 10.1155/2018/4809673

Physical modification of three-dimensional structures composed of 3D/BSA, 3D/FN, 3D/Col1, and 3D/LN by hMSCs. (a) Representative phase contrast images of 3D/BSA, 3D/FN, 3D/Col1, and 3D/LN structures on day 1, 7, and 14. Scale bar = 50 μ m. (b) Line graph showing changes in 3D/BSA, 3D/FN, 3D/Col1, and 3D/LN structure size over a two-week period. (c) Bar graph showing the average structure size after 14 days in culture. Error bars depict SD associated with n = 2 experimental replicates with at least 3 different prisms analyzed per experimental replicate. ∗ P
Figure Legend Snippet: Physical modification of three-dimensional structures composed of 3D/BSA, 3D/FN, 3D/Col1, and 3D/LN by hMSCs. (a) Representative phase contrast images of 3D/BSA, 3D/FN, 3D/Col1, and 3D/LN structures on day 1, 7, and 14. Scale bar = 50 μ m. (b) Line graph showing changes in 3D/BSA, 3D/FN, 3D/Col1, and 3D/LN structure size over a two-week period. (c) Bar graph showing the average structure size after 14 days in culture. Error bars depict SD associated with n = 2 experimental replicates with at least 3 different prisms analyzed per experimental replicate. ∗ P

Techniques Used: Modification

GO analysis of hMSCs in 3D bioprinted prisms, especially related to the differentiation state. (a) Counts of GO terms associated with commonly examined stem cell responses. Protein degradation, development, and differentiation are the most significantly expressed pathways. (b) Development and differentiation pathways separated by 3D ECM structures. Most influential 3D structures rank from 3D/LN > 3D/Col1 > 3D/BSA > 3D/FN, with 3D/FN structures predominantly influencing vasculature development. Expression of endothelial cell markers (c), smooth muscle cell markers (d), and sphingosine phosphate lyase (e) in hMSCs cultured in 3D/FN structures. Error bars depict SD associated with n = 3 experimental replicates with at least 2 different prisms analyzed per experimental replicate. hMSCs cultured in 3D/FN structures expressed markers for both endothelial and smooth muscle cells and had a reduction in SGPL1 expression.
Figure Legend Snippet: GO analysis of hMSCs in 3D bioprinted prisms, especially related to the differentiation state. (a) Counts of GO terms associated with commonly examined stem cell responses. Protein degradation, development, and differentiation are the most significantly expressed pathways. (b) Development and differentiation pathways separated by 3D ECM structures. Most influential 3D structures rank from 3D/LN > 3D/Col1 > 3D/BSA > 3D/FN, with 3D/FN structures predominantly influencing vasculature development. Expression of endothelial cell markers (c), smooth muscle cell markers (d), and sphingosine phosphate lyase (e) in hMSCs cultured in 3D/FN structures. Error bars depict SD associated with n = 3 experimental replicates with at least 2 different prisms analyzed per experimental replicate. hMSCs cultured in 3D/FN structures expressed markers for both endothelial and smooth muscle cells and had a reduction in SGPL1 expression.

Techniques Used: Expressing, Cell Culture

Fabrication of 3D ECM-based, bioprinted prisms. (a) Fabrication Schematic. Multiphoton excitation was used to polymerize a focal volume containing individual ECM proteins (e.g., FN, Col1, and LN) and associated photocrosslinking agent. 3D printing of this type was used so that a three-dimensional construct, in this case a rectangular prism, could be generated even with ECM types that do not form hydrogels spontaneously ex vivo. (b) Geometric template (above; dimensions of micron scale) and associated bioprinted ECM-based rectangular prism containing BSA and LN (BSA/LN; below). Scale bar = 50 μ m. (c) Representative SEM images of prisms fabricated with BSA, FN, Col1, and LN. Scale bar = 10 μ m. (d) Average fractal dimension of each ECM-based, 3D bioprinted prism (above); volumetric swelling ratio of each ECM-based bioprinted prism (below). Error bars depict standard deviation (SD), ∗ P
Figure Legend Snippet: Fabrication of 3D ECM-based, bioprinted prisms. (a) Fabrication Schematic. Multiphoton excitation was used to polymerize a focal volume containing individual ECM proteins (e.g., FN, Col1, and LN) and associated photocrosslinking agent. 3D printing of this type was used so that a three-dimensional construct, in this case a rectangular prism, could be generated even with ECM types that do not form hydrogels spontaneously ex vivo. (b) Geometric template (above; dimensions of micron scale) and associated bioprinted ECM-based rectangular prism containing BSA and LN (BSA/LN; below). Scale bar = 50 μ m. (c) Representative SEM images of prisms fabricated with BSA, FN, Col1, and LN. Scale bar = 10 μ m. (d) Average fractal dimension of each ECM-based, 3D bioprinted prism (above); volumetric swelling ratio of each ECM-based bioprinted prism (below). Error bars depict standard deviation (SD), ∗ P

Techniques Used: Construct, Generated, Ex Vivo, Standard Deviation

RNA sequencing analysis of hMSCs in 3D bioprinted prisms at 0 and 28 days. (a) Hierarchical clustering of all genes with FPKM > 1 for 3D bioprinted prisms containing either BSA alone (3D/BSA), 3D/FN, 3D/Col1, or 3D/LN at day 28 and associated 2D control cultures at day 28. (b) qPCR validation of gene expression of a subset of genes. These genes were selected as they represent families of ECM proteins and differentiation markers associated with hMSC progeny. (c) PCA analysis of all genes with FPKM > 1. (d–f) Comparison of individual 3D bioprinted prisms containing ECM to bioprinted prisms with BSA only.
Figure Legend Snippet: RNA sequencing analysis of hMSCs in 3D bioprinted prisms at 0 and 28 days. (a) Hierarchical clustering of all genes with FPKM > 1 for 3D bioprinted prisms containing either BSA alone (3D/BSA), 3D/FN, 3D/Col1, or 3D/LN at day 28 and associated 2D control cultures at day 28. (b) qPCR validation of gene expression of a subset of genes. These genes were selected as they represent families of ECM proteins and differentiation markers associated with hMSC progeny. (c) PCA analysis of all genes with FPKM > 1. (d–f) Comparison of individual 3D bioprinted prisms containing ECM to bioprinted prisms with BSA only.

Techniques Used: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Expressing

48) Product Images from "The Interactions between ZnO Nanoparticles (NPs) and α-Linolenic Acid (LNA) Complexed to BSA Did Not Influence the Toxicity of ZnO NPs on HepG2 Cells"

Article Title: The Interactions between ZnO Nanoparticles (NPs) and α-Linolenic Acid (LNA) Complexed to BSA Did Not Influence the Toxicity of ZnO NPs on HepG2 Cells

Journal: Nanomaterials

doi: 10.3390/nano7040091

The accumulation of intracellular Zn ions. HepG2 cells were exposed to various concentrations of ZnO NPs (code: XFI06) with the presence of bovine serum albumin (BSA) or α-linolenic acid (LNA complexed to BSA; referred to as LNA) for 3 h, and the accumulation of intracellular Zn ions was measured by using a fluorescent probe. *, p
Figure Legend Snippet: The accumulation of intracellular Zn ions. HepG2 cells were exposed to various concentrations of ZnO NPs (code: XFI06) with the presence of bovine serum albumin (BSA) or α-linolenic acid (LNA complexed to BSA; referred to as LNA) for 3 h, and the accumulation of intracellular Zn ions was measured by using a fluorescent probe. *, p

Techniques Used:

The UV-Vis spectra of bovine serum albumin (BSA), α-linolenic acid (LNA complexed to BSA; referred as LNA) and ZnO NPs (code: XFI06) suspended in distilled and deionized water (DDW), 0.5% BSA, or 200 µM LNA. Distilled and deionized water (DDW) was used as a blank.
Figure Legend Snippet: The UV-Vis spectra of bovine serum albumin (BSA), α-linolenic acid (LNA complexed to BSA; referred as LNA) and ZnO NPs (code: XFI06) suspended in distilled and deionized water (DDW), 0.5% BSA, or 200 µM LNA. Distilled and deionized water (DDW) was used as a blank.

Techniques Used:

The intracellular superoxide. HepG2 cells were exposed to various concentrations of ZnO NPs (code: XFI06) with the presence of bovine serum albumin (BSA) or α-linolenic acid (LNA complexed to BSA; referred to as LNA) for 3 h, and the intracellular superoxide was measured by using dihydroethidium (DHE).
Figure Legend Snippet: The intracellular superoxide. HepG2 cells were exposed to various concentrations of ZnO NPs (code: XFI06) with the presence of bovine serum albumin (BSA) or α-linolenic acid (LNA complexed to BSA; referred to as LNA) for 3 h, and the intracellular superoxide was measured by using dihydroethidium (DHE).

Techniques Used:

The damage of lysosomes as assessed by neutral red uptake assay ( A ) or acridine orange staining ( B ). HepG2 cells were exposed to various concentrations of ZnO NPs (code: XFI06) with the presence of bovine serum albumin (BSA) or α-linolenic acid (LNA; complexed to BSA; referred to as LNA) for 24 h ( A ) or 3 h ( B ), and neutral red uptake assay ( A ) or acridine orange staining ( B ) were used to indicate the integrity of lysosomes. *, p
Figure Legend Snippet: The damage of lysosomes as assessed by neutral red uptake assay ( A ) or acridine orange staining ( B ). HepG2 cells were exposed to various concentrations of ZnO NPs (code: XFI06) with the presence of bovine serum albumin (BSA) or α-linolenic acid (LNA; complexed to BSA; referred to as LNA) for 24 h ( A ) or 3 h ( B ), and neutral red uptake assay ( A ) or acridine orange staining ( B ) were used to indicate the integrity of lysosomes. *, p

Techniques Used: Staining

Cellular viability of HepG2 cells as assessed by cell counting kit-8 (CCK-8) assay. The cells were exposed to various concentrations of ZnO NPs (code: XFI06) with the presence of bovine serum albumin (BSA) or 200 µM α-linolenic acid (LNA; complexed to BSA; referred to as LNA) for 24 h, and the cellular viability was measured by CCK-8 assay. *, p
Figure Legend Snippet: Cellular viability of HepG2 cells as assessed by cell counting kit-8 (CCK-8) assay. The cells were exposed to various concentrations of ZnO NPs (code: XFI06) with the presence of bovine serum albumin (BSA) or 200 µM α-linolenic acid (LNA; complexed to BSA; referred to as LNA) for 24 h, and the cellular viability was measured by CCK-8 assay. *, p

Techniques Used: Cell Counting, CCK-8 Assay

The hydrodynamic size ( A , B ) and zeta potential ( C , D ) of freshly prepared ( A , C ) and aged for 24 h ( B , D ) ZnO NPs (code: XFI06) suspended in distilled and deionized water (DDW), 0.5% BSA, or 200 µM α-linolenic acid (LNA; complexed to BSA; referred as LNA). The sizes smaller than 50 nm are not shown in this figure.
Figure Legend Snippet: The hydrodynamic size ( A , B ) and zeta potential ( C , D ) of freshly prepared ( A , C ) and aged for 24 h ( B , D ) ZnO NPs (code: XFI06) suspended in distilled and deionized water (DDW), 0.5% BSA, or 200 µM α-linolenic acid (LNA; complexed to BSA; referred as LNA). The sizes smaller than 50 nm are not shown in this figure.

Techniques Used:

The release of inflammatory cytokines interleukin-1β (IL-1β; ( A )) or IL-6 ( B ). HepG2 cells were exposed to various concentrations of ZnO NPs (code: XFI06) with the presence of bovine serum albumin (BSA) or α-linolenic acid (LNA; complexed to BSA; referred to as LNA) for 24 h, and the release of IL-1β and IL-6 was measured by ELISA. *, p
Figure Legend Snippet: The release of inflammatory cytokines interleukin-1β (IL-1β; ( A )) or IL-6 ( B ). HepG2 cells were exposed to various concentrations of ZnO NPs (code: XFI06) with the presence of bovine serum albumin (BSA) or α-linolenic acid (LNA; complexed to BSA; referred to as LNA) for 24 h, and the release of IL-1β and IL-6 was measured by ELISA. *, p

Techniques Used: Enzyme-linked Immunosorbent Assay

The fluorescence spectra (λ = 280 nm) of bovine serum albumin (BSA), α-linolenic acid (LNA; complexed to BSA; referred as LNA) and ZnO NPs (code: XFI06) suspended in distilled and deionized water (DDW), 0.5% BSA, or 200 µM LNA.
Figure Legend Snippet: The fluorescence spectra (λ = 280 nm) of bovine serum albumin (BSA), α-linolenic acid (LNA; complexed to BSA; referred as LNA) and ZnO NPs (code: XFI06) suspended in distilled and deionized water (DDW), 0.5% BSA, or 200 µM LNA.

Techniques Used: Fluorescence

Synchronous fluorescence spectra of bovine serum albumin (BSA), α-linolenic acid (LNA; complexed to BSA; referred as LNA) and ZnO NPs (code: XFI06) suspended in distilled and deionized water (DDW), 0.5% BSA, or 200 µM LNA. ( A ) Δλ = 15 nm; ( B ) Δλ = 60 nm.
Figure Legend Snippet: Synchronous fluorescence spectra of bovine serum albumin (BSA), α-linolenic acid (LNA; complexed to BSA; referred as LNA) and ZnO NPs (code: XFI06) suspended in distilled and deionized water (DDW), 0.5% BSA, or 200 µM LNA. ( A ) Δλ = 15 nm; ( B ) Δλ = 60 nm.

Techniques Used: Fluorescence

49) Product Images from "Extracellular HSP60 triggers tissue regeneration and wound healing by regulating inflammation and cell proliferation"

Article Title: Extracellular HSP60 triggers tissue regeneration and wound healing by regulating inflammation and cell proliferation

Journal: NPJ Regenerative medicine

doi: 10.1038/npjregenmed.2016.13

Exogenous HSP60 promotes hair cell regeneration and signalling inhibitor L-37pA inhibits regeneration. ( a ) GroEL injected into the trunk attracted mpx -positive neutrophils similar to brain ventricle injections. The areas framed with red dotted lines in the left panels indicate the areas of injection (the panel on the left) or quantification (three panels on the right). The red colour shown within the dotted lines of the first panel is from phenol red in the injection buffer. BSA protein is used as a control for GroEL protein and also a control for assessing the needle-induced injury. Red arrows point to the sites of injection. ( b ) GroEL injected into the trunk promotes hair cell regeneration. The top panel shows a 7 dpf zebrafish larvae stained with Yopro-1, with each fluorescent dot indicating a hair cell containing neuromast. The injection site is pointed. The bottom panels show representative images of hair cells in a neuromast for each condition. ( c ) Quantification of hair cell sensitivity to copper (left) and rate of regeneration (right). GroEL does not provide protection from cell death ( n = 16, P = 0.471), but does significantly boost regeneration rates ( n = 15, P
Figure Legend Snippet: Exogenous HSP60 promotes hair cell regeneration and signalling inhibitor L-37pA inhibits regeneration. ( a ) GroEL injected into the trunk attracted mpx -positive neutrophils similar to brain ventricle injections. The areas framed with red dotted lines in the left panels indicate the areas of injection (the panel on the left) or quantification (three panels on the right). The red colour shown within the dotted lines of the first panel is from phenol red in the injection buffer. BSA protein is used as a control for GroEL protein and also a control for assessing the needle-induced injury. Red arrows point to the sites of injection. ( b ) GroEL injected into the trunk promotes hair cell regeneration. The top panel shows a 7 dpf zebrafish larvae stained with Yopro-1, with each fluorescent dot indicating a hair cell containing neuromast. The injection site is pointed. The bottom panels show representative images of hair cells in a neuromast for each condition. ( c ) Quantification of hair cell sensitivity to copper (left) and rate of regeneration (right). GroEL does not provide protection from cell death ( n = 16, P = 0.471), but does significantly boost regeneration rates ( n = 15, P

Techniques Used: Injection, Staining

Ectopic application of HSP60 stimulates wound healing in diabetic mice and stimulates M2 macrophages in human peripheral blood cells. ( a ) Representative images of skin puncture wounds of db/db mice on the back at 7, 14 and 21 days after the initial injury. Dotted black lines demarcate the wound opening. Bar = 1 mm. ( b ) Quantification of wound healing in the untreated control, BSA-treated or GroEL-treated wounds over a 21-day test period. Wound size is expressed as a percentage of the initial wound area. The number of wounds and mice used for the treatment and quantification: 12 wounds from 6 mice for the untreated control and 9 wounds from 9 mice for the BS or GroEL-treated. The difference is significant at 14 and 21 days between untreated control and GroEL-treated, or between BSA-treated and GroEL-treated mice ( P
Figure Legend Snippet: Ectopic application of HSP60 stimulates wound healing in diabetic mice and stimulates M2 macrophages in human peripheral blood cells. ( a ) Representative images of skin puncture wounds of db/db mice on the back at 7, 14 and 21 days after the initial injury. Dotted black lines demarcate the wound opening. Bar = 1 mm. ( b ) Quantification of wound healing in the untreated control, BSA-treated or GroEL-treated wounds over a 21-day test period. Wound size is expressed as a percentage of the initial wound area. The number of wounds and mice used for the treatment and quantification: 12 wounds from 6 mice for the untreated control and 9 wounds from 9 mice for the BS or GroEL-treated. The difference is significant at 14 and 21 days between untreated control and GroEL-treated, or between BSA-treated and GroEL-treated mice ( P

Techniques Used: Mouse Assay

Extracellular HSP60 promotes caudal fin regeneration. ( a ) Schematic of injection site and amputation site. ( b ) Caudal fin area measured in the injected embryos at 4 dpa. Quantified areas are framed with dotted red lines, starting from the anterior end of the ventral pigmentation break. ( c ) Quantification of caudal fin regeneration in GroEL- and BSA-injected embryos. A significant increase (indicated by an asterisk) in the fin area is detected in GroEL-injected embryos ( n = 10, P = 0.003). Bars = 500 µm in a , 100 µm in b . BSA, bovine serum albumin; dpa, day post amputation.
Figure Legend Snippet: Extracellular HSP60 promotes caudal fin regeneration. ( a ) Schematic of injection site and amputation site. ( b ) Caudal fin area measured in the injected embryos at 4 dpa. Quantified areas are framed with dotted red lines, starting from the anterior end of the ventral pigmentation break. ( c ) Quantification of caudal fin regeneration in GroEL- and BSA-injected embryos. A significant increase (indicated by an asterisk) in the fin area is detected in GroEL-injected embryos ( n = 10, P = 0.003). Bars = 500 µm in a , 100 µm in b . BSA, bovine serum albumin; dpa, day post amputation.

Techniques Used: Injection

50) Product Images from "Human RECQL5? stimulates flap endonuclease 1"

Article Title: Human RECQL5? stimulates flap endonuclease 1

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkp1217

RECQL5β stimulates FEN1 cleavage of duplex DNA containing 30 nt 5′-flap. Reactions (10 μl) containing 1 nM 30 nt 5′-flap substrate, 0.1 nM FEN1 and the indicated amounts of RECQL5β, or 0.8 nM BSA, were incubated at 37°C for 15 min. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10–18). ( A ) A phosphorimage of a typical gel. ( B ) Percent incision from the data shown in (A ) , data points are the mean of three independent experiments with SDs indicated by error bars.
Figure Legend Snippet: RECQL5β stimulates FEN1 cleavage of duplex DNA containing 30 nt 5′-flap. Reactions (10 μl) containing 1 nM 30 nt 5′-flap substrate, 0.1 nM FEN1 and the indicated amounts of RECQL5β, or 0.8 nM BSA, were incubated at 37°C for 15 min. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10–18). ( A ) A phosphorimage of a typical gel. ( B ) Percent incision from the data shown in (A ) , data points are the mean of three independent experiments with SDs indicated by error bars.

Techniques Used: Incubation

RECQL5β stimulates FEN1 cleavage of duplex DNA containing a nick. Reactions (10 μl) containing 1 nM nicked duplex substrate, 1 nM FEN1 and the indicated amounts of RECQL5β, or 2 nM BSA, were incubated at 37°C for 15 min. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10–18). ( A ) A phosphorimage of a typical gel. ( B ) Percent incision from the data shown in (A), data points are the mean of three independent experiments with SDs indicated by error bars. Open circles, plus ATP; filled circles, minus ATP.
Figure Legend Snippet: RECQL5β stimulates FEN1 cleavage of duplex DNA containing a nick. Reactions (10 μl) containing 1 nM nicked duplex substrate, 1 nM FEN1 and the indicated amounts of RECQL5β, or 2 nM BSA, were incubated at 37°C for 15 min. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10–18). ( A ) A phosphorimage of a typical gel. ( B ) Percent incision from the data shown in (A), data points are the mean of three independent experiments with SDs indicated by error bars. Open circles, plus ATP; filled circles, minus ATP.

Techniques Used: Incubation

RECQL5β stimulates FEN1 cleavage of 1 nt 5′-flap DNA. Reactions (10 μl) containing 1 nM 1 nt 5′-flap DNA substrate, 5 nM FEN1 and the indicated amounts of RECQL5β, or 20 nM BSA, were incubated at 37°C for 15 min under conditions described in the ‘Materials and Methods’ section. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10-18). ( A ) A phosphorimage of a typical gel. ( B ) Percent incision from the data shown in (A), data points are the mean of three independent experiments with SDs indicated by error bars. Open circles, plus ATP; filled circles, minus ATP.
Figure Legend Snippet: RECQL5β stimulates FEN1 cleavage of 1 nt 5′-flap DNA. Reactions (10 μl) containing 1 nM 1 nt 5′-flap DNA substrate, 5 nM FEN1 and the indicated amounts of RECQL5β, or 20 nM BSA, were incubated at 37°C for 15 min under conditions described in the ‘Materials and Methods’ section. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10-18). ( A ) A phosphorimage of a typical gel. ( B ) Percent incision from the data shown in (A), data points are the mean of three independent experiments with SDs indicated by error bars. Open circles, plus ATP; filled circles, minus ATP.

Techniques Used: Incubation

RECQL5β effects on FEN1’s GEN activity on double-stranded flap substrates. Reactions (10 μl) containing 1 nM 3′ double-stranded flap substrates ( A , Leading) or 5′ double-stranded flap substrate ( B , Lagging), the indicated amounts of FEN1 and RECQL5β, or BSA, were incubated at 37°C for 15 min. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10–18). ( C ) Percent incision from the data shown in (B), data points are the mean of three independent experiments with SDs indicated by error bars.
Figure Legend Snippet: RECQL5β effects on FEN1’s GEN activity on double-stranded flap substrates. Reactions (10 μl) containing 1 nM 3′ double-stranded flap substrates ( A , Leading) or 5′ double-stranded flap substrate ( B , Lagging), the indicated amounts of FEN1 and RECQL5β, or BSA, were incubated at 37°C for 15 min. The presence of 2 mM ATP in reaction mixtures is indicated (lanes 10–18). ( C ) Percent incision from the data shown in (B), data points are the mean of three independent experiments with SDs indicated by error bars.

Techniques Used: Activity Assay, Incubation

51) Product Images from "Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells"

Article Title: Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells

Journal: Arthritis Research & Therapy

doi: 10.1186/ar2807

RANKL and osteoprotegerin expression of dermal fibroblasts after incubation with AGE-BSA. (a and c) Contrary to fibroblast-like synovial cells (FLS), advanced glycation end products-modified (AGE)-BSA significantly upregulated the receptor activator of nuclear factor kappa B ligand (RANKL) mRNA and protein expression of dermal fibroblasts. (* P
Figure Legend Snippet: RANKL and osteoprotegerin expression of dermal fibroblasts after incubation with AGE-BSA. (a and c) Contrary to fibroblast-like synovial cells (FLS), advanced glycation end products-modified (AGE)-BSA significantly upregulated the receptor activator of nuclear factor kappa B ligand (RANKL) mRNA and protein expression of dermal fibroblasts. (* P

Techniques Used: Expressing, Incubation, Modification

Stimulation of TNF-α and Il-6 by AGE-BSA. (a) Transcripts for TNF-α were significantly increased after one and two days (* P
Figure Legend Snippet: Stimulation of TNF-α and Il-6 by AGE-BSA. (a) Transcripts for TNF-α were significantly increased after one and two days (* P

Techniques Used:

Reduction of RANKL and osteoprotegerin expression in FLS after incubation with AGE-BSA. (a) Receptor activator of nuclear factor kappa B ligand (RANKL) mRNA expression was significantly lower after treatment with advanced glycation end products-modified (AGE)-BSA (* P
Figure Legend Snippet: Reduction of RANKL and osteoprotegerin expression in FLS after incubation with AGE-BSA. (a) Receptor activator of nuclear factor kappa B ligand (RANKL) mRNA expression was significantly lower after treatment with advanced glycation end products-modified (AGE)-BSA (* P

Techniques Used: Expressing, Incubation, Modification

Inhibition of p27 Kip1 expression by an anti-RAGE antibody. Fibroblast-like synovial cells (FLS) were incubated for 24 hours with either control-BSA (Co-BSA), advanced glycation end products-modified (AGE)-BSA or AGE-BSA together with the anti- receptor for AGEs (RAGE) antibody (a) The significant increase of p27 Kip1 mRNA expression was inhibited when RAGE was blocked (AGE-BSA + anti-RAGE antibody versus AGE-BSA: P
Figure Legend Snippet: Inhibition of p27 Kip1 expression by an anti-RAGE antibody. Fibroblast-like synovial cells (FLS) were incubated for 24 hours with either control-BSA (Co-BSA), advanced glycation end products-modified (AGE)-BSA or AGE-BSA together with the anti- receptor for AGEs (RAGE) antibody (a) The significant increase of p27 Kip1 mRNA expression was inhibited when RAGE was blocked (AGE-BSA + anti-RAGE antibody versus AGE-BSA: P

Techniques Used: Inhibition, Expressing, Incubation, Modification

Characterisation of FLS and AGE uptake. (a) Immunohistochemical staining of fibroblast-like synovial cells (FLS) cultured from osteoarthritic synovial tissues. FLS were stimulated with control-BSA (Co-BSA) or advanced glycation end products-modified (AGE)-BSA (5 mg/ml) for 24 hours. FLS stained positive for the fibroblast marker CD90 and AGE-BSA incubation had no influence on CD90+ expression. The intensive intracellular staining for N ε -carboxymethyllysine (CML) and imidazolone in AGE-BSA treated cells in comparison with Co-BSA suggests active uptake of AGE. (b) Western blot for CML. FLS treated with AGE-BSA expressed more CML protein than cells incubated with Co-BSA.
Figure Legend Snippet: Characterisation of FLS and AGE uptake. (a) Immunohistochemical staining of fibroblast-like synovial cells (FLS) cultured from osteoarthritic synovial tissues. FLS were stimulated with control-BSA (Co-BSA) or advanced glycation end products-modified (AGE)-BSA (5 mg/ml) for 24 hours. FLS stained positive for the fibroblast marker CD90 and AGE-BSA incubation had no influence on CD90+ expression. The intensive intracellular staining for N ε -carboxymethyllysine (CML) and imidazolone in AGE-BSA treated cells in comparison with Co-BSA suggests active uptake of AGE. (b) Western blot for CML. FLS treated with AGE-BSA expressed more CML protein than cells incubated with Co-BSA.

Techniques Used: Immunohistochemistry, Staining, Cell Culture, Modification, Marker, Incubation, Expressing, Western Blot

EMSA for NFκB. (a) A control experiment was performed to demonstrate the specificity of the assay. Aliquots of the nuclear extracts of TNF-α-activated fibroblast-like synovial cells (FLS; 10 ng/ml TNF-α for two hours) were incubated without (-) or with the indicated unlabelled oligonucleotides in the competition assays. The DNA-binding was reduced in the presence of cold nuclear factor kappa B (NFκB) probe, but not with NFκB mutant or AP1 oligonucleotides. An anti-NFκB p65 antibody induced a supershift. Data are representative of three separate experiments. (b) Advanced glycation end products-modified (AGE)-BSA, but not control-BSA (Co-BSA) treatment results in NFκB activation and the formation of NFκB-DNA complexes. Nuclear proteins isolated from AGE-BSA treated FLS in the presence of a receptor for AGEs (RAGE)-neutralising antibody showed a weaker NFκB binding in comparison with AGE-BSA stimulation alone. This demonstrates that the AGE-induced NFκB activation was caused by AGE-RAGE interactions. The specificity of NFκB binding was confirmed by supershifts using the NFκB p65 antibody. The shown electrophoretic mobility shift assay (EMSA) is representative of two independent experiments with similar results.
Figure Legend Snippet: EMSA for NFκB. (a) A control experiment was performed to demonstrate the specificity of the assay. Aliquots of the nuclear extracts of TNF-α-activated fibroblast-like synovial cells (FLS; 10 ng/ml TNF-α for two hours) were incubated without (-) or with the indicated unlabelled oligonucleotides in the competition assays. The DNA-binding was reduced in the presence of cold nuclear factor kappa B (NFκB) probe, but not with NFκB mutant or AP1 oligonucleotides. An anti-NFκB p65 antibody induced a supershift. Data are representative of three separate experiments. (b) Advanced glycation end products-modified (AGE)-BSA, but not control-BSA (Co-BSA) treatment results in NFκB activation and the formation of NFκB-DNA complexes. Nuclear proteins isolated from AGE-BSA treated FLS in the presence of a receptor for AGEs (RAGE)-neutralising antibody showed a weaker NFκB binding in comparison with AGE-BSA stimulation alone. This demonstrates that the AGE-induced NFκB activation was caused by AGE-RAGE interactions. The specificity of NFκB binding was confirmed by supershifts using the NFκB p65 antibody. The shown electrophoretic mobility shift assay (EMSA) is representative of two independent experiments with similar results.

Techniques Used: Incubation, Binding Assay, Mutagenesis, Modification, Activation Assay, Isolation, Electrophoretic Mobility Shift Assay

52) Product Images from "PLLA-PEG-TCH-labeled bioactive molecule nanofibers for tissue engineering"

Article Title: PLLA-PEG-TCH-labeled bioactive molecule nanofibers for tissue engineering

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S23688

Functionalization procedure of PLLA/PLLA-PEG-NH 2 nanofibers (brown). Step 1: Amino groups are activated by exposing nanofibers to saturated water vapor. Step 2: Carboxyl groups are activated by hydrolysis. Blue represents the loaded drug; red is rhodamine BSA; and green is FITC-BSA. Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PEG, poly(ethylene glycol); PLLA, poly(L-lactide).
Figure Legend Snippet: Functionalization procedure of PLLA/PLLA-PEG-NH 2 nanofibers (brown). Step 1: Amino groups are activated by exposing nanofibers to saturated water vapor. Step 2: Carboxyl groups are activated by hydrolysis. Blue represents the loaded drug; red is rhodamine BSA; and green is FITC-BSA. Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PEG, poly(ethylene glycol); PLLA, poly(L-lactide).

Techniques Used:

Confocal images of PLLA/PLLA-PEG-NH 2 nanofibers functionalized with both FITC-BSA and rhodamine-BSA. ( A ) Image showing FITC-BSA, ( B ) image showing rhodamine-BSA, and ( C ) merged A and B. Bar: 20 μm. Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PEG, poly(ethylene glycol); PLLA, poly(L-lactide).
Figure Legend Snippet: Confocal images of PLLA/PLLA-PEG-NH 2 nanofibers functionalized with both FITC-BSA and rhodamine-BSA. ( A ) Image showing FITC-BSA, ( B ) image showing rhodamine-BSA, and ( C ) merged A and B. Bar: 20 μm. Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; PEG, poly(ethylene glycol); PLLA, poly(L-lactide).

Techniques Used:

53) Product Images from "An effector from the Huanglongbing-associated pathogen targets citrus proteases"

Article Title: An effector from the Huanglongbing-associated pathogen targets citrus proteases

Journal: Nature Communications

doi: 10.1038/s41467-018-04140-9

SDE1 inhibits PLCP activity in vitro and in plant cells. a Proteolytic activity of papain measured by digestion of a fluorescent casein substrate in the presence of E-64, purified SDE1 protein, or BSA (as a negative control). Fluorescence was measured at 485/530 nm excitation/emission. Mean ± standard deviation ( n = 3) is shown. Asterisks (*) indicate statistically significant differences based on the two-tailed Student’s t -test. p
Figure Legend Snippet: SDE1 inhibits PLCP activity in vitro and in plant cells. a Proteolytic activity of papain measured by digestion of a fluorescent casein substrate in the presence of E-64, purified SDE1 protein, or BSA (as a negative control). Fluorescence was measured at 485/530 nm excitation/emission. Mean ± standard deviation ( n = 3) is shown. Asterisks (*) indicate statistically significant differences based on the two-tailed Student’s t -test. p

Techniques Used: Activity Assay, In Vitro, Purification, Negative Control, Fluorescence, Standard Deviation, Two Tailed Test

54) Product Images from "Norfloxacin and N-Donor Mixed-Ligand Copper(II) Complexes: Synthesis, Albumin Interaction, and Anti-Trypanosoma cruzi Activity"

Article Title: Norfloxacin and N-Donor Mixed-Ligand Copper(II) Complexes: Synthesis, Albumin Interaction, and Anti-Trypanosoma cruzi Activity

Journal: Bioinorganic Chemistry and Applications

doi: 10.1155/2016/5027404

Stern-Volmer plots for the quenching of albumins' intrinsic fluorescence by the copper complexes: (a) HSA-complex ( 1 ), (b) HSA-complex ( 2 ), (c) BSA-complex ( 1 ), and (d) BSA-complex ( 2 ). Control results of the effects of NOR and Cu(II) are also presented. F 0 and F are the fluorescence intensities at the peak in the absence and presence of the complexes. HSA and BSA concentrations equal to 4.0 × 10 −6 mol L −1 ; temperature: 296 K.
Figure Legend Snippet: Stern-Volmer plots for the quenching of albumins' intrinsic fluorescence by the copper complexes: (a) HSA-complex ( 1 ), (b) HSA-complex ( 2 ), (c) BSA-complex ( 1 ), and (d) BSA-complex ( 2 ). Control results of the effects of NOR and Cu(II) are also presented. F 0 and F are the fluorescence intensities at the peak in the absence and presence of the complexes. HSA and BSA concentrations equal to 4.0 × 10 −6 mol L −1 ; temperature: 296 K.

Techniques Used: Fluorescence

X-band EPR spectra for complexes 1 , Cu(II)-bipy, 2, and Cu(II)-phen with equimolar amounts of HSA or BSA (concentration of the complexes: 0.5 mM, phosphate buffer 20 mM, pH 7.4, and temperature 77 K). (a) 1 :HSA; (b) Cu(II)-bipy:HSA; (c) 1 :BSA; (d) Cu(II)-bipy:BSA; (e) 2 :HSA; (f) Cu(II)-phen:HSA; (g) 2 :BSA; (h) Cu(II)-phen:BSA.
Figure Legend Snippet: X-band EPR spectra for complexes 1 , Cu(II)-bipy, 2, and Cu(II)-phen with equimolar amounts of HSA or BSA (concentration of the complexes: 0.5 mM, phosphate buffer 20 mM, pH 7.4, and temperature 77 K). (a) 1 :HSA; (b) Cu(II)-bipy:HSA; (c) 1 :BSA; (d) Cu(II)-bipy:BSA; (e) 2 :HSA; (f) Cu(II)-phen:HSA; (g) 2 :BSA; (h) Cu(II)-phen:BSA.

Techniques Used: Electron Paramagnetic Resonance, Concentration Assay

X-band EPR spectra for HSA and BSA Cu(II) complexes ((a) and (b), resp.) and for Cu(II)-bipy and Cu(II)-phen complexes with HSA ((c) and (d), resp.). Experimental conditions: Cu, HSA, and BSA 0.5 mM, phosphate buffer 20 mM, and pH 7.4, at 77 K. (b′) and (c′) are the simulated spectra of (b) and (c), using EasySpin [ 20 ] with parameters in Table 4 .
Figure Legend Snippet: X-band EPR spectra for HSA and BSA Cu(II) complexes ((a) and (b), resp.) and for Cu(II)-bipy and Cu(II)-phen complexes with HSA ((c) and (d), resp.). Experimental conditions: Cu, HSA, and BSA 0.5 mM, phosphate buffer 20 mM, and pH 7.4, at 77 K. (b′) and (c′) are the simulated spectra of (b) and (c), using EasySpin [ 20 ] with parameters in Table 4 .

Techniques Used: Electron Paramagnetic Resonance

55) Product Images from "Biodegradable double nanocapsule as a novel multifunctional carrier for drug delivery and cell imaging"

Article Title: Biodegradable double nanocapsule as a novel multifunctional carrier for drug delivery and cell imaging

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S83731

Fluorescence intensities and cytotoxicity of nanocapsules in Hela cells. Notes: Fluorescence intensities of green ( A ) and red ( B ) of cells increased along with the increment of nanocapsule concentrations. FITC–BSA and TRITC–BSA were used as controls, respectively. ( C ) Cytotoxicity of the nanocapsules with different concentrations by AlamarBlue cell viability assay. The data were expressed as mean ± SEM (n%4). Abbreviations: FITC–BSA, fluorescein isothiocyanate–bovine serum albumin; TRITC–BSA, tetramethylrhodamine–bovine serum albumin; SEM, standard error of mean.
Figure Legend Snippet: Fluorescence intensities and cytotoxicity of nanocapsules in Hela cells. Notes: Fluorescence intensities of green ( A ) and red ( B ) of cells increased along with the increment of nanocapsule concentrations. FITC–BSA and TRITC–BSA were used as controls, respectively. ( C ) Cytotoxicity of the nanocapsules with different concentrations by AlamarBlue cell viability assay. The data were expressed as mean ± SEM (n%4). Abbreviations: FITC–BSA, fluorescein isothiocyanate–bovine serum albumin; TRITC–BSA, tetramethylrhodamine–bovine serum albumin; SEM, standard error of mean.

Techniques Used: Fluorescence, Viability Assay

Intercellular delivery of NLS–GFP by the nanocapsules. Notes: ( A ) The scheme of preparation of the nanocapsules containing NLS–GFP. ( B ) Fluorescence distribution in Hela cells after exposure to nanocapsules. NLS could carry GFP into the nucleus after NLS–GFP was transduced into the cytosol. TRITC–BSA on the surface of the nanocapsules was left in the cytosol. Scale bar 20 μm. Abbreviations: NLS, nuclear location signal; GFP, green fluorescence protein; TRITC–BSA, tetramethylrhodamine–bovine serum albumin.
Figure Legend Snippet: Intercellular delivery of NLS–GFP by the nanocapsules. Notes: ( A ) The scheme of preparation of the nanocapsules containing NLS–GFP. ( B ) Fluorescence distribution in Hela cells after exposure to nanocapsules. NLS could carry GFP into the nucleus after NLS–GFP was transduced into the cytosol. TRITC–BSA on the surface of the nanocapsules was left in the cytosol. Scale bar 20 μm. Abbreviations: NLS, nuclear location signal; GFP, green fluorescence protein; TRITC–BSA, tetramethylrhodamine–bovine serum albumin.

Techniques Used: Fluorescence

Cell uptake of the nanocapsules in Hela cells. Notes: ( A ) Cell uptake process of the nanocapsules consisted of FITC–BSA coating and TRITC–BSA core. ( B ) Fluorescence images under confocal microscope after 60 minutes of incubation. Nuclei were stained with Hoechst 33258 (blue). Scale bar 20 μm. Abbreviations: FITC–BSA, fluorescein isothiocyanate–bovine serum albumin; TRITC–BSA, tetramethylrhodamine–bovine serum albumin.
Figure Legend Snippet: Cell uptake of the nanocapsules in Hela cells. Notes: ( A ) Cell uptake process of the nanocapsules consisted of FITC–BSA coating and TRITC–BSA core. ( B ) Fluorescence images under confocal microscope after 60 minutes of incubation. Nuclei were stained with Hoechst 33258 (blue). Scale bar 20 μm. Abbreviations: FITC–BSA, fluorescein isothiocyanate–bovine serum albumin; TRITC–BSA, tetramethylrhodamine–bovine serum albumin.

Techniques Used: Fluorescence, Microscopy, Incubation, Staining

56) Product Images from "Effects of miR-33a-5P on ABCA1/G1-Mediated Cholesterol Efflux under Inflammatory Stress in THP-1 Macrophages"

Article Title: Effects of miR-33a-5P on ABCA1/G1-Mediated Cholesterol Efflux under Inflammatory Stress in THP-1 Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109722

Effects of overexpression of miR-33a-5P and Anti-miR-33a-5P on intracellular lipid accumulation in THP-1 macrophages in the absence or presence of LDL. THP-1 macrophages were infected using Con-miR, miR-33a-5P, Con-Anti-miR, and Anti-miR-33a-5P, respectively, after 24 h PMA stimulation. After 48 h infection, THP-1 macrophages we incubated in serum-free medium at 37°C for 24 h. Then, the medium was respectively replaced by fresh serum-free medium (0.2% BSA) containing blank control ( A , I, Con-miR), blank control ( A , II, miR-33a-5P), 40 ng/ml IL-6 ( A , III, Con- Anti-miR plus IL-6), 40 ng/ml IL-6 ( A , IV, anti-miR-33a-5P plus IL-6), 25 µg/ml LDL ( B , I, Con-miR plus LDL), 25 µg/ml LDL ( B , II, miR-33a-5P plus LDL), 25 µg/ml LDL plus 40 ng/ml IL-6 ( B , III, Con-Anti-miR plus IL-6 plus LDL), or 25 µg/ml LDL plus 40 ng/ml IL-6 ( B , IV, Anti-miR-33a-5P plus IL-6 plus LDL), followed by incubation at 37°C for 24 h. ( A and B ) The cells were examined for lipid inclusions by oil red O staining. The results are representative of those observed in six separate experiments (×400). ( C and D ) Semi-quantitative analysis of oil red O positive staining. Data are means ± SD from 6 separate fields. ( E and F ) Quantification of levels of intracellular cholesterol contents. Data are means ± SD of duplicate wells from 6 experiments. *, P
Figure Legend Snippet: Effects of overexpression of miR-33a-5P and Anti-miR-33a-5P on intracellular lipid accumulation in THP-1 macrophages in the absence or presence of LDL. THP-1 macrophages were infected using Con-miR, miR-33a-5P, Con-Anti-miR, and Anti-miR-33a-5P, respectively, after 24 h PMA stimulation. After 48 h infection, THP-1 macrophages we incubated in serum-free medium at 37°C for 24 h. Then, the medium was respectively replaced by fresh serum-free medium (0.2% BSA) containing blank control ( A , I, Con-miR), blank control ( A , II, miR-33a-5P), 40 ng/ml IL-6 ( A , III, Con- Anti-miR plus IL-6), 40 ng/ml IL-6 ( A , IV, anti-miR-33a-5P plus IL-6), 25 µg/ml LDL ( B , I, Con-miR plus LDL), 25 µg/ml LDL ( B , II, miR-33a-5P plus LDL), 25 µg/ml LDL plus 40 ng/ml IL-6 ( B , III, Con-Anti-miR plus IL-6 plus LDL), or 25 µg/ml LDL plus 40 ng/ml IL-6 ( B , IV, Anti-miR-33a-5P plus IL-6 plus LDL), followed by incubation at 37°C for 24 h. ( A and B ) The cells were examined for lipid inclusions by oil red O staining. The results are representative of those observed in six separate experiments (×400). ( C and D ) Semi-quantitative analysis of oil red O positive staining. Data are means ± SD from 6 separate fields. ( E and F ) Quantification of levels of intracellular cholesterol contents. Data are means ± SD of duplicate wells from 6 experiments. *, P

Techniques Used: Over Expression, Infection, Incubation, Staining

Effects of overexpression of miR-33a-5P and anti-miR-33a-5P on apoA-I mediated cholesterol efflux in THP-1 macrophages in the absence or presence of LDL. THP-1 macrophages were infected using Con-miR, miR-33a-5P, Con-Anti-miR, and anti-miR-33a-5P, respectively, after 24 h PMA stimulation. After 48 h infection, THP-1 macrophages were incubated in serum-free medium at 37°C for 24 h. The medium was then respectively replaced by fresh serum-free medium (0.2% BSA) containing ( A ) blank control, blank control, 40 ng/ml IL-6, or 40 ng/ml IL-6; ( B ) 25 µg/ml LDL, 25 µg/ml LDL, 25 µg/ml LDL plus 40 ng/ml IL-6, or 25 µg/ml LDL plus 40 ng/ml IL-6, followed by incubation at 37°C for 18 h. Next, the cells were cultured in serum-free medium containing treatment factors and 10 µg/ml apoA-I for 4 h. ApoA-I-mediated cholesterol efflux was assayed as described in the Materials and Methods section. Data are means ± SD of duplicate wells from 6 experiments. *, P
Figure Legend Snippet: Effects of overexpression of miR-33a-5P and anti-miR-33a-5P on apoA-I mediated cholesterol efflux in THP-1 macrophages in the absence or presence of LDL. THP-1 macrophages were infected using Con-miR, miR-33a-5P, Con-Anti-miR, and anti-miR-33a-5P, respectively, after 24 h PMA stimulation. After 48 h infection, THP-1 macrophages were incubated in serum-free medium at 37°C for 24 h. The medium was then respectively replaced by fresh serum-free medium (0.2% BSA) containing ( A ) blank control, blank control, 40 ng/ml IL-6, or 40 ng/ml IL-6; ( B ) 25 µg/ml LDL, 25 µg/ml LDL, 25 µg/ml LDL plus 40 ng/ml IL-6, or 25 µg/ml LDL plus 40 ng/ml IL-6, followed by incubation at 37°C for 18 h. Next, the cells were cultured in serum-free medium containing treatment factors and 10 µg/ml apoA-I for 4 h. ApoA-I-mediated cholesterol efflux was assayed as described in the Materials and Methods section. Data are means ± SD of duplicate wells from 6 experiments. *, P

Techniques Used: Over Expression, Infection, Incubation, Cell Culture

Effects of overexpression of miR-33a-5P and Anti-miR-33a-5P on the expression of miR-33a-5P, ABCA1 and ABCG1 in THP-1 macrophages in the absence or presence of LDL. THP-1 macrophages were infected using Con-miR, miR-33a-5P, Con-Anti-miR, and Anti-miR-33a-5P, respectively, after 24 h PMA stimulation. After 48 h infection, THP-1 macrophages were incubated in serum-free medium at 37°C for 24 h. The medium was then respectively replaced by fresh serum-free medium (0.2% BSA) containing ( A ) blank control, blank control, 40 ng/ml IL-6, or 40 ng/ml IL-6, ( B ) 25 µg/ml LDL, 25 µg/ml LDL, 25 µg/ml LDL plus 40 ng/ml IL-6, or 25 µg/ml LDL plus 40 ng/ml IL-6, followed by incubation at 37°C for 24 h. mRNA levels of miR-33a-5P, ABCA1 and ABCG1 were determined using RT-PCR. U6 or β-actin served as a reference gene. Data are means ± SD from 6 experiments. ( C and D ) Protein levels of ABCA1 and ABCG1 determined by Western blotting. ( E and F ) Quantification of densitometric values of ABCA1 and ABCG1 protein bands from four experiments, which were normalized to β-actin and expressed as a percentage of control. Data are means ± SD from 4 experiments. *, P
Figure Legend Snippet: Effects of overexpression of miR-33a-5P and Anti-miR-33a-5P on the expression of miR-33a-5P, ABCA1 and ABCG1 in THP-1 macrophages in the absence or presence of LDL. THP-1 macrophages were infected using Con-miR, miR-33a-5P, Con-Anti-miR, and Anti-miR-33a-5P, respectively, after 24 h PMA stimulation. After 48 h infection, THP-1 macrophages were incubated in serum-free medium at 37°C for 24 h. The medium was then respectively replaced by fresh serum-free medium (0.2% BSA) containing ( A ) blank control, blank control, 40 ng/ml IL-6, or 40 ng/ml IL-6, ( B ) 25 µg/ml LDL, 25 µg/ml LDL, 25 µg/ml LDL plus 40 ng/ml IL-6, or 25 µg/ml LDL plus 40 ng/ml IL-6, followed by incubation at 37°C for 24 h. mRNA levels of miR-33a-5P, ABCA1 and ABCG1 were determined using RT-PCR. U6 or β-actin served as a reference gene. Data are means ± SD from 6 experiments. ( C and D ) Protein levels of ABCA1 and ABCG1 determined by Western blotting. ( E and F ) Quantification of densitometric values of ABCA1 and ABCG1 protein bands from four experiments, which were normalized to β-actin and expressed as a percentage of control. Data are means ± SD from 4 experiments. *, P

Techniques Used: Over Expression, Expressing, Infection, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot

57) Product Images from "Drug Development in Conformational Diseases: A Novel Family of Chemical Chaperones that Bind and Stabilise Several Polymorphic Amyloid Structures"

Article Title: Drug Development in Conformational Diseases: A Novel Family of Chemical Chaperones that Bind and Stabilise Several Polymorphic Amyloid Structures

Journal: PLoS ONE

doi: 10.1371/journal.pone.0135292

A . Transmission Electron Microscopy (TEM) micrographs of BSA fibrilogenesis process (75 μmol/L), with and without N-(2-aminoethyl)-N'-1-naphthylsuccinamide A; methyl (2-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}ethyl) dithiocarbamate B; (2R)-2-(6-methoxy-2-naphthyl)propanoic acid (Naproxen) C; N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine D and 6-{[4-(1-naphthylamino)-4-oxobutanoyl]amino} hexanoic acid E, at 70°C. B : Transmission Electron Microscopy (TEM) micrographs of hIAPP 20–29 fibrilogenesis process with and without N-(2-aminoethyl)-N'-1-naphthylsuccinamide A; methyl (2-{[4-(1-naphthylamino)-4-oxobutanoyl] amino} ethyl) dithiocarbamate B and (2R)-2-(6-methoxy-2-naphthyl)propanoic acid (Naproxen) C; at 25°C. Molar ratio hIAPP 20–29 : chaperones was 1:1. C : Atomic Force Microscopy (AFM) micrographs of BSA fibrilogenesis process (75 μmol/L), with and without B, at 70°C. Molar ratio BSA:chaperones was 1:1.
Figure Legend Snippet: A . Transmission Electron Microscopy (TEM) micrographs of BSA fibrilogenesis process (75 μmol/L), with and without N-(2-aminoethyl)-N'-1-naphthylsuccinamide A; methyl (2-{[4-(1-naphthylamino)-4-oxobutanoyl]amino}ethyl) dithiocarbamate B; (2R)-2-(6-methoxy-2-naphthyl)propanoic acid (Naproxen) C; N-[4-(1-naphthylamino)-4-oxobutanoyl]-β-alanine D and 6-{[4-(1-naphthylamino)-4-oxobutanoyl]amino} hexanoic acid E, at 70°C. B : Transmission Electron Microscopy (TEM) micrographs of hIAPP 20–29 fibrilogenesis process with and without N-(2-aminoethyl)-N'-1-naphthylsuccinamide A; methyl (2-{[4-(1-naphthylamino)-4-oxobutanoyl] amino} ethyl) dithiocarbamate B and (2R)-2-(6-methoxy-2-naphthyl)propanoic acid (Naproxen) C; at 25°C. Molar ratio hIAPP 20–29 : chaperones was 1:1. C : Atomic Force Microscopy (AFM) micrographs of BSA fibrilogenesis process (75 μmol/L), with and without B, at 70°C. Molar ratio BSA:chaperones was 1:1.

Techniques Used: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Microscopy

58) Product Images from "A-FABP mediates adaptive thermogenesis by promoting intracellular activation of thyroid hormones in brown adipocytes"

Article Title: A-FABP mediates adaptive thermogenesis by promoting intracellular activation of thyroid hormones in brown adipocytes

Journal: Nature Communications

doi: 10.1038/ncomms14147

Circulating A-FABP facilitates the uptake of free fatty acid into adipocytes. ( a ) Circulating A-FABP and ( b ) FFA profile of male 4-week-old C57BL/6N mice fed with HFD for 24 weeks ( n=8 ). ( c ) Circulating A-FABP and ( d ) FFA level of male 8-week-old C57BL/6N mice during cold exposure (6 °C) for 4 h ( n= 8). ( e ) Circulating A-FABP and ( f ) FFA level of male 8-week-old C57BL/6N mice intraperitoneally injected with norepinephrine (NE; 1 mg kg −1 ) or PBS (vehicle) for 4 h under fasting condition ( n= 6). ( g ) Co-immunoprecipitation (Co-IP) of A-FABP and 3 H-palmitate in serum of male 8-week-old C57BL/6N mice after administration of 3 H-palmitate (2 μCi) for 4 h. Right panel is the 3 H-palmitate radioactivity of the co-immunoprecipitated A-FABP protein ( n= 6). ( h , i ) 3 H-palmitate uptake in BAT and WAT of 8-week-old A-FABP KO mice and their WT littermates infused with PBS or ( h ) recombinant A-FABP (rA-FABP; 1 μg h −1 ) or ( i ) mutant R126Q (1 μg h −1 ) ( n= 6). ( j ) BODIPY-FA uptake in WT or A-FABP-deficient brown adipocytes treated with PBS or rA-FABP (2 μg ml −1 ) for 10 min (min) ( n= 6). ( k ) 3 H-palmitate uptake in A-FABP-deficient adipocytes incubated with PBS, bovine serum albumin (BSA; 3 μg ml −1 ) or rA-FABP (2 μg ml −1 ) ( n= 6). ( l ) In vitro fluorescent imaging analysis of brown adipocytes treated with BODIPY-FA (2 μM) with or without pre-incubation with fluorescent-labelled rA-FABP (2 μg ml −1 ). Images were taken at 5, 10 and 30 min after treatment. Control image was taken at 30 min in which A-FABP-deficient brown adipocytes were incubated with BODIPY-FA without pre-incubation with rA-FABP. Scale bar, 20 μm, with magnification of 400 × . Representative images from three independent experiments are shown ( n= 6). ( m ) Oxygen consumption rate (OCR) and its mean value (lower panel) of A-FABP-deficient brown adipocytes treated with palmitate (PA: 200 nM) with or without pre-incubation with rA-FABP (2 μg ml −1 ) ( n= 6). CPMA, count per minutes for beta particles; RFU, relative fluorescence units; OCR, oxygen consumption rate; FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; R/A, rotenone/antimycin A. Uncropped image for co-immunoprecipitation is shown in Supplementary Fig. 13 . Data are represented as mean±s.e.m. *P
Figure Legend Snippet: Circulating A-FABP facilitates the uptake of free fatty acid into adipocytes. ( a ) Circulating A-FABP and ( b ) FFA profile of male 4-week-old C57BL/6N mice fed with HFD for 24 weeks ( n=8 ). ( c ) Circulating A-FABP and ( d ) FFA level of male 8-week-old C57BL/6N mice during cold exposure (6 °C) for 4 h ( n= 8). ( e ) Circulating A-FABP and ( f ) FFA level of male 8-week-old C57BL/6N mice intraperitoneally injected with norepinephrine (NE; 1 mg kg −1 ) or PBS (vehicle) for 4 h under fasting condition ( n= 6). ( g ) Co-immunoprecipitation (Co-IP) of A-FABP and 3 H-palmitate in serum of male 8-week-old C57BL/6N mice after administration of 3 H-palmitate (2 μCi) for 4 h. Right panel is the 3 H-palmitate radioactivity of the co-immunoprecipitated A-FABP protein ( n= 6). ( h , i ) 3 H-palmitate uptake in BAT and WAT of 8-week-old A-FABP KO mice and their WT littermates infused with PBS or ( h ) recombinant A-FABP (rA-FABP; 1 μg h −1 ) or ( i ) mutant R126Q (1 μg h −1 ) ( n= 6). ( j ) BODIPY-FA uptake in WT or A-FABP-deficient brown adipocytes treated with PBS or rA-FABP (2 μg ml −1 ) for 10 min (min) ( n= 6). ( k ) 3 H-palmitate uptake in A-FABP-deficient adipocytes incubated with PBS, bovine serum albumin (BSA; 3 μg ml −1 ) or rA-FABP (2 μg ml −1 ) ( n= 6). ( l ) In vitro fluorescent imaging analysis of brown adipocytes treated with BODIPY-FA (2 μM) with or without pre-incubation with fluorescent-labelled rA-FABP (2 μg ml −1 ). Images were taken at 5, 10 and 30 min after treatment. Control image was taken at 30 min in which A-FABP-deficient brown adipocytes were incubated with BODIPY-FA without pre-incubation with rA-FABP. Scale bar, 20 μm, with magnification of 400 × . Representative images from three independent experiments are shown ( n= 6). ( m ) Oxygen consumption rate (OCR) and its mean value (lower panel) of A-FABP-deficient brown adipocytes treated with palmitate (PA: 200 nM) with or without pre-incubation with rA-FABP (2 μg ml −1 ) ( n= 6). CPMA, count per minutes for beta particles; RFU, relative fluorescence units; OCR, oxygen consumption rate; FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; R/A, rotenone/antimycin A. Uncropped image for co-immunoprecipitation is shown in Supplementary Fig. 13 . Data are represented as mean±s.e.m. *P

Techniques Used: Mouse Assay, Injection, Immunoprecipitation, Co-Immunoprecipitation Assay, Radioactivity, Recombinant, Mutagenesis, Incubation, In Vitro, Imaging, Fluorescence

59) Product Images from "Pregnancy-specific Glycoprotein 1 Induces Endothelial Tubulogenesis through Interaction with Cell Surface Proteoglycans *"

Article Title: Pregnancy-specific Glycoprotein 1 Induces Endothelial Tubulogenesis through Interaction with Cell Surface Proteoglycans *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.161810

PSG1 binds to immobilized GAGs. A , 96-well ELISA plates were coated with 200 μg/well heparin, heparan sulfate, chondroitin sulfate, or BSA and treated with PSG1-Fc or the control protein FLAGFc (200 ng/well). Bound proteins were detected with
Figure Legend Snippet: PSG1 binds to immobilized GAGs. A , 96-well ELISA plates were coated with 200 μg/well heparin, heparan sulfate, chondroitin sulfate, or BSA and treated with PSG1-Fc or the control protein FLAGFc (200 ng/well). Bound proteins were detected with

Techniques Used: Enzyme-linked Immunosorbent Assay

60) Product Images from "CTLA-4 Is a Direct Target of Wnt/?-Catenin Signaling and Is Expressed in Human Melanoma Tumors"

Article Title: CTLA-4 Is a Direct Target of Wnt/?-Catenin Signaling and Is Expressed in Human Melanoma Tumors

Journal: The Journal of investigative dermatology

doi: 10.1038/jid.2008.170

CTLA-4 transcript is induced by Wnt/β-catenin signaling in two melanoma cell lines ( a ) RT-PCR analysis of CTLA-4 transcript splice variants in A2058 melanoma cells transiently transfected with empty vector (EV) or full-length CTLA-4 ( CTLA-4 ) and UACC 1273 and A2058 cells treated with recombinant Wnt-3a for 6 hours. ( b ) Real-time quantitative PCR analysis of CTLA-4 expression in UACC 1273 and A2058 cells treated with recombinant Wnt-3a or vehicle (BSA) for 6 hours. ( c ) Real-time quantitative PCR analysis of CTLA-4 expression in UACC 1273 and A2058 cells treated for 6 hours with Wnt-3a-conditioned media (Wnt-3a) or vehicle (control) alone or in the presence of DKK-1 conditioned media or vehicle (green fluorescent protein). Expression of CTLA-4 was normalized by comparison to GAPDH and 18S (not shown) as internal controls. Fold change is calculated relative to ( b ) BSA or ( c ) control. Gray, UACC 1273 cells; striped, A2058 cells. Data represent one of three identical experiments.
Figure Legend Snippet: CTLA-4 transcript is induced by Wnt/β-catenin signaling in two melanoma cell lines ( a ) RT-PCR analysis of CTLA-4 transcript splice variants in A2058 melanoma cells transiently transfected with empty vector (EV) or full-length CTLA-4 ( CTLA-4 ) and UACC 1273 and A2058 cells treated with recombinant Wnt-3a for 6 hours. ( b ) Real-time quantitative PCR analysis of CTLA-4 expression in UACC 1273 and A2058 cells treated with recombinant Wnt-3a or vehicle (BSA) for 6 hours. ( c ) Real-time quantitative PCR analysis of CTLA-4 expression in UACC 1273 and A2058 cells treated for 6 hours with Wnt-3a-conditioned media (Wnt-3a) or vehicle (control) alone or in the presence of DKK-1 conditioned media or vehicle (green fluorescent protein). Expression of CTLA-4 was normalized by comparison to GAPDH and 18S (not shown) as internal controls. Fold change is calculated relative to ( b ) BSA or ( c ) control. Gray, UACC 1273 cells; striped, A2058 cells. Data represent one of three identical experiments.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Recombinant, Real-time Polymerase Chain Reaction, Expressing

61) Product Images from "Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag Times"

Article Title: Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag Times

Journal: Biophysical Journal

doi: 10.1016/j.bpj.2013.06.016

Illustration of the microfluidic device used for cell-virus fusion. ( a ) Fluids are withdrawn through the channel network from a reservoir with a syringe pump. Posts inside the channel act as filters to retain large agglomerates of cells and debris, whereas single cells are captured in cell traps (see brightfield image). ( b ) The working principle of cell traps. Without a trapped cell, the fluid can pass under the traps. After a cell has been trapped, the flow is diverted (around the cell), making it unlikely to trap more than one cell. Microcontact-printed BSA-FITC is used to monitor the pH inside the channel directly next to the cell. ( c ) Side view of the set-up. The cell is slightly pressed onto the glass by the buffer flow. Single virus particles are imaged by two-color TIRF microscopy with an evanescent field of 250 nm.
Figure Legend Snippet: Illustration of the microfluidic device used for cell-virus fusion. ( a ) Fluids are withdrawn through the channel network from a reservoir with a syringe pump. Posts inside the channel act as filters to retain large agglomerates of cells and debris, whereas single cells are captured in cell traps (see brightfield image). ( b ) The working principle of cell traps. Without a trapped cell, the fluid can pass under the traps. After a cell has been trapped, the flow is diverted (around the cell), making it unlikely to trap more than one cell. Microcontact-printed BSA-FITC is used to monitor the pH inside the channel directly next to the cell. ( c ) Side view of the set-up. The cell is slightly pressed onto the glass by the buffer flow. Single virus particles are imaged by two-color TIRF microscopy with an evanescent field of 250 nm.

Techniques Used: Activated Clotting Time Assay, Flow Cytometry, Microscopy

62) Product Images from "Developmental Pathways Pervade Stem Cell Responses to Evolving Extracellular Matrices of 3D Bioprinted Microenvironments"

Article Title: Developmental Pathways Pervade Stem Cell Responses to Evolving Extracellular Matrices of 3D Bioprinted Microenvironments

Journal: Stem Cells International

doi: 10.1155/2018/4809673

GO analysis of hMSCs in 3D bioprinted prisms, especially related to the differentiation state. (a) Counts of GO terms associated with commonly examined stem cell responses. Protein degradation, development, and differentiation are the most significantly expressed pathways. (b) Development and differentiation pathways separated by 3D ECM structures. Most influential 3D structures rank from 3D/LN > 3D/Col1 > 3D/BSA > 3D/FN, with 3D/FN structures predominantly influencing vasculature development. Expression of endothelial cell markers (c), smooth muscle cell markers (d), and sphingosine phosphate lyase (e) in hMSCs cultured in 3D/FN structures. Error bars depict SD associated with n = 3 experimental replicates with at least 2 different prisms analyzed per experimental replicate. hMSCs cultured in 3D/FN structures expressed markers for both endothelial and smooth muscle cells and had a reduction in SGPL1 expression.
Figure Legend Snippet: GO analysis of hMSCs in 3D bioprinted prisms, especially related to the differentiation state. (a) Counts of GO terms associated with commonly examined stem cell responses. Protein degradation, development, and differentiation are the most significantly expressed pathways. (b) Development and differentiation pathways separated by 3D ECM structures. Most influential 3D structures rank from 3D/LN > 3D/Col1 > 3D/BSA > 3D/FN, with 3D/FN structures predominantly influencing vasculature development. Expression of endothelial cell markers (c), smooth muscle cell markers (d), and sphingosine phosphate lyase (e) in hMSCs cultured in 3D/FN structures. Error bars depict SD associated with n = 3 experimental replicates with at least 2 different prisms analyzed per experimental replicate. hMSCs cultured in 3D/FN structures expressed markers for both endothelial and smooth muscle cells and had a reduction in SGPL1 expression.

Techniques Used: Expressing, Cell Culture

RNA sequencing analysis of hMSCs in 3D bioprinted prisms at 0 and 28 days. (a) Hierarchical clustering of all genes with FPKM > 1 for 3D bioprinted prisms containing either BSA alone (3D/BSA), 3D/FN, 3D/Col1, or 3D/LN at day 28 and associated 2D control cultures at day 28. (b) qPCR validation of gene expression of a subset of genes. These genes were selected as they represent families of ECM proteins and differentiation markers associated with hMSC progeny. (c) PCA analysis of all genes with FPKM > 1. (d–f) Comparison of individual 3D bioprinted prisms containing ECM to bioprinted prisms with BSA only.
Figure Legend Snippet: RNA sequencing analysis of hMSCs in 3D bioprinted prisms at 0 and 28 days. (a) Hierarchical clustering of all genes with FPKM > 1 for 3D bioprinted prisms containing either BSA alone (3D/BSA), 3D/FN, 3D/Col1, or 3D/LN at day 28 and associated 2D control cultures at day 28. (b) qPCR validation of gene expression of a subset of genes. These genes were selected as they represent families of ECM proteins and differentiation markers associated with hMSC progeny. (c) PCA analysis of all genes with FPKM > 1. (d–f) Comparison of individual 3D bioprinted prisms containing ECM to bioprinted prisms with BSA only.

Techniques Used: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Expressing

Fabrication of 3D ECM-based, bioprinted prisms. (a) Fabrication Schematic. Multiphoton excitation was used to polymerize a focal volume containing individual ECM proteins (e.g., FN, Col1, and LN) and associated photocrosslinking agent. 3D printing of this type was used so that a three-dimensional construct, in this case a rectangular prism, could be generated even with ECM types that do not form hydrogels spontaneously ex vivo. (b) Geometric template (above; dimensions of micron scale) and associated bioprinted ECM-based rectangular prism containing BSA and LN (BSA/LN; below). Scale bar = 50 μ m. (c) Representative SEM images of prisms fabricated with BSA, FN, Col1, and LN. Scale bar = 10 μ m. (d) Average fractal dimension of each ECM-based, 3D bioprinted prism (above); volumetric swelling ratio of each ECM-based bioprinted prism (below). Error bars depict standard deviation (SD), ∗ P
Figure Legend Snippet: Fabrication of 3D ECM-based, bioprinted prisms. (a) Fabrication Schematic. Multiphoton excitation was used to polymerize a focal volume containing individual ECM proteins (e.g., FN, Col1, and LN) and associated photocrosslinking agent. 3D printing of this type was used so that a three-dimensional construct, in this case a rectangular prism, could be generated even with ECM types that do not form hydrogels spontaneously ex vivo. (b) Geometric template (above; dimensions of micron scale) and associated bioprinted ECM-based rectangular prism containing BSA and LN (BSA/LN; below). Scale bar = 50 μ m. (c) Representative SEM images of prisms fabricated with BSA, FN, Col1, and LN. Scale bar = 10 μ m. (d) Average fractal dimension of each ECM-based, 3D bioprinted prism (above); volumetric swelling ratio of each ECM-based bioprinted prism (below). Error bars depict standard deviation (SD), ∗ P

Techniques Used: Construct, Generated, Ex Vivo, Standard Deviation

63) Product Images from "Developmental Pathways Pervade Stem Cell Responses to Evolving Extracellular Matrices of 3D Bioprinted Microenvironments"

Article Title: Developmental Pathways Pervade Stem Cell Responses to Evolving Extracellular Matrices of 3D Bioprinted Microenvironments

Journal: Stem Cells International

doi: 10.1155/2018/4809673

GO analysis of hMSCs in 3D bioprinted prisms, especially related to the differentiation state. (a) Counts of GO terms associated with commonly examined stem cell responses. Protein degradation, development, and differentiation are the most significantly expressed pathways. (b) Development and differentiation pathways separated by 3D ECM structures. Most influential 3D structures rank from 3D/LN > 3D/Col1 > 3D/BSA > 3D/FN, with 3D/FN structures predominantly influencing vasculature development. Expression of endothelial cell markers (c), smooth muscle cell markers (d), and sphingosine phosphate lyase (e) in hMSCs cultured in 3D/FN structures. Error bars depict SD associated with n = 3 experimental replicates with at least 2 different prisms analyzed per experimental replicate. hMSCs cultured in 3D/FN structures expressed markers for both endothelial and smooth muscle cells and had a reduction in SGPL1 expression.
Figure Legend Snippet: GO analysis of hMSCs in 3D bioprinted prisms, especially related to the differentiation state. (a) Counts of GO terms associated with commonly examined stem cell responses. Protein degradation, development, and differentiation are the most significantly expressed pathways. (b) Development and differentiation pathways separated by 3D ECM structures. Most influential 3D structures rank from 3D/LN > 3D/Col1 > 3D/BSA > 3D/FN, with 3D/FN structures predominantly influencing vasculature development. Expression of endothelial cell markers (c), smooth muscle cell markers (d), and sphingosine phosphate lyase (e) in hMSCs cultured in 3D/FN structures. Error bars depict SD associated with n = 3 experimental replicates with at least 2 different prisms analyzed per experimental replicate. hMSCs cultured in 3D/FN structures expressed markers for both endothelial and smooth muscle cells and had a reduction in SGPL1 expression.

Techniques Used: Expressing, Cell Culture

RNA sequencing analysis of hMSCs in 3D bioprinted prisms at 0 and 28 days. (a) Hierarchical clustering of all genes with FPKM > 1 for 3D bioprinted prisms containing either BSA alone (3D/BSA), 3D/FN, 3D/Col1, or 3D/LN at day 28 and associated 2D control cultures at day 28. (b) qPCR validation of gene expression of a subset of genes. These genes were selected as they represent families of ECM proteins and differentiation markers associated with hMSC progeny. (c) PCA analysis of all genes with FPKM > 1. (d–f) Comparison of individual 3D bioprinted prisms containing ECM to bioprinted prisms with BSA only.
Figure Legend Snippet: RNA sequencing analysis of hMSCs in 3D bioprinted prisms at 0 and 28 days. (a) Hierarchical clustering of all genes with FPKM > 1 for 3D bioprinted prisms containing either BSA alone (3D/BSA), 3D/FN, 3D/Col1, or 3D/LN at day 28 and associated 2D control cultures at day 28. (b) qPCR validation of gene expression of a subset of genes. These genes were selected as they represent families of ECM proteins and differentiation markers associated with hMSC progeny. (c) PCA analysis of all genes with FPKM > 1. (d–f) Comparison of individual 3D bioprinted prisms containing ECM to bioprinted prisms with BSA only.

Techniques Used: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Expressing

Fabrication of 3D ECM-based, bioprinted prisms. (a) Fabrication Schematic. Multiphoton excitation was used to polymerize a focal volume containing individual ECM proteins (e.g., FN, Col1, and LN) and associated photocrosslinking agent. 3D printing of this type was used so that a three-dimensional construct, in this case a rectangular prism, could be generated even with ECM types that do not form hydrogels spontaneously ex vivo. (b) Geometric template (above; dimensions of micron scale) and associated bioprinted ECM-based rectangular prism containing BSA and LN (BSA/LN; below). Scale bar = 50 μ m. (c) Representative SEM images of prisms fabricated with BSA, FN, Col1, and LN. Scale bar = 10 μ m. (d) Average fractal dimension of each ECM-based, 3D bioprinted prism (above); volumetric swelling ratio of each ECM-based bioprinted prism (below). Error bars depict standard deviation (SD), ∗ P
Figure Legend Snippet: Fabrication of 3D ECM-based, bioprinted prisms. (a) Fabrication Schematic. Multiphoton excitation was used to polymerize a focal volume containing individual ECM proteins (e.g., FN, Col1, and LN) and associated photocrosslinking agent. 3D printing of this type was used so that a three-dimensional construct, in this case a rectangular prism, could be generated even with ECM types that do not form hydrogels spontaneously ex vivo. (b) Geometric template (above; dimensions of micron scale) and associated bioprinted ECM-based rectangular prism containing BSA and LN (BSA/LN; below). Scale bar = 50 μ m. (c) Representative SEM images of prisms fabricated with BSA, FN, Col1, and LN. Scale bar = 10 μ m. (d) Average fractal dimension of each ECM-based, 3D bioprinted prism (above); volumetric swelling ratio of each ECM-based bioprinted prism (below). Error bars depict standard deviation (SD), ∗ P

Techniques Used: Construct, Generated, Ex Vivo, Standard Deviation

64) Product Images from "Terahertz-infrared spectroscopy of Shewanella oneidensis MR-1 extracellular matrix"

Article Title: Terahertz-infrared spectroscopy of Shewanella oneidensis MR-1 extracellular matrix

Journal: Journal of Biological Physics

doi: 10.1007/s10867-018-9497-4

Room-temperature infrared spectra of imaginary part of dielectric permittivity of EMF, CytC, and BSA. The vertical dotted lines indicate the positions of intramolecular ν 1 , ν 2 , and ν 3 vibrations of free H 2 ]
Figure Legend Snippet: Room-temperature infrared spectra of imaginary part of dielectric permittivity of EMF, CytC, and BSA. The vertical dotted lines indicate the positions of intramolecular ν 1 , ν 2 , and ν 3 vibrations of free H 2 ]

Techniques Used:

Temperature dependence of imaginary part of dielectric permittivity of EMF, CytC, and BSA at a frequency 10 cm −1
Figure Legend Snippet: Temperature dependence of imaginary part of dielectric permittivity of EMF, CytC, and BSA at a frequency 10 cm −1

Techniques Used:

65) Product Images from "Effects of pentoxifylline on the histological and ultra-structural features of vitrified mouse ovarian tissue: An experimental study"

Article Title: Effects of pentoxifylline on the histological and ultra-structural features of vitrified mouse ovarian tissue: An experimental study

Journal: International Journal of Reproductive Biomedicine

doi:

Experimental design, phosphate buffer solution (PBS); bovine serum albumin (BSA); pentoxifylline (PTX).
Figure Legend Snippet: Experimental design, phosphate buffer solution (PBS); bovine serum albumin (BSA); pentoxifylline (PTX).

Techniques Used:

66) Product Images from "Understanding the Impact of Extracellular Polymeric Substances on Lead Release in Drinking Water Systems"

Article Title: Understanding the Impact of Extracellular Polymeric Substances on Lead Release in Drinking Water Systems

Journal: ACS Omega

doi: 10.1021/acsomega.8b02363

Representative chromatograms and UV–vis absorption spectra showing the complexation of metal ions and reduction of cytochrome c. Panel (A) shows the complexation of Pb by sEPS1 (alginate and BSA) on the anode (orange), the complexation of Fe by sEPS1 on the cathode (blue), and the complexation of Pb when Cyto. c (alginate, BSA, and cytochrome c) was on the anode (red). Panel (B) shows the contrast of UV–vis absorption spectra from cytochrome c before and after corrosion tests on the anode and cathode in which anaerobic or aerobic states the condition on the cathode side and those after the colon indicate the location of Cyto. c.
Figure Legend Snippet: Representative chromatograms and UV–vis absorption spectra showing the complexation of metal ions and reduction of cytochrome c. Panel (A) shows the complexation of Pb by sEPS1 (alginate and BSA) on the anode (orange), the complexation of Fe by sEPS1 on the cathode (blue), and the complexation of Pb when Cyto. c (alginate, BSA, and cytochrome c) was on the anode (red). Panel (B) shows the contrast of UV–vis absorption spectra from cytochrome c before and after corrosion tests on the anode and cathode in which anaerobic or aerobic states the condition on the cathode side and those after the colon indicate the location of Cyto. c.

Techniques Used:

67) Product Images from "Optimization of Islet Microencapsulation with Thin Polymer Membranes for Long-Term Stability"

Article Title: Optimization of Islet Microencapsulation with Thin Polymer Membranes for Long-Term Stability

Journal: Micromachines

doi: 10.3390/mi10110755

Microencapsulation of human erythrocytes with different 4-arm PEG-Mal. ( a ) Confocal microscopic images of microencapsulation of human erythrocytes with 4-arm PEG(10k, 20k, and 40k)-Mal. FITC-BSA-SH was used for the visualization of the polymer membrane. Scale bar: 20 µm. ( b ) Quantitative analysis of polymer membrane on human erythrocytes using flow cytometry at 0 and 44 days. Error bars indicate standard deviation; n = 3. * = p
Figure Legend Snippet: Microencapsulation of human erythrocytes with different 4-arm PEG-Mal. ( a ) Confocal microscopic images of microencapsulation of human erythrocytes with 4-arm PEG(10k, 20k, and 40k)-Mal. FITC-BSA-SH was used for the visualization of the polymer membrane. Scale bar: 20 µm. ( b ) Quantitative analysis of polymer membrane on human erythrocytes using flow cytometry at 0 and 44 days. Error bars indicate standard deviation; n = 3. * = p

Techniques Used: Flow Cytometry, Cytometry, Standard Deviation

Microencapsulation of beta-TC-6 cells. with different 4-arm PEG-Mal. ( a ) Confocal microscopic images of microencapsulation of beta-TC-6 cells with 4-arm PEG (10k, 20k, and 40k)-Mal. FITC-BSA-SH was used for the visualization of the polymer membrane. Scale bar: 20 µm. ( b ) Quantitative analysis of polymer membranes on beta-TC-6 cells using flow cytometry. Error bars indicate standard deviation; n = 3. ( c ) Glucose-responsive insulin secretion from microencapsulated and non-treated beta-TC-6 cells measured by ELISA. Error bars indicate standard deviation; n = 3. *** = p
Figure Legend Snippet: Microencapsulation of beta-TC-6 cells. with different 4-arm PEG-Mal. ( a ) Confocal microscopic images of microencapsulation of beta-TC-6 cells with 4-arm PEG (10k, 20k, and 40k)-Mal. FITC-BSA-SH was used for the visualization of the polymer membrane. Scale bar: 20 µm. ( b ) Quantitative analysis of polymer membranes on beta-TC-6 cells using flow cytometry. Error bars indicate standard deviation; n = 3. ( c ) Glucose-responsive insulin secretion from microencapsulated and non-treated beta-TC-6 cells measured by ELISA. Error bars indicate standard deviation; n = 3. *** = p

Techniques Used: Flow Cytometry, Cytometry, Standard Deviation, Enzyme-linked Immunosorbent Assay

68) Product Images from "Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions"

Article Title: Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.002425

Multimerization of NpDps1, NpDps2, and NpDps3 and E. coli Dps (EcDps) analyzed by nondenaturing PAGE. The theoretical molecular masses of putative dodecamers were 253 kDa for NpDps1, 233 kDa for NpDps2, 265 kDa for NpDps3, and 236 kDa for EcDps. Additionally, the different native forms of BSA were used to estimate the approximate molecular masses. The pH of the gel was 8.5 and the pH of the running buffer was 8.0. The gel was stained with colloidal Coomassie.
Figure Legend Snippet: Multimerization of NpDps1, NpDps2, and NpDps3 and E. coli Dps (EcDps) analyzed by nondenaturing PAGE. The theoretical molecular masses of putative dodecamers were 253 kDa for NpDps1, 233 kDa for NpDps2, 265 kDa for NpDps3, and 236 kDa for EcDps. Additionally, the different native forms of BSA were used to estimate the approximate molecular masses. The pH of the gel was 8.5 and the pH of the running buffer was 8.0. The gel was stained with colloidal Coomassie.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining

EMSA to analyze the DNA-binding properties of the NpDps proteins under different pH conditions. At pH 6.0 and 7.0, 125 ng of plasmid DNA (pSB1A3 vector) was incubated with 1 μg of each Dps protein, and separated on an agarose gel (1%). E. coli Dps (EcDps) and BSA served as a positive and negative control, respectively. Plasmid DNA is shown in lane DNA . Thiazole orange staining was performed after gel electrophoresis for DNA detection. The gel documentations are shown in inverted colors.
Figure Legend Snippet: EMSA to analyze the DNA-binding properties of the NpDps proteins under different pH conditions. At pH 6.0 and 7.0, 125 ng of plasmid DNA (pSB1A3 vector) was incubated with 1 μg of each Dps protein, and separated on an agarose gel (1%). E. coli Dps (EcDps) and BSA served as a positive and negative control, respectively. Plasmid DNA is shown in lane DNA . Thiazole orange staining was performed after gel electrophoresis for DNA detection. The gel documentations are shown in inverted colors.

Techniques Used: Binding Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Negative Control, Staining, Nucleic Acid Electrophoresis

69) Product Images from "Preformed CD40L Is Stored in Th1, Th2, Th17, and T Follicular Helper Cells as Well as CD4+8− Thymocytes and Invariant NKT Cells but Not in Treg Cells"

Article Title: Preformed CD40L Is Stored in Th1, Th2, Th17, and T Follicular Helper Cells as Well as CD4+8− Thymocytes and Invariant NKT Cells but Not in Treg Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0031296

CD4 SP thymocytes, but not Treg cells, store and mobilize pCD40L. A , Gating strategy for thymic nTreg cells and CD4 SP thymocytes. B , Generation of in vivo iTreg cells. DO11.10 CD4 + T cells were recovered from OVA- or BSA-fed recipient mice on day 6. C , Gating strategy for in vivo iTreg cells and effector CD4 + T cells. A BSA-fed mouse is shown as a negative control. D , thymic nTreg cells; E , CD4 SP thymocytes; F , in vivo iTreg cells; and G , effector CD4 + T cells, are analyzed by the mobilization assay and intracellular staining for pCD40L and CTLA-4. Data are representative of three ( D and E ) or two ( F and G ) independent experiments.
Figure Legend Snippet: CD4 SP thymocytes, but not Treg cells, store and mobilize pCD40L. A , Gating strategy for thymic nTreg cells and CD4 SP thymocytes. B , Generation of in vivo iTreg cells. DO11.10 CD4 + T cells were recovered from OVA- or BSA-fed recipient mice on day 6. C , Gating strategy for in vivo iTreg cells and effector CD4 + T cells. A BSA-fed mouse is shown as a negative control. D , thymic nTreg cells; E , CD4 SP thymocytes; F , in vivo iTreg cells; and G , effector CD4 + T cells, are analyzed by the mobilization assay and intracellular staining for pCD40L and CTLA-4. Data are representative of three ( D and E ) or two ( F and G ) independent experiments.

Techniques Used: In Vivo, Mouse Assay, Negative Control, Staining

70) Product Images from "Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display"

Article Title: Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms13066582

Recombinant protein expression and purification for use as biopanning bait. ( a ) Heated E. coli lysates of SMP1; ( b ) Heated fraction 15 GmPM28 recombinant protein; ( c ) Both SMP1 and Gm PM28 were recovered from lysed E. coli and the hexahistidyl tagged proteins purified on a nickel-charged column. SDS-PAGE (15%) separate the proteins in 10 μL of: (1) marker; (2) protein before filtration; (3) unbound proteins; (4) start of, and; (5) end of the column wash; (6–9) every third fraction; Lanes (10–16) fraction 11 through 17; lanes (17–19) fractions 20, 23 and 26; ( d ) ELISA assays showing the LEA proteins attach to the microtiter plate wells. Mean ± standard error at pH 7.5 depicted; ( e ) In solution G6PDH protection assays with LEA proteins, BSA, commercial blocking reagent or water; ( f ) G6PDH protection assays using various proteins or block bound to the microtiter plate well. Asterisks in d - f denotes significant differences among: ( d ) ELISA readings for SMP1 or GmPM28 and BSA/buffer; ( e , f ) percent activity retained relative to the block.
Figure Legend Snippet: Recombinant protein expression and purification for use as biopanning bait. ( a ) Heated E. coli lysates of SMP1; ( b ) Heated fraction 15 GmPM28 recombinant protein; ( c ) Both SMP1 and Gm PM28 were recovered from lysed E. coli and the hexahistidyl tagged proteins purified on a nickel-charged column. SDS-PAGE (15%) separate the proteins in 10 μL of: (1) marker; (2) protein before filtration; (3) unbound proteins; (4) start of, and; (5) end of the column wash; (6–9) every third fraction; Lanes (10–16) fraction 11 through 17; lanes (17–19) fractions 20, 23 and 26; ( d ) ELISA assays showing the LEA proteins attach to the microtiter plate wells. Mean ± standard error at pH 7.5 depicted; ( e ) In solution G6PDH protection assays with LEA proteins, BSA, commercial blocking reagent or water; ( f ) G6PDH protection assays using various proteins or block bound to the microtiter plate well. Asterisks in d - f denotes significant differences among: ( d ) ELISA readings for SMP1 or GmPM28 and BSA/buffer; ( e , f ) percent activity retained relative to the block.

Techniques Used: Recombinant, Expressing, Purification, SDS Page, Marker, Filtration, Enzyme-linked Immunosorbent Assay, Blocking Assay, Activity Assay

Phage titer (plaque forming units (PFU)·mL −1 ) at each of the four rounds of biopanning at 25 °C ( a and c ) and 41 °C ( b and d ) for seed library 1 (SL1; a and b ) and SL2 ( c and d ). Asterisks over bars for SMP1 or Gm PM28 denote significant differences between titers for BSA relative to either SMP1 or Gm PM28 in each biopanning round. The p -values for SMP1 and GmPM28, respectively for rounds 1–4 (R1–R4) are: ( a ) all less than 0.01; ( b ) R1 0.02 and 0.005, R2 0.006 and 0.006, R3 0.03 and 0.002 and, R4 0.007 and 0.049; ( c ) R1
Figure Legend Snippet: Phage titer (plaque forming units (PFU)·mL −1 ) at each of the four rounds of biopanning at 25 °C ( a and c ) and 41 °C ( b and d ) for seed library 1 (SL1; a and b ) and SL2 ( c and d ). Asterisks over bars for SMP1 or Gm PM28 denote significant differences between titers for BSA relative to either SMP1 or Gm PM28 in each biopanning round. The p -values for SMP1 and GmPM28, respectively for rounds 1–4 (R1–R4) are: ( a ) all less than 0.01; ( b ) R1 0.02 and 0.005, R2 0.006 and 0.006, R3 0.03 and 0.002 and, R4 0.007 and 0.049; ( c ) R1

Techniques Used:

71) Product Images from "p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes"

Article Title: p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201206006

HMGB1 depletion or overexpression induces senescence. (A) Lysates from IMR-90 cells infected with lentiviruses carrying no insert (Vector) or HMGB1 shRNAs (shRNA1, 2) were analyzed for HMGB1 and actin (control) by Western blotting. Shown is expression level relative to Vector. (B) Cells in A were immunostained for HMGB1 (red). Nuclei were stained with DAPI (blue). REP SEN cells are shown for comparison. Bar, 10 µM. (C) Cells in B were scored for SA-β-Gal, BrdU (24 h pulse), and SAHF. Error bars = SEM of two independent experiments. (D) Lysates from IMR-90 cells infected with lentivirus expressing no insert (Vector) or HMGB1 (OE) were analyzed for HMGB1 and actin (control) by Western blotting. Shown is expression level relative to Vector. (E) Cells in D were immunostained for HMGB1 (red). Nuclei were stained with DAPI (blue). REP SEN cells are shown for comparison. Bar, 10 µM. (F) Cells in E were scored for SA-β-Gal, BrdU (24 h pulse), and SAHFs. REP SEN cells are shown for comparison. Error bars = SEM of two independent experiments. (G) Lysates from PRE IMR-90 cells or cells induced to senesce by XRA (XRA), HMGB1-overexpression (OE), or HMGB1-depletion (KD) were analyzed for p53, p16 INK4a , and actin (control) by Western blotting. (H) CM from PRE or SEN (REP, XRA, and HMGB1 OE or KD) IMR-90 cells were analyzed for IL-6 by ELISA. Shown is a representative of two experiments. Error bars = SEM of duplicate determinations. (I) HCA2 cells expressing an NF-κB luciferase reporter were infected with lentiviruses carrying GFP shRNA (control), RelA shRNA, HMGB1 cDNA (OE), or HMGB1 shRNA (KD). 7 d after selection, shGFP and shRel A cells were given TNF for 24 h in serum-free media. Cells were lysed and luciferase activity measured (fold change over cells given BSA (control) protein). One-way ANOVA was used to analyze groups; P
Figure Legend Snippet: HMGB1 depletion or overexpression induces senescence. (A) Lysates from IMR-90 cells infected with lentiviruses carrying no insert (Vector) or HMGB1 shRNAs (shRNA1, 2) were analyzed for HMGB1 and actin (control) by Western blotting. Shown is expression level relative to Vector. (B) Cells in A were immunostained for HMGB1 (red). Nuclei were stained with DAPI (blue). REP SEN cells are shown for comparison. Bar, 10 µM. (C) Cells in B were scored for SA-β-Gal, BrdU (24 h pulse), and SAHF. Error bars = SEM of two independent experiments. (D) Lysates from IMR-90 cells infected with lentivirus expressing no insert (Vector) or HMGB1 (OE) were analyzed for HMGB1 and actin (control) by Western blotting. Shown is expression level relative to Vector. (E) Cells in D were immunostained for HMGB1 (red). Nuclei were stained with DAPI (blue). REP SEN cells are shown for comparison. Bar, 10 µM. (F) Cells in E were scored for SA-β-Gal, BrdU (24 h pulse), and SAHFs. REP SEN cells are shown for comparison. Error bars = SEM of two independent experiments. (G) Lysates from PRE IMR-90 cells or cells induced to senesce by XRA (XRA), HMGB1-overexpression (OE), or HMGB1-depletion (KD) were analyzed for p53, p16 INK4a , and actin (control) by Western blotting. (H) CM from PRE or SEN (REP, XRA, and HMGB1 OE or KD) IMR-90 cells were analyzed for IL-6 by ELISA. Shown is a representative of two experiments. Error bars = SEM of duplicate determinations. (I) HCA2 cells expressing an NF-κB luciferase reporter were infected with lentiviruses carrying GFP shRNA (control), RelA shRNA, HMGB1 cDNA (OE), or HMGB1 shRNA (KD). 7 d after selection, shGFP and shRel A cells were given TNF for 24 h in serum-free media. Cells were lysed and luciferase activity measured (fold change over cells given BSA (control) protein). One-way ANOVA was used to analyze groups; P

Techniques Used: Over Expression, Infection, Plasmid Preparation, Western Blot, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Luciferase, shRNA, Selection, Activity Assay

72) Product Images from "Lysosomal trafficking functions of mucolipin-1 in murine macrophages"

Article Title: Lysosomal trafficking functions of mucolipin-1 in murine macrophages

Journal: BMC Cell Biology

doi: 10.1186/1471-2121-8-54

MCOLN1 RNAi clones . A) Northern blot done on 15 μg of total RNA isolated from RAW264.7, LS9, or LS10 cells. The same filter was probed for MCOLN1 , stripped, and re-probed for GAPDH , stripped again and re-probed for MCOLN2 . B) Confocal images of RAW264.7, LS9, and LS10 cells stained with Acridine Orange (AO). Bar is 6 μm. C) Quantitation of the intensity of staining of AO-stained compartments and of dextran-Oregon Green (OG)-stained compartments. Bars represent standard deviations. D) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with Dextran-Cascade Blue (DEX-CB, red). BSA-Bodipy LacCer (B-LACCER, green) was added for 30 minutes and chased for the indicated times before fixation. Arrows indicate co-localization of the two markers. Bar is 5 μm.
Figure Legend Snippet: MCOLN1 RNAi clones . A) Northern blot done on 15 μg of total RNA isolated from RAW264.7, LS9, or LS10 cells. The same filter was probed for MCOLN1 , stripped, and re-probed for GAPDH , stripped again and re-probed for MCOLN2 . B) Confocal images of RAW264.7, LS9, and LS10 cells stained with Acridine Orange (AO). Bar is 6 μm. C) Quantitation of the intensity of staining of AO-stained compartments and of dextran-Oregon Green (OG)-stained compartments. Bars represent standard deviations. D) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with Dextran-Cascade Blue (DEX-CB, red). BSA-Bodipy LacCer (B-LACCER, green) was added for 30 minutes and chased for the indicated times before fixation. Arrows indicate co-localization of the two markers. Bar is 5 μm.

Techniques Used: Northern Blot, Isolation, Staining, Quantitation Assay

Trafficking and endocytosis defect of proteins in MCOLN1 RNAi clones . A) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with BSA-AlexaFluor 594 (BSA-AF 594, red). BSA-AlexaFluor 488 (BSA-AF 488, green) was added for 10 minutes to the cells and the cells were chased for the indicated times before fixation. Bar is 5 μm. B) Western blots of HEL that was endocytosed for 5 minutes and chased for the indicated times. C) Quantitation of the HEL that remains in cells relative to the 0 time point. Bars represent standard deviations from two independent assays.
Figure Legend Snippet: Trafficking and endocytosis defect of proteins in MCOLN1 RNAi clones . A) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with BSA-AlexaFluor 594 (BSA-AF 594, red). BSA-AlexaFluor 488 (BSA-AF 488, green) was added for 10 minutes to the cells and the cells were chased for the indicated times before fixation. Bar is 5 μm. B) Western blots of HEL that was endocytosed for 5 minutes and chased for the indicated times. C) Quantitation of the HEL that remains in cells relative to the 0 time point. Bars represent standard deviations from two independent assays.

Techniques Used: Western Blot, Quantitation Assay

73) Product Images from "P21-activated kinase 1: convergence point in PDGF- and LPA-stimulated collagen matrix contraction by human fibroblasts"

Article Title: P21-activated kinase 1: convergence point in PDGF- and LPA-stimulated collagen matrix contraction by human fibroblasts

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200505175

Cofilin1 is downstream of PAK1 in LPA and PDGF regulation of collagen matrix contraction. (A) PAK1-silenced and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for the times shown in DME containing 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA, as indicated. At the end of the incubations, extracts of the samples were prepared and subjected to immunoblotting with antibodies directed against phospho-cofilin1 and tubulin. (B) Cells were transfected for 36 h with 500 nM of cofilin1 siRNA or sense RNA only (Mock) and then cultured an additional 24 h in growth medium without siRNA. Extracts were prepared and subjected to SDS-PAGE and immunoblotted to analyze levels of cofilin1 (arrow) and tubulin. (C) Nontransfected (CTL), cofilin1-silenced, and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA added as shown. At the end of the incuba-tions, samples were fixed and the extent of matrix contraction was meas-ured as the decrease in matrix diameter. Data shown are arithmetic means ± SD for three separate experiments.
Figure Legend Snippet: Cofilin1 is downstream of PAK1 in LPA and PDGF regulation of collagen matrix contraction. (A) PAK1-silenced and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for the times shown in DME containing 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA, as indicated. At the end of the incubations, extracts of the samples were prepared and subjected to immunoblotting with antibodies directed against phospho-cofilin1 and tubulin. (B) Cells were transfected for 36 h with 500 nM of cofilin1 siRNA or sense RNA only (Mock) and then cultured an additional 24 h in growth medium without siRNA. Extracts were prepared and subjected to SDS-PAGE and immunoblotted to analyze levels of cofilin1 (arrow) and tubulin. (C) Nontransfected (CTL), cofilin1-silenced, and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA added as shown. At the end of the incuba-tions, samples were fixed and the extent of matrix contraction was meas-ured as the decrease in matrix diameter. Data shown are arithmetic means ± SD for three separate experiments.

Techniques Used: Transfection, Incubation, Cell Culture, SDS Page, CTL Assay

mDia1 cooperates with PAK1 in LPA regulation of collagen matrix contraction silencing in human fibroblasts. (A) Cells were transfected for 12 h with 700 nM siRNA or sense RNA only (Mock) and cultured an additional 24 h in growth medium without siRNA. Extracts were prepared and subjected to SDS-PAGE and immunoblotted to analyze levels of mDia1 and actin. (B) mDia1-silenced and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA added as shown. At the end of the incubations, samples were fixed, and the extent of matrix contraction was measured as the decrease in matrix diameter. Data shown are arithmetic means ± SD for three separate experiments. (C) At the end of the transfection period, mock- and siRNA-transfected cells were incubated in serum-free medium for 36 h, followed by 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA added as shown. Stable microtubules were detected as previously described ( Gundersen et al., 1994 ; Cook et al., 1998 ). Subsequently, the indicated samples were treated with 2 μM nocodazole for 2 h . After two rinses with microtubule-stabilizing buffer (MSB; 85 mM Pipes, pH 6.9, 1 mM EGTA, 1 mM MgCl 2 , 2 M glycerol, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride), samples were treated with 1 ml of MSB containing 200 μg/ml saponin for 5 min at 37°C to extract tubulin monomer, rinsed with MSB, and fixed with methanol (−20°C) for 10 min, and then stained with anti-tubulin antibody. Bar, 50 μm.
Figure Legend Snippet: mDia1 cooperates with PAK1 in LPA regulation of collagen matrix contraction silencing in human fibroblasts. (A) Cells were transfected for 12 h with 700 nM siRNA or sense RNA only (Mock) and cultured an additional 24 h in growth medium without siRNA. Extracts were prepared and subjected to SDS-PAGE and immunoblotted to analyze levels of mDia1 and actin. (B) mDia1-silenced and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA added as shown. At the end of the incubations, samples were fixed, and the extent of matrix contraction was measured as the decrease in matrix diameter. Data shown are arithmetic means ± SD for three separate experiments. (C) At the end of the transfection period, mock- and siRNA-transfected cells were incubated in serum-free medium for 36 h, followed by 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA added as shown. Stable microtubules were detected as previously described ( Gundersen et al., 1994 ; Cook et al., 1998 ). Subsequently, the indicated samples were treated with 2 μM nocodazole for 2 h . After two rinses with microtubule-stabilizing buffer (MSB; 85 mM Pipes, pH 6.9, 1 mM EGTA, 1 mM MgCl 2 , 2 M glycerol, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride), samples were treated with 1 ml of MSB containing 200 μg/ml saponin for 5 min at 37°C to extract tubulin monomer, rinsed with MSB, and fixed with methanol (−20°C) for 10 min, and then stained with anti-tubulin antibody. Bar, 50 μm.

Techniques Used: Transfection, Cell Culture, SDS Page, Incubation, Staining

Pertussis toxin inhibits LPA-stimulated, but not PDGF-stimulated, collagen matrix contraction. (A) Fibroblasts in monolayer culture were incubated overnight with 25 ng/ml pertussis toxin. Subsequently, the cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA added where indicated. At the end the incubations, samples were fixed and the extent of matrix contraction was measured as the decrease in matrix diameter. Data shown are arithmetic means ± SD for three separate experiments. (B) Selected samples from A were fixed and stained with anti-tubulin antibody for microtubules (green) and rhodamine-phalloidin for actin (red). (right) Enlarged view of dendritic extension tips in the boxed areas. Bars, 20 μm.
Figure Legend Snippet: Pertussis toxin inhibits LPA-stimulated, but not PDGF-stimulated, collagen matrix contraction. (A) Fibroblasts in monolayer culture were incubated overnight with 25 ng/ml pertussis toxin. Subsequently, the cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA added where indicated. At the end the incubations, samples were fixed and the extent of matrix contraction was measured as the decrease in matrix diameter. Data shown are arithmetic means ± SD for three separate experiments. (B) Selected samples from A were fixed and stained with anti-tubulin antibody for microtubules (green) and rhodamine-phalloidin for actin (red). (right) Enlarged view of dendritic extension tips in the boxed areas. Bars, 20 μm.

Techniques Used: Incubation, Staining

Inhibition of collagen matrix contraction in PAK1-silenced cells. Nontransfected (Control), PAK1-silenced, and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA added as shown. At the end of the incubations samples were fixed and the extent of matrix contraction was measured as the decrease in matrix diameter. Data shown are arithmetic mean ± SD for three separate experiments.
Figure Legend Snippet: Inhibition of collagen matrix contraction in PAK1-silenced cells. Nontransfected (Control), PAK1-silenced, and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA added as shown. At the end of the incubations samples were fixed and the extent of matrix contraction was measured as the decrease in matrix diameter. Data shown are arithmetic mean ± SD for three separate experiments.

Techniques Used: Inhibition, Transfection, Incubation

Dendritic extensions and ruffling by PAK1-silenced human fibroblasts incubated for 1 h in collagen matrices. (A) PAK1-silenced and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 1 h in DME containing 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA, as indicated. At the end of the incubations, samples were fixed and stained with anti-tubulin antibody for microtubules and rhodamine-phalloidin for actin. (B) Enlarged view of dendritic extension tips in the boxed areas. Green, microtubules; red, actin. Bar, 20 μm.
Figure Legend Snippet: Dendritic extensions and ruffling by PAK1-silenced human fibroblasts incubated for 1 h in collagen matrices. (A) PAK1-silenced and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 1 h in DME containing 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA, as indicated. At the end of the incubations, samples were fixed and stained with anti-tubulin antibody for microtubules and rhodamine-phalloidin for actin. (B) Enlarged view of dendritic extension tips in the boxed areas. Green, microtubules; red, actin. Bar, 20 μm.

Techniques Used: Incubation, Transfection, Staining

Dendritic extensions and ruffling by PAK1-silenced fibroblasts incubated for 4 h in collagen matrices. (A) PAK1-silenced and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME containing 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA, as indicated. At the end of the incubations, samples were fixed and stained with anti-tubulin antibody for microtubules and rhodamine-phalloidin for actin. (B) Enlarged view of dendritic extension tips in the boxed areas. Green, microtubules; red, actin. Bar, 20 μm.
Figure Legend Snippet: Dendritic extensions and ruffling by PAK1-silenced fibroblasts incubated for 4 h in collagen matrices. (A) PAK1-silenced and mock-transfected cells were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME containing 5 mg/ml BSA and 50 ng/ml PDGF or 10 μM LPA, as indicated. At the end of the incubations, samples were fixed and stained with anti-tubulin antibody for microtubules and rhodamine-phalloidin for actin. (B) Enlarged view of dendritic extension tips in the boxed areas. Green, microtubules; red, actin. Bar, 20 μm.

Techniques Used: Incubation, Transfection, Staining

PAK1 silencing in human fibroblasts and cell morphology. (A) Cells were transfected for 12 h with 700 nM siRNA or sense RNA only (Mock) and cultured for an additional 24 h in growth medium without siRNA. Extracts were prepared and subjected to SDS-PAGE and immunoblotted to analyze levels of PAK1, PAK2, and tubulin. (B) PAK1-silenced and mock-transfected cells were harvested and incubated for 1 h on collagencoated glass coverslips in DME containing 5 mg/ml BSA and 10 μM LPA or 50 ng/ml PDGF as indicated. At the end of the incubation, samples were fixed and stained for actin. Bar, 50 μm.
Figure Legend Snippet: PAK1 silencing in human fibroblasts and cell morphology. (A) Cells were transfected for 12 h with 700 nM siRNA or sense RNA only (Mock) and cultured for an additional 24 h in growth medium without siRNA. Extracts were prepared and subjected to SDS-PAGE and immunoblotted to analyze levels of PAK1, PAK2, and tubulin. (B) PAK1-silenced and mock-transfected cells were harvested and incubated for 1 h on collagencoated glass coverslips in DME containing 5 mg/ml BSA and 10 μM LPA or 50 ng/ml PDGF as indicated. At the end of the incubation, samples were fixed and stained for actin. Bar, 50 μm.

Techniques Used: Transfection, Cell Culture, SDS Page, Incubation, Staining

PI3 kinase and Rho kinase are required for PDGF-stimulated, but not LPA-stimulated, collagen matrix contraction. (A) Fibroblasts were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF, 10 μM LPA, 20 μM LY294002 (LY; PI3 kinase inhibitor), and 10 μM Y27634 (Y; Rho kinase inhibitor) added where indicated. At the end the incubations, samples were fixed and the extent of matrix contraction was measured as the decrease in matrix diameter. Data shown are arithmetic means ± SD for three separate experiments. (B) Selected samples from A were fixed and stained with anti-tubulin antibody for microtubules (green) and rhodaminephalloidin for actin (red). Enlarged view of dendritic extension tips in the boxed areas. Bar, 20 μm.
Figure Legend Snippet: PI3 kinase and Rho kinase are required for PDGF-stimulated, but not LPA-stimulated, collagen matrix contraction. (A) Fibroblasts were harvested and used to prepare floating collagen matrices. Samples were incubated for 4 h in DME with 5 mg/ml BSA and 50 ng/ml PDGF, 10 μM LPA, 20 μM LY294002 (LY; PI3 kinase inhibitor), and 10 μM Y27634 (Y; Rho kinase inhibitor) added where indicated. At the end the incubations, samples were fixed and the extent of matrix contraction was measured as the decrease in matrix diameter. Data shown are arithmetic means ± SD for three separate experiments. (B) Selected samples from A were fixed and stained with anti-tubulin antibody for microtubules (green) and rhodaminephalloidin for actin (red). Enlarged view of dendritic extension tips in the boxed areas. Bar, 20 μm.

Techniques Used: Incubation, Staining

74) Product Images from "Muscle cells challenged with saturated fatty acids mount an autonomous inflammatory response that activates macrophages"

Article Title: Muscle cells challenged with saturated fatty acids mount an autonomous inflammatory response that activates macrophages

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/1478-811X-10-30

Palmitate activates ROS and signalling pathways in muscle cells. L6 muscle cells were treated for 24h with 0.2 mM palmitate (PA), palmitoleate (PO) or BSA control. A ) Reactive oxygen species were analyzed using DCFDA, normalized to the protein content and expressed as percent of the BSA control. Results are mean ±SEM (n=4) analyzed using 1-way ANOVA, **p
Figure Legend Snippet: Palmitate activates ROS and signalling pathways in muscle cells. L6 muscle cells were treated for 24h with 0.2 mM palmitate (PA), palmitoleate (PO) or BSA control. A ) Reactive oxygen species were analyzed using DCFDA, normalized to the protein content and expressed as percent of the BSA control. Results are mean ±SEM (n=4) analyzed using 1-way ANOVA, **p

Techniques Used:

Palmitate treatment does not induce insulin-resistance in muscle cells. A-B ) The direct effect of PA was measured by treating L6 cells with 0.2 mM palmitate (PA), palmitoleate (PO) or BSA control for 24 h, followed by 3 h serum starvation and insulin stimulation (20 min, 100 nM) in the absence of PA. C - D ) The indirect effect of palmitate through a possible autocrine effect was tested by generating conditioned media from muscle and applying it on naive untreated muscle cells. Surface GLUT4 myc and glucose uptake were measured as described in Methods. Results are normalized to the CM-BSA control and expressed as mean ± SEM (n=4), **p
Figure Legend Snippet: Palmitate treatment does not induce insulin-resistance in muscle cells. A-B ) The direct effect of PA was measured by treating L6 cells with 0.2 mM palmitate (PA), palmitoleate (PO) or BSA control for 24 h, followed by 3 h serum starvation and insulin stimulation (20 min, 100 nM) in the absence of PA. C - D ) The indirect effect of palmitate through a possible autocrine effect was tested by generating conditioned media from muscle and applying it on naive untreated muscle cells. Surface GLUT4 myc and glucose uptake were measured as described in Methods. Results are normalized to the CM-BSA control and expressed as mean ± SEM (n=4), **p

Techniques Used:

Low level palmitate treatment activates inflammatory cytokine chemokine gene expression in muscle cells. L6 muscle cells were treated with 0.2 mM palmitate (PA), palmitoleate (PO) or BSA control for (A) 24 h or (B) at the times indicated. A ) mRNA expression was analyzed by qRT-PCR using primers for TNFα IL-6 and CCL2. B ) mRNA expression was analyzed by qRT-PCR using primers for IL-6. Results are normalized to the reference gene hprt1 and then to the BSA control and reported as fold change ±SEM (n≥3), **p
Figure Legend Snippet: Low level palmitate treatment activates inflammatory cytokine chemokine gene expression in muscle cells. L6 muscle cells were treated with 0.2 mM palmitate (PA), palmitoleate (PO) or BSA control for (A) 24 h or (B) at the times indicated. A ) mRNA expression was analyzed by qRT-PCR using primers for TNFα IL-6 and CCL2. B ) mRNA expression was analyzed by qRT-PCR using primers for IL-6. Results are normalized to the reference gene hprt1 and then to the BSA control and reported as fold change ±SEM (n≥3), **p

Techniques Used: Expressing, Quantitative RT-PCR

75) Product Images from "Recombinant Major Vault Protein Is Targeted to Neuritic Tips of PC12 Cells "

Article Title: Recombinant Major Vault Protein Is Targeted to Neuritic Tips of PC12 Cells

Journal: The Journal of Cell Biology

doi:

Recombinant MVP is assembled into vault particles: subcellular fractionation (A) and immunoprecipitation of VSVG-tagged major vault protein (B and C). (A) Western blot analysis of the sedimentation behavior of vaults derived from CHO cells on glycerol gradients is shown. The following are shown: (top) Immunodetection of endogenous and recombinant MVP (MVP*) of pvMVP-transfected CHO cells using the anti–rat vault antibody; (middle) immunodetection of vMVP using the anti-VSVG antibody. In the top and middle frames identical fractions were applied per lane. The bottom frame shows immunodetection of endogenous MVP derived from nontransfected cells using the anti–rat vault antibody. Note that both the amount of material loaded per lane as well as the exposure time was doubled in the bottom frame blot as compared to the top two (50:100 μg protein per gradient, 2:4 min exposure time). Fractions of 300 μl were collected from the gradient and analyzed by immunoblotting. The number of fraction is indicated below the graph: starting fraction on top of the gradient (1) vault-enriched fraction (8) and bottom fraction (15). (B) Western blot and (C) silver staining of immunoprecipitate using anti-VSVG mAb and anti–mouse IgG-coated magnetic beads as a solid carrier (lane 1). Bands comprising minor vault proteins are not visible. Parent fraction of transfected cells before immunoisolation (lane 2). Immunoprecipitate of nontransfected CHO cells applying an identical experimental procedure as for transfected cells (lane 3). Parent fraction of nontransfected cells before immunoisolation (lane 4). Positions of vMVP, BSA, and heavy chains of mouse immunoglobulins (IgG HC ) are indicated.
Figure Legend Snippet: Recombinant MVP is assembled into vault particles: subcellular fractionation (A) and immunoprecipitation of VSVG-tagged major vault protein (B and C). (A) Western blot analysis of the sedimentation behavior of vaults derived from CHO cells on glycerol gradients is shown. The following are shown: (top) Immunodetection of endogenous and recombinant MVP (MVP*) of pvMVP-transfected CHO cells using the anti–rat vault antibody; (middle) immunodetection of vMVP using the anti-VSVG antibody. In the top and middle frames identical fractions were applied per lane. The bottom frame shows immunodetection of endogenous MVP derived from nontransfected cells using the anti–rat vault antibody. Note that both the amount of material loaded per lane as well as the exposure time was doubled in the bottom frame blot as compared to the top two (50:100 μg protein per gradient, 2:4 min exposure time). Fractions of 300 μl were collected from the gradient and analyzed by immunoblotting. The number of fraction is indicated below the graph: starting fraction on top of the gradient (1) vault-enriched fraction (8) and bottom fraction (15). (B) Western blot and (C) silver staining of immunoprecipitate using anti-VSVG mAb and anti–mouse IgG-coated magnetic beads as a solid carrier (lane 1). Bands comprising minor vault proteins are not visible. Parent fraction of transfected cells before immunoisolation (lane 2). Immunoprecipitate of nontransfected CHO cells applying an identical experimental procedure as for transfected cells (lane 3). Parent fraction of nontransfected cells before immunoisolation (lane 4). Positions of vMVP, BSA, and heavy chains of mouse immunoglobulins (IgG HC ) are indicated.

Techniques Used: Recombinant, Fractionation, Immunoprecipitation, Western Blot, Sedimentation, Derivative Assay, Immunodetection, Transfection, Silver Staining, Magnetic Beads

Related Articles

Synthesized:

Article Title: Tailoring sub-micron PLGA particle release profiles via centrifugal fractioning
Article Snippet: Sub-micron PLGA particles were synthesized via a W/O/W emulsion technique adopted from a method further described by McCall et al. .. The first emulsion was generated by vortexing the organic phase with a phosphate-buffered saline (PBS) solution containing 20 mg/ mL BSA (total protein content of 2.0% w/w of PLGA; Sigma-Aldrich, St. Louis, MO).

BIA-KA:

Article Title: Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture
Article Snippet: Paragraph title: Bicinchoninic Acid (BCA) Protein Quantification ... Known concentrations of bovine serum albumin (BSA, Sigma-Aldrich, 1076192 USP) were used as standard curve.

Blocking Assay:

Article Title: Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display
Article Snippet: .. Additionally, equimolar concentrations of BSA, a protein known to protect G6PDH activity [ ] or the commercial blocking reagent (EMD Chemicals Inc., Gibbstown, NJ, USA), used to coat the wells prior to panning, were tested for their capacities to protect G6PDH. .. Three units of G6PDH (144 pmol) or the same volume of water (neg. control) were added to 500 μL 10 mM Tris-HCl, pH 7.5 which did or did not contain (10 μg·mL−1 ) recombinant SMP1, Gm PM28, the molar equivalent to SMP1 of commercial BSA, or commercial blocking reagent (EMD Chemicals Inc.) (all 530 pmol, 3.6 molar excess of G6PDH).

Enzyme-linked Immunosorbent Assay:

Article Title: Inherent Anti-amyloidogenic Activity of Human Immunoglobulin ? Heavy Chains *
Article Snippet: HC and antibody reactivity with amyloid fibrils, CAPS, and elastin fibrils was determined at 37 °C using a europium (Eu3+ )-based fluoroimmunoassay (EuLISA) ( ) or ELISA. .. Antibodies or HCs were serially diluted in assay buffer (1% BSA in PBSA) and tested (100 μl/well) in activated, high binding microtiter plate wells (COSTAR, Corning, NY) that were coated with 400 ng of target protein and blocked with 1% BSA (Sigma-Aldrich) in PBSA.

Incubation:

Article Title: The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling
Article Snippet: Proteins were transferred to nitrocellulose membranes which were then blocked in 5% skim milk (Invitrogen) or 5% BSA (Sigma, for phosphorylated proteins). .. The membranes were incubated with primary antibody overnight followed by horseradish peroxidase‐conjugated secondary antibodies for 1 h at room temperature.

Article Title: Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells
Article Snippet: .. Briefly, approximately 1 × 109 carboxylate-modified, fluorescent latex beads (1 μm in diameter, Yellow Green fluorescent Fluo-Spheres, Molecular Probes) were incubated with 300 μg of purified protein or with 1 mg of BSA (Sigma) in a MES buffer at pH 6.0 at room temperature for 15 min. EDAC [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, hydrochloride] (Molecular Probes) was added to a final concentration of 1.6 mg ml-1 to activate surface carboxyl groups and the pH was adjusted to 6.5 with 0.2 M NaOH. ..

Article Title: Decellularization and Solubilization of Porcine Liver for Use as a Substrate for Porcine Hepatocyte Culture
Article Snippet: Extracted protein (25 µL) was mixed with 200 µL BCA working reagent and incubated at 37 °C for 30 min. Absorbance was measured at 562 nm using a Synergy HTX Multi-Mode Reader (Biotek). .. Known concentrations of bovine serum albumin (BSA, Sigma-Aldrich, 1076192 USP) were used as standard curve.

Activity Assay:

Article Title: Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display
Article Snippet: .. Additionally, equimolar concentrations of BSA, a protein known to protect G6PDH activity [ ] or the commercial blocking reagent (EMD Chemicals Inc., Gibbstown, NJ, USA), used to coat the wells prior to panning, were tested for their capacities to protect G6PDH. .. Three units of G6PDH (144 pmol) or the same volume of water (neg. control) were added to 500 μL 10 mM Tris-HCl, pH 7.5 which did or did not contain (10 μg·mL−1 ) recombinant SMP1, Gm PM28, the molar equivalent to SMP1 of commercial BSA, or commercial blocking reagent (EMD Chemicals Inc.) (all 530 pmol, 3.6 molar excess of G6PDH).

Bradford Assay:

Article Title: Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins
Article Snippet: .. The protein concentration of TSP extracts was determined by Bradford assay using the Bio-Rad Protein Assay and BSA (Sigma-Aldrich) as a standard. .. For analysis by 12% SDS/PAGE and Coomassie staining using PageBlue Protein Staining Solution (Fermentas), protein extracts were denatured at 95 °C for 5 min before loading.

Western Blot:

Article Title: The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling
Article Snippet: Paragraph title: Protein extraction and western blot analysis ... Proteins were transferred to nitrocellulose membranes which were then blocked in 5% skim milk (Invitrogen) or 5% BSA (Sigma, for phosphorylated proteins).

Flow Cytometry:

Article Title: The R882H DNMT3A Mutation Associated with AML Dominantly Inhibits WT DNMT3A by Blocking its Ability to Form Active Tetramers
Article Snippet: The flow rate was kept constant at 0.5 mL/min using a running buffer of 25 mM Tris-HCl, 150 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA pH 7.2. .. To generate a standard molecular weight curve, 100 μg of each of five proteins were used (urease, BSA, chicken egg albumin, carbonic anhydrase, and α-lactalbumin; Sigma MWND500).

Cell Culture:

Article Title: Interleukin-22 Promotes Intestinal Stem Cell-Mediated Epithelial Regeneration
Article Snippet: .. For mouse large intestine, crypts were cultured in “WENR” medium containing 50% of Wnt3a-conditioned medium in addition to the aforementioned proteins and 1% BSA (Sigma) and supplemented with SB202190 (10uM, Sigma), ALK5 inhibitor A83-01(500nM, Tocris Bioscience) and Nicotinamide (10mM, Sigma). .. Along with medium changes, treatment wells received different concentrations of recombinant murine (rm)IL-22 (Genscript).

Article Title: Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment
Article Snippet: Adhesion assay The leukemia cells treated with and without 1 μg/mL PMA were cultured in flasks in advance for 48 hours. .. The cells were then counted with a hemocytometer to a concentration of 1×106 cells/mL, and 100 μL of the culture medium containing the cells was added to each well of the 96-well microplates, which were precoated with 4 μg/mL BSA proteins, fibronectin, collagen I, and Anti-E-cadherin (Sigma-Aldrich, St Louis, MO, USA).

Generated:

Article Title: Tailoring sub-micron PLGA particle release profiles via centrifugal fractioning
Article Snippet: .. The first emulsion was generated by vortexing the organic phase with a phosphate-buffered saline (PBS) solution containing 20 mg/ mL BSA (total protein content of 2.0% w/w of PLGA; Sigma-Aldrich, St. Louis, MO). .. The above mixture was added dropwise to a 3.6× volume excess of an aqueous solution containing 2% (w/v) d-α tocopheryl polyethylene glycol 1000 succinate (TPGS; Sigma-Aldrich) under heavy vortex.

Protein Concentration:

Article Title: Snake Venom Hemotoxic Enzymes: Biochemical Comparison between Crotalus Species from Central Mexico
Article Snippet: .. For all the analyses, the venom was dissolved in ddH2 O and protein concentration in venom was determined [ ] using BSA as a standard (Sigma-Aldrich, St. Louis, MO, USA). .. SDS-PAGE Protein Banding Pattern Twenty microgram of lyophilized venom protein was loaded into a 10 or 12% SDS-PAGE gel and electrophoresed in a Mini Protean II unit (Bio-Rad Hercules, CA, USA) or Hoefer MiniVE system (Hoefer, Holliston, MA, USA) under reducing (4% of β-mercaptoethanol and 5 min at boiling water temperature) and non-reducing conditions [ ].

Article Title: Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins
Article Snippet: .. The protein concentration of TSP extracts was determined by Bradford assay using the Bio-Rad Protein Assay and BSA (Sigma-Aldrich) as a standard. .. For analysis by 12% SDS/PAGE and Coomassie staining using PageBlue Protein Staining Solution (Fermentas), protein extracts were denatured at 95 °C for 5 min before loading.

Sonication:

Article Title: The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling
Article Snippet: Protein extraction and western blot analysis RPE‐choroid preparations from freshly dissected mice were sonicated in RIPA lysis buffer (Millipore, Billerica, MA, 20‐188) plus 1% protease and phosphatase inhibitors (Sigma). .. Proteins were transferred to nitrocellulose membranes which were then blocked in 5% skim milk (Invitrogen) or 5% BSA (Sigma, for phosphorylated proteins).

Recombinant:

Article Title: Interleukin-22 Promotes Intestinal Stem Cell-Mediated Epithelial Regeneration
Article Snippet: For mouse large intestine, crypts were cultured in “WENR” medium containing 50% of Wnt3a-conditioned medium in addition to the aforementioned proteins and 1% BSA (Sigma) and supplemented with SB202190 (10uM, Sigma), ALK5 inhibitor A83-01(500nM, Tocris Bioscience) and Nicotinamide (10mM, Sigma). .. Along with medium changes, treatment wells received different concentrations of recombinant murine (rm)IL-22 (Genscript).

Article Title: Preformed CD40L Is Stored in Th1, Th2, Th17, and T Follicular Helper Cells as Well as CD4+8− Thymocytes and Invariant NKT Cells but Not in Treg Cells
Article Snippet: Recombinant cytokines were purchased from Peprotech (Rocky Hill, NJ). .. All-trans retinoic acid, BSA, OVA protein, PMA and ionomycin were from Sigma-Aldrich (St Louis, MO).

Article Title: Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display
Article Snippet: GLUCOSE 6 PHOSPHATE DEHYDROGENASE (G6PDH) Protection Assays Recombinant LEA protein was tested for biological activity by co-incubation with the labile enzyme GLUCOSE 6 PHOSPHATE DEHYDROGENASE (G6PDH; EC 1.1.1.49). .. Additionally, equimolar concentrations of BSA, a protein known to protect G6PDH activity [ ] or the commercial blocking reagent (EMD Chemicals Inc., Gibbstown, NJ, USA), used to coat the wells prior to panning, were tested for their capacities to protect G6PDH.

Article Title: Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins
Article Snippet: The protein concentration of TSP extracts was determined by Bradford assay using the Bio-Rad Protein Assay and BSA (Sigma-Aldrich) as a standard. .. The estimation of the percentage of recombinant colicins of TSP was done by comparison of TSP extracts with known amounts of BSA (Sigma-Aldrich) on Coomassie-stained SDS/PAGE gels.

Molecular Weight:

Article Title: The R882H DNMT3A Mutation Associated with AML Dominantly Inhibits WT DNMT3A by Blocking its Ability to Form Active Tetramers
Article Snippet: .. To generate a standard molecular weight curve, 100 μg of each of five proteins were used (urease, BSA, chicken egg albumin, carbonic anhydrase, and α-lactalbumin; Sigma MWND500). .. Molecular weights of DNMT3A complexes were calculated by non-linear regression based on Kav values determined from elution values, the column void volume, and the total column volume (GraphPad Prism 6.0).

MTT Assay:

Article Title: Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment
Article Snippet: The cells were then counted with a hemocytometer to a concentration of 1×106 cells/mL, and 100 μL of the culture medium containing the cells was added to each well of the 96-well microplates, which were precoated with 4 μg/mL BSA proteins, fibronectin, collagen I, and Anti-E-cadherin (Sigma-Aldrich, St Louis, MO, USA). .. The attached cells were measured using the MTT assay at 570 nm wavelength absorbance in an Asys UVM 340 microplate reader (Biochrom, Cambridge, UK).

Size-exclusion Chromatography:

Article Title: The R882H DNMT3A Mutation Associated with AML Dominantly Inhibits WT DNMT3A by Blocking its Ability to Form Active Tetramers
Article Snippet: Paragraph title: Size-exclusion Chromatography ... To generate a standard molecular weight curve, 100 μg of each of five proteins were used (urease, BSA, chicken egg albumin, carbonic anhydrase, and α-lactalbumin; Sigma MWND500).

Purification:

Article Title: Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells
Article Snippet: .. Briefly, approximately 1 × 109 carboxylate-modified, fluorescent latex beads (1 μm in diameter, Yellow Green fluorescent Fluo-Spheres, Molecular Probes) were incubated with 300 μg of purified protein or with 1 mg of BSA (Sigma) in a MES buffer at pH 6.0 at room temperature for 15 min. EDAC [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, hydrochloride] (Molecular Probes) was added to a final concentration of 1.6 mg ml-1 to activate surface carboxyl groups and the pH was adjusted to 6.5 with 0.2 M NaOH. ..

Article Title: The R882H DNMT3A Mutation Associated with AML Dominantly Inhibits WT DNMT3A by Blocking its Ability to Form Active Tetramers
Article Snippet: Purified DNMT3A proteins (100 μL of 0.5 mg/mL protein) were loaded onto a Superose 6 HR 10/30 column controlled by an AKTApurifier with UNICORN software (GE v5.11). .. To generate a standard molecular weight curve, 100 μg of each of five proteins were used (urease, BSA, chicken egg albumin, carbonic anhydrase, and α-lactalbumin; Sigma MWND500).

Protein Extraction:

Article Title: The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling
Article Snippet: Paragraph title: Protein extraction and western blot analysis ... Proteins were transferred to nitrocellulose membranes which were then blocked in 5% skim milk (Invitrogen) or 5% BSA (Sigma, for phosphorylated proteins).

Cell Adhesion Assay:

Article Title: Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment
Article Snippet: Paragraph title: Adhesion assay ... The cells were then counted with a hemocytometer to a concentration of 1×106 cells/mL, and 100 μL of the culture medium containing the cells was added to each well of the 96-well microplates, which were precoated with 4 μg/mL BSA proteins, fibronectin, collagen I, and Anti-E-cadherin (Sigma-Aldrich, St Louis, MO, USA).

Staining:

Article Title: Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins
Article Snippet: The protein concentration of TSP extracts was determined by Bradford assay using the Bio-Rad Protein Assay and BSA (Sigma-Aldrich) as a standard. .. For analysis by 12% SDS/PAGE and Coomassie staining using PageBlue Protein Staining Solution (Fermentas), protein extracts were denatured at 95 °C for 5 min before loading.

Mouse Assay:

Article Title: The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling
Article Snippet: Protein extraction and western blot analysis RPE‐choroid preparations from freshly dissected mice were sonicated in RIPA lysis buffer (Millipore, Billerica, MA, 20‐188) plus 1% protease and phosphatase inhibitors (Sigma). .. Proteins were transferred to nitrocellulose membranes which were then blocked in 5% skim milk (Invitrogen) or 5% BSA (Sigma, for phosphorylated proteins).

SDS Page:

Article Title: Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins
Article Snippet: The protein concentration of TSP extracts was determined by Bradford assay using the Bio-Rad Protein Assay and BSA (Sigma-Aldrich) as a standard. .. For analysis by 12% SDS/PAGE and Coomassie staining using PageBlue Protein Staining Solution (Fermentas), protein extracts were denatured at 95 °C for 5 min before loading.

Software:

Article Title: The R882H DNMT3A Mutation Associated with AML Dominantly Inhibits WT DNMT3A by Blocking its Ability to Form Active Tetramers
Article Snippet: Purified DNMT3A proteins (100 μL of 0.5 mg/mL protein) were loaded onto a Superose 6 HR 10/30 column controlled by an AKTApurifier with UNICORN software (GE v5.11). .. To generate a standard molecular weight curve, 100 μg of each of five proteins were used (urease, BSA, chicken egg albumin, carbonic anhydrase, and α-lactalbumin; Sigma MWND500).

Binding Assay:

Article Title: Inherent Anti-amyloidogenic Activity of Human Immunoglobulin ? Heavy Chains *
Article Snippet: .. Antibodies or HCs were serially diluted in assay buffer (1% BSA in PBSA) and tested (100 μl/well) in activated, high binding microtiter plate wells (COSTAR, Corning, NY) that were coated with 400 ng of target protein and blocked with 1% BSA (Sigma-Aldrich) in PBSA. .. For competition studies involving Aβ monomers as an inhibitor of HC or antibody binding to the plate-immobilized homologous Aβ conformer or fibrils, 1 μ m HC F1 or 5 n m MAB1560 was preincubated with the competitor for 40 min.

Evaporation:

Article Title: Tailoring sub-micron PLGA particle release profiles via centrifugal fractioning
Article Snippet: The first emulsion was generated by vortexing the organic phase with a phosphate-buffered saline (PBS) solution containing 20 mg/ mL BSA (total protein content of 2.0% w/w of PLGA; Sigma-Aldrich, St. Louis, MO). .. The emulsion was then quickly transferred to a stirring aqueous bath containing 0.2% TPGS (10× volume excess; 300 rpm) and left undisturbed for 3 hrs to undergo solvent evaporation.

Concentration Assay:

Article Title: Interleukin-22 Promotes Intestinal Stem Cell-Mediated Epithelial Regeneration
Article Snippet: For experiments evaluating organoid budding, the concentration of R-spondin was lowered to 1.25-5%. .. For mouse large intestine, crypts were cultured in “WENR” medium containing 50% of Wnt3a-conditioned medium in addition to the aforementioned proteins and 1% BSA (Sigma) and supplemented with SB202190 (10uM, Sigma), ALK5 inhibitor A83-01(500nM, Tocris Bioscience) and Nicotinamide (10mM, Sigma).

Article Title: Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment
Article Snippet: .. The cells were then counted with a hemocytometer to a concentration of 1×106 cells/mL, and 100 μL of the culture medium containing the cells was added to each well of the 96-well microplates, which were precoated with 4 μg/mL BSA proteins, fibronectin, collagen I, and Anti-E-cadherin (Sigma-Aldrich, St Louis, MO, USA). ..

Article Title: Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells
Article Snippet: .. Briefly, approximately 1 × 109 carboxylate-modified, fluorescent latex beads (1 μm in diameter, Yellow Green fluorescent Fluo-Spheres, Molecular Probes) were incubated with 300 μg of purified protein or with 1 mg of BSA (Sigma) in a MES buffer at pH 6.0 at room temperature for 15 min. EDAC [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, hydrochloride] (Molecular Probes) was added to a final concentration of 1.6 mg ml-1 to activate surface carboxyl groups and the pH was adjusted to 6.5 with 0.2 M NaOH. ..

Lysis:

Article Title: The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling
Article Snippet: Protein extraction and western blot analysis RPE‐choroid preparations from freshly dissected mice were sonicated in RIPA lysis buffer (Millipore, Billerica, MA, 20‐188) plus 1% protease and phosphatase inhibitors (Sigma). .. Proteins were transferred to nitrocellulose membranes which were then blocked in 5% skim milk (Invitrogen) or 5% BSA (Sigma, for phosphorylated proteins).

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    Millipore bsa
    Penetration of C. neoformans through reconstituted ECM. The ECM invasion chambers are composed of matrigel (basement membrane) layered on membranes with 8 µm pores. Strain JEC21 was incubated with plasminogen in phosphate-buffered saline with <t>BSA</t> in the presence or absence of tissue-derived plasminogen activator (tPA), incubated in the upper chamber of the transwell for 24 hours at <t>37°C</t> prior to analysis of colony counts from the lower well (* (p = 0.0093); ** (p = 0.0084)).
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    Penetration of C. neoformans through reconstituted ECM. The ECM invasion chambers are composed of matrigel (basement membrane) layered on membranes with 8 µm pores. Strain JEC21 was incubated with plasminogen in phosphate-buffered saline with BSA in the presence or absence of tissue-derived plasminogen activator (tPA), incubated in the upper chamber of the transwell for 24 hours at 37°C prior to analysis of colony counts from the lower well (* (p = 0.0093); ** (p = 0.0084)).

    Journal: PLoS ONE

    Article Title: Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

    doi: 10.1371/journal.pone.0005780

    Figure Lengend Snippet: Penetration of C. neoformans through reconstituted ECM. The ECM invasion chambers are composed of matrigel (basement membrane) layered on membranes with 8 µm pores. Strain JEC21 was incubated with plasminogen in phosphate-buffered saline with BSA in the presence or absence of tissue-derived plasminogen activator (tPA), incubated in the upper chamber of the transwell for 24 hours at 37°C prior to analysis of colony counts from the lower well (* (p = 0.0093); ** (p = 0.0084)).

    Article Snippet: Surface plasminogen activation Approximately 1×108 log phase cells were washed in PBS and incubated with 100 µg plasminogen for 2–4 hrs at 37°C in PBS with 1.5% BSA, in the presence or absence of 100 ng tissue plasminogen activator (tPA) (Calbiochem) or aprotinin (Roche).

    Techniques: Incubation, Derivative Assay

    Plasminogen binds selectively and specifically to the cell-surface of intact C. neoformans strains. (A–B) Conversion of plasminogen (Plg) into plasmin heavy chain (Pla H ) and light chain (Pla L ) on the surface of intact C. neoformans serotype D and A strains. (A) Serotype D strain JEC21 was incubated in the presence or absence of plasminogen, tissue plasminogen activator (tPA), and/or the plasmin inhibitor aprotinin in phosphate-buffered saline with BSA. Cell wall proteins were released by boiling labeled cells in SDS-extraction buffer and fractionated by SDS-PAGE, transferred to PVDF, and Western blotted with polyclonal anti-plasminogen antibody. Lane descriptions as follow: cells (JEC21) only (1), 100 µg plasminogen (2), plasminogen and 100 ng tPA (3), plasminogen, tPA, and 1 unit aprotinin (4). (B) Serotype A strains C23 and A1 38-2 were incubated in the presence or absence of plasminogen and/or tPA for 4 hrs at 37°C prior to Western blot analysis as described above. Lanes: cells (C23) only (1), C23 with 15 µg plasminogen (2), C23 with plasminogen and 100 ng tPA (3), cells (A1 38-2) only (4), A1 38-2 with 15 µg plasminogen (5), and A1 38-2 with plasminogen and tPA (6). (C) Plasminogen associates with the cell wall of intact cells. Cells (1×10 10 ) from log phase cultures (JEC21) were incubated 4 hr at 37°C in the presence (lane 1) or absence (lane 3) of 50 µg plasminogen and separated into cell wall and cytosol components, as described in Methods . Membranes (lane 2, 4) from cell walls were extracted and each fraction examined for the presence of plasminogen by Western blot analysis. Sample loading was uniform at 5 µg per well. (D) Sulfo-NHS-biotin and plasminogen compete for cell-surface binding sites. Log phase cells (JEC21) were initially labeled with sulfo-NHS-biotin in 0-, 1-, 10-, 100-fold molar equivalents of plasminogen then labeled 1 hr at 37°C with 50 µg plasminogen (lanes 1–4, respectively).

    Journal: PLoS ONE

    Article Title: Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

    doi: 10.1371/journal.pone.0005780

    Figure Lengend Snippet: Plasminogen binds selectively and specifically to the cell-surface of intact C. neoformans strains. (A–B) Conversion of plasminogen (Plg) into plasmin heavy chain (Pla H ) and light chain (Pla L ) on the surface of intact C. neoformans serotype D and A strains. (A) Serotype D strain JEC21 was incubated in the presence or absence of plasminogen, tissue plasminogen activator (tPA), and/or the plasmin inhibitor aprotinin in phosphate-buffered saline with BSA. Cell wall proteins were released by boiling labeled cells in SDS-extraction buffer and fractionated by SDS-PAGE, transferred to PVDF, and Western blotted with polyclonal anti-plasminogen antibody. Lane descriptions as follow: cells (JEC21) only (1), 100 µg plasminogen (2), plasminogen and 100 ng tPA (3), plasminogen, tPA, and 1 unit aprotinin (4). (B) Serotype A strains C23 and A1 38-2 were incubated in the presence or absence of plasminogen and/or tPA for 4 hrs at 37°C prior to Western blot analysis as described above. Lanes: cells (C23) only (1), C23 with 15 µg plasminogen (2), C23 with plasminogen and 100 ng tPA (3), cells (A1 38-2) only (4), A1 38-2 with 15 µg plasminogen (5), and A1 38-2 with plasminogen and tPA (6). (C) Plasminogen associates with the cell wall of intact cells. Cells (1×10 10 ) from log phase cultures (JEC21) were incubated 4 hr at 37°C in the presence (lane 1) or absence (lane 3) of 50 µg plasminogen and separated into cell wall and cytosol components, as described in Methods . Membranes (lane 2, 4) from cell walls were extracted and each fraction examined for the presence of plasminogen by Western blot analysis. Sample loading was uniform at 5 µg per well. (D) Sulfo-NHS-biotin and plasminogen compete for cell-surface binding sites. Log phase cells (JEC21) were initially labeled with sulfo-NHS-biotin in 0-, 1-, 10-, 100-fold molar equivalents of plasminogen then labeled 1 hr at 37°C with 50 µg plasminogen (lanes 1–4, respectively).

    Article Snippet: Surface plasminogen activation Approximately 1×108 log phase cells were washed in PBS and incubated with 100 µg plasminogen for 2–4 hrs at 37°C in PBS with 1.5% BSA, in the presence or absence of 100 ng tissue plasminogen activator (tPA) (Calbiochem) or aprotinin (Roche).

    Techniques: Proximity Ligation Assay, Incubation, Labeling, SDS Page, Western Blot, Binding Assay

    Effects of bromodomain inhibitor in DN. MS417, a bromodomain inhibitor, suppressed acetylation of Stat3 and NF-κB in podocytes treated with AGE. Podocytes were incubated with either BSA or AGE-BSA together with DMSO or MS417 (1.0 μmol/L) for 24 h. Western blot analysis was performed in these cells for acetyl, phosphor- and total p65 ( A ) and STAT3 ( C ). The representative blots of three independent experiments are shown. The densitometry analyses of these Western blots are shown for p65 ( B ) and STAT3 ( D ). The ratios of acetyl-protein or phosphor-protein to total protein were calculated for p65 and STAT3. The fold changes relative to cells treated with BSA + DMSO are shown. * P

    Journal: Diabetes

    Article Title: Role of Transcription Factor Acetylation in Diabetic Kidney Disease

    doi: 10.2337/db13-1810

    Figure Lengend Snippet: Effects of bromodomain inhibitor in DN. MS417, a bromodomain inhibitor, suppressed acetylation of Stat3 and NF-κB in podocytes treated with AGE. Podocytes were incubated with either BSA or AGE-BSA together with DMSO or MS417 (1.0 μmol/L) for 24 h. Western blot analysis was performed in these cells for acetyl, phosphor- and total p65 ( A ) and STAT3 ( C ). The representative blots of three independent experiments are shown. The densitometry analyses of these Western blots are shown for p65 ( B ) and STAT3 ( D ). The ratios of acetyl-protein or phosphor-protein to total protein were calculated for p65 and STAT3. The fold changes relative to cells treated with BSA + DMSO are shown. * P

    Article Snippet: Chromatin Immunoprecipitation Assay Human podocytes were infected with the following viruses: green fluorescent protein, wild-type and K658R STAT3 or green fluorescent protein, wild-type and K310R p65 (1:100 V/V) for 3 days and then treated with either AGE-BSA (100 μg/mL) or BSA (100 μg/mL) for an additional 24 h. Chromatin immunoprecipitation assay was performed according to the EZ-CHIP protocol (Millipore catalog no. 17–371) ( ).

    Techniques: Incubation, Western Blot

    AGE induced acetylation of p65 and STAT3, which is required for their transcriptional activation. A : Human podocytes were cultured with AGE or BSA at the indicated doses for 24 h. Cell lysates were subject to Western blot analysis for acetyl-, phosphor-, and total p65 and STAT3. The representative blots of three independent experiments are shown. B : The Western blots from all experiments were quantified by densitometry analysis. The ratios of acetyl-protein or phosphor-protein to total protein were calculated for p65 and STAT3. The fold changes relative to BSA-treated cells are shown. * P

    Journal: Diabetes

    Article Title: Role of Transcription Factor Acetylation in Diabetic Kidney Disease

    doi: 10.2337/db13-1810

    Figure Lengend Snippet: AGE induced acetylation of p65 and STAT3, which is required for their transcriptional activation. A : Human podocytes were cultured with AGE or BSA at the indicated doses for 24 h. Cell lysates were subject to Western blot analysis for acetyl-, phosphor-, and total p65 and STAT3. The representative blots of three independent experiments are shown. B : The Western blots from all experiments were quantified by densitometry analysis. The ratios of acetyl-protein or phosphor-protein to total protein were calculated for p65 and STAT3. The fold changes relative to BSA-treated cells are shown. * P

    Article Snippet: Chromatin Immunoprecipitation Assay Human podocytes were infected with the following viruses: green fluorescent protein, wild-type and K658R STAT3 or green fluorescent protein, wild-type and K310R p65 (1:100 V/V) for 3 days and then treated with either AGE-BSA (100 μg/mL) or BSA (100 μg/mL) for an additional 24 h. Chromatin immunoprecipitation assay was performed according to the EZ-CHIP protocol (Millipore catalog no. 17–371) ( ).

    Techniques: Activation Assay, Cell Culture, Western Blot

    Acetylation of p65 and STAT3 is required for AGE-induced transcriptional activation of their target genes. Podocytes were infected with p65 or STAT3 constructs with mutated acetyl residues for 3 days then stimulated with AGE or BSA at the indicated doses for an additional 24 h. Total RNA was isolated from these cells for real-time PCR analysis of NF-κB target genes ( CXCL5 [ A ] and CCL17 [ B ]) and STAT3 target genes ( SOC3 [ C ] and Casp9 [ D ]). For chromatin immunoprecipitation (ChiP) assay for NF-κB and STAT3 target genes, podocytes were infected with p65 or STAT3 constructs with mutated acetyl residues for 3 days then stimulated with AGE or BSA at the indicated doses for an additional 24 h. ChiP assay also was performed in these cells to determine the binding of p65 and STAT3 on the promoter sites of their target genes ( CXCL5 [ E ], CCL17 [ F ], SOC3 [ G ], and Casp9 [ H ]). Immunoprecipitated DNA samples were subjected to PCR analysis and are expressed as a percentage of input. * P

    Journal: Diabetes

    Article Title: Role of Transcription Factor Acetylation in Diabetic Kidney Disease

    doi: 10.2337/db13-1810

    Figure Lengend Snippet: Acetylation of p65 and STAT3 is required for AGE-induced transcriptional activation of their target genes. Podocytes were infected with p65 or STAT3 constructs with mutated acetyl residues for 3 days then stimulated with AGE or BSA at the indicated doses for an additional 24 h. Total RNA was isolated from these cells for real-time PCR analysis of NF-κB target genes ( CXCL5 [ A ] and CCL17 [ B ]) and STAT3 target genes ( SOC3 [ C ] and Casp9 [ D ]). For chromatin immunoprecipitation (ChiP) assay for NF-κB and STAT3 target genes, podocytes were infected with p65 or STAT3 constructs with mutated acetyl residues for 3 days then stimulated with AGE or BSA at the indicated doses for an additional 24 h. ChiP assay also was performed in these cells to determine the binding of p65 and STAT3 on the promoter sites of their target genes ( CXCL5 [ E ], CCL17 [ F ], SOC3 [ G ], and Casp9 [ H ]). Immunoprecipitated DNA samples were subjected to PCR analysis and are expressed as a percentage of input. * P

    Article Snippet: Chromatin Immunoprecipitation Assay Human podocytes were infected with the following viruses: green fluorescent protein, wild-type and K658R STAT3 or green fluorescent protein, wild-type and K310R p65 (1:100 V/V) for 3 days and then treated with either AGE-BSA (100 μg/mL) or BSA (100 μg/mL) for an additional 24 h. Chromatin immunoprecipitation assay was performed according to the EZ-CHIP protocol (Millipore catalog no. 17–371) ( ).

    Techniques: Activation Assay, Infection, Construct, Isolation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction

    Sca-1 repression leads to increased adhesion to collagen I, collagen IV and fibronectin. Cell adhesion of 100,000 cells/well coated with fibronectin, vitronectin, laminin, collagen I or collagen IV comparing shLuc (open bars), shSca-1 (grey bars) and shSca-1+Sca-1 (black bars). (A). Relative adhesion was normalized to a BSA control, (* p

    Journal: PLoS ONE

    Article Title: Stem Cell Antigen-1 (Sca-1) Regulates Mammary Tumor Development and Cell Migration

    doi: 10.1371/journal.pone.0027841

    Figure Lengend Snippet: Sca-1 repression leads to increased adhesion to collagen I, collagen IV and fibronectin. Cell adhesion of 100,000 cells/well coated with fibronectin, vitronectin, laminin, collagen I or collagen IV comparing shLuc (open bars), shSca-1 (grey bars) and shSca-1+Sca-1 (black bars). (A). Relative adhesion was normalized to a BSA control, (* p

    Article Snippet: Adhesion Assay 105 cells were seeded onto CytoMatrix™ Cell Adhesion Strips coated with BSA, Collagen Type I and IV, Fibronectin, Laminin, Vitronectin (Millipore) according to the manufacturer's guidelines.

    Techniques:

    Overexpression of ADAM10 promotes proliferation and migration of HASMCs in low glucose, high glucose and high glucose medium with addition of AGE-BSA (100 and 200 µg/ml). A , vector-transduced, ADAM10-overexpressing and ADAM10-silenced HASMCs were generated by retrovirus-mediated gene transfer and selection. These HASMCs were grown in low glucose, high glucose and high glucose with addition of AGE-BSA (100 and 200 ug/ml). MTT assay was performed to test the cell viability. *P

    Journal: PLoS ONE

    Article Title: Increase of ADAM10 Level in Coronary Artery In-Stent Restenosis Segments in Diabetic Minipigs: High ADAM10 Expression Promoting Growth and Migration in Human Vascular Smooth Muscle Cells via Notch 1 and 3

    doi: 10.1371/journal.pone.0083853

    Figure Lengend Snippet: Overexpression of ADAM10 promotes proliferation and migration of HASMCs in low glucose, high glucose and high glucose medium with addition of AGE-BSA (100 and 200 µg/ml). A , vector-transduced, ADAM10-overexpressing and ADAM10-silenced HASMCs were generated by retrovirus-mediated gene transfer and selection. These HASMCs were grown in low glucose, high glucose and high glucose with addition of AGE-BSA (100 and 200 ug/ml). MTT assay was performed to test the cell viability. *P

    Article Snippet: AGE-BSA was from Calbiochem (Merck, Darmstadt, Germany).

    Techniques: Over Expression, Migration, Plasmid Preparation, Generated, Selection, MTT Assay