Structured Review

Millipore mscs
Generation and characterization of human <t>ESC-MSCs.</t> (A) section. Sequential morphological changes of epithelial ESCs into mesenchymal cells during differentiation are shown by inverted phase contrast microscope. (B) Immunophenotyping of RH6-MSCs, RH5-MSCs, and human bone marrow MSCs (BM-MSCs). The three cell types (passages 3–5) are examined by flow cytometry. Analysis of MSC markers, including CD44, CD73, CD90, and CD105, as well as hematopoietic markers CD34 and CD45 (double staining by anti-CD34-PE and anti-CD45-FITC) was performed by Flowing software (version 2.5.1). The white histogram represents the cells stained with isotype control antibodies. Expression of CD markers is presented as the mean ± SD of three different passages. (C) In vitro differentiation of MSCs into mesodermal lineages (adipocytes and osteoblasts). Adipodifferentiation was performed by the serum withdrawal/hypoxia method. Lipid droplets in cells stained with Oil Red O ( upper panel ) after 2 weeks. For <t>osteodifferentiation</t> of MSCs, we cultured the cells in osteogenic media for 3 weeks. Alizarin Red staining showed deposits of calcium crystals ( lower panel ). Scale bars: 100 μm. (D) The in vitro differentiation potential of MSCs (BM-MSC and RH6-MSC) was confirmed by studying the expressions of LPL and PPARG for adipodifferentiation and RUNX and COL1 for osteodifferentiation by real-time RT-PCR. BM-MSCs and ESC-MSCs after differentiation were compared with control groups (MSCs cultured in nondifferentiated medium). (E)
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Images

1) Product Images from "Effect of Secreted Molecules of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells on Acute Hepatic Failure Model"

Article Title: Effect of Secreted Molecules of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells on Acute Hepatic Failure Model

Journal: Stem Cells and Development

doi: 10.1089/scd.2016.0244

Generation and characterization of human ESC-MSCs. (A) section. Sequential morphological changes of epithelial ESCs into mesenchymal cells during differentiation are shown by inverted phase contrast microscope. (B) Immunophenotyping of RH6-MSCs, RH5-MSCs, and human bone marrow MSCs (BM-MSCs). The three cell types (passages 3–5) are examined by flow cytometry. Analysis of MSC markers, including CD44, CD73, CD90, and CD105, as well as hematopoietic markers CD34 and CD45 (double staining by anti-CD34-PE and anti-CD45-FITC) was performed by Flowing software (version 2.5.1). The white histogram represents the cells stained with isotype control antibodies. Expression of CD markers is presented as the mean ± SD of three different passages. (C) In vitro differentiation of MSCs into mesodermal lineages (adipocytes and osteoblasts). Adipodifferentiation was performed by the serum withdrawal/hypoxia method. Lipid droplets in cells stained with Oil Red O ( upper panel ) after 2 weeks. For osteodifferentiation of MSCs, we cultured the cells in osteogenic media for 3 weeks. Alizarin Red staining showed deposits of calcium crystals ( lower panel ). Scale bars: 100 μm. (D) The in vitro differentiation potential of MSCs (BM-MSC and RH6-MSC) was confirmed by studying the expressions of LPL and PPARG for adipodifferentiation and RUNX and COL1 for osteodifferentiation by real-time RT-PCR. BM-MSCs and ESC-MSCs after differentiation were compared with control groups (MSCs cultured in nondifferentiated medium). (E)
Figure Legend Snippet: Generation and characterization of human ESC-MSCs. (A) section. Sequential morphological changes of epithelial ESCs into mesenchymal cells during differentiation are shown by inverted phase contrast microscope. (B) Immunophenotyping of RH6-MSCs, RH5-MSCs, and human bone marrow MSCs (BM-MSCs). The three cell types (passages 3–5) are examined by flow cytometry. Analysis of MSC markers, including CD44, CD73, CD90, and CD105, as well as hematopoietic markers CD34 and CD45 (double staining by anti-CD34-PE and anti-CD45-FITC) was performed by Flowing software (version 2.5.1). The white histogram represents the cells stained with isotype control antibodies. Expression of CD markers is presented as the mean ± SD of three different passages. (C) In vitro differentiation of MSCs into mesodermal lineages (adipocytes and osteoblasts). Adipodifferentiation was performed by the serum withdrawal/hypoxia method. Lipid droplets in cells stained with Oil Red O ( upper panel ) after 2 weeks. For osteodifferentiation of MSCs, we cultured the cells in osteogenic media for 3 weeks. Alizarin Red staining showed deposits of calcium crystals ( lower panel ). Scale bars: 100 μm. (D) The in vitro differentiation potential of MSCs (BM-MSC and RH6-MSC) was confirmed by studying the expressions of LPL and PPARG for adipodifferentiation and RUNX and COL1 for osteodifferentiation by real-time RT-PCR. BM-MSCs and ESC-MSCs after differentiation were compared with control groups (MSCs cultured in nondifferentiated medium). (E)

Techniques Used: Microscopy, Flow Cytometry, Cytometry, Double Staining, Software, Staining, Expressing, In Vitro, Cell Culture, Quantitative RT-PCR

2) Product Images from "Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors"

Article Title: Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors

Journal: Stem Cells International

doi: 10.1155/2017/1513281

Induced osteoprogenitors (iOPs) cells differentiated from hOB-iPSC. (a) Flow cytometry analysis of iOPs and hMSCs revealed that the cells were positive for mesenchymal markers (CD29, CD44, CD90, CD105, and CD166) and negative for hematopoietic markers (CD14, CD31, and CD45) (black: CD marker expression, red: isotype control, and blue: unstained control). (b) Significantly ( P
Figure Legend Snippet: Induced osteoprogenitors (iOPs) cells differentiated from hOB-iPSC. (a) Flow cytometry analysis of iOPs and hMSCs revealed that the cells were positive for mesenchymal markers (CD29, CD44, CD90, CD105, and CD166) and negative for hematopoietic markers (CD14, CD31, and CD45) (black: CD marker expression, red: isotype control, and blue: unstained control). (b) Significantly ( P

Techniques Used: Flow Cytometry, Cytometry, Marker, Expressing

3) Product Images from "Biological Functions of miR-29b Contribute to Positive Regulation of Osteoblast Differentiation *"

Article Title: Biological Functions of miR-29b Contribute to Positive Regulation of Osteoblast Differentiation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M809787200

miR-29b expression profile during MC3T3 osteoblast differentiation. A , MC3T3 preosteoblasts cells cultured in differentiation medium for 28 days. Histochemical staining of alkaline phosphatase ( AP ) activity and von Kossa ( VK ) for mineral deposition at 10, 14, 21, and 28 days is shown. B , Western blot for Runx2 protein which increases during osteoblast differentiation. β-Actin protein was used as control. C , quantitative mRNA normalized by GAPDH for osteoblastic markers Runx2, AlkP, and osteocalcin on selected days during MC3T3 osteoblast differentiation used in miR profiling studies. D , significantly changed microRNAs that putatively target extracellular cellular matrix genes. Total RNA of MC3T3 cells during differentiation time points (0, 7, 14, 21, and 28 days) was used for miRNA microarray analysis. Relative fold changes of the microRNAs were hierarchically clustered by using dChip software. E and F , representative Northern blot analysis of miR-29b using total RNA isolated from mouse (MC3T3) ( E ) and rat primary osteoblasts ( F ), which were induced to differentiate. U6 RNA was used as a loading control. G , densitometric quantitation of miR-29b in indicated osteoblasts normalized to U6. The average volumes from two different MC3T3 studies are shown, and one time course from primary osteoblasts is shown.
Figure Legend Snippet: miR-29b expression profile during MC3T3 osteoblast differentiation. A , MC3T3 preosteoblasts cells cultured in differentiation medium for 28 days. Histochemical staining of alkaline phosphatase ( AP ) activity and von Kossa ( VK ) for mineral deposition at 10, 14, 21, and 28 days is shown. B , Western blot for Runx2 protein which increases during osteoblast differentiation. β-Actin protein was used as control. C , quantitative mRNA normalized by GAPDH for osteoblastic markers Runx2, AlkP, and osteocalcin on selected days during MC3T3 osteoblast differentiation used in miR profiling studies. D , significantly changed microRNAs that putatively target extracellular cellular matrix genes. Total RNA of MC3T3 cells during differentiation time points (0, 7, 14, 21, and 28 days) was used for miRNA microarray analysis. Relative fold changes of the microRNAs were hierarchically clustered by using dChip software. E and F , representative Northern blot analysis of miR-29b using total RNA isolated from mouse (MC3T3) ( E ) and rat primary osteoblasts ( F ), which were induced to differentiate. U6 RNA was used as a loading control. G , densitometric quantitation of miR-29b in indicated osteoblasts normalized to U6. The average volumes from two different MC3T3 studies are shown, and one time course from primary osteoblasts is shown.

Techniques Used: Expressing, Cell Culture, Staining, Activity Assay, Western Blot, Microarray, Software, Northern Blot, Isolation, Quantitation Assay

4) Product Images from "Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *"

Article Title: Transcriptional Co-activator LEDGF Interacts with Cdc7-Activator of S-phase Kinase (ASK) and Stimulates Its Enzymatic Activity *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.036491

Identification of the LEDGF-Cdc7-ASK interaction. A , schematics showing the domain organization of LEDGF and the cTAP-tagged LEDGF-(326–530) construct. Locations of the PWWP domain, NLS, AT-hooks, and IBD of LEDGF, calmodulin binding peptide ( CBP ), tobacco etch virus ( TEV ) protease site, and the IgG binding module from S. aureus protein A ( protA ) of the cTAP tag are indicated. B , co-IP experiments. HeLa cells were transiently transfected with HA-tagged mouse LEDGF (mLEDGF), human LEDGF, LEDGF-(326–530), HRP2, mouse p52 ( mp52 ), or an empty vector. Whole cell extracts ( WCE ; lanes 1–6 ) or proteins pulled down with anti-HA affinity matrix from whole cell extracts ( lanes 7–12 ) were tested by Western blotting using anti-HA, anti-Cdc7, and anti-β-actin antibodies. Migration positions of protein molecular mass standards (kDa), and the heavy chain of mouse IgG ( IgG H ) are indicated. C , IP of endogenous proteins. Extracts from untransfected 293T cells were incubated with rabbit anti-LEDGF antibody ( lane 3 ) or control rabbit IgG ( lane 4 ) and protein G-agarose, and the recovered proteins were analyzed by Western blotting with anti-Cdc7 and anti-ASK antibodies. Lanes 1 and 2 contained whole cell extract. To improve detection of ASK, the samples in lanes 2–4 were treated with λ-protein phosphatase (λ PPase ). The bands corresponding to ASK are indicated with asterisks .
Figure Legend Snippet: Identification of the LEDGF-Cdc7-ASK interaction. A , schematics showing the domain organization of LEDGF and the cTAP-tagged LEDGF-(326–530) construct. Locations of the PWWP domain, NLS, AT-hooks, and IBD of LEDGF, calmodulin binding peptide ( CBP ), tobacco etch virus ( TEV ) protease site, and the IgG binding module from S. aureus protein A ( protA ) of the cTAP tag are indicated. B , co-IP experiments. HeLa cells were transiently transfected with HA-tagged mouse LEDGF (mLEDGF), human LEDGF, LEDGF-(326–530), HRP2, mouse p52 ( mp52 ), or an empty vector. Whole cell extracts ( WCE ; lanes 1–6 ) or proteins pulled down with anti-HA affinity matrix from whole cell extracts ( lanes 7–12 ) were tested by Western blotting using anti-HA, anti-Cdc7, and anti-β-actin antibodies. Migration positions of protein molecular mass standards (kDa), and the heavy chain of mouse IgG ( IgG H ) are indicated. C , IP of endogenous proteins. Extracts from untransfected 293T cells were incubated with rabbit anti-LEDGF antibody ( lane 3 ) or control rabbit IgG ( lane 4 ) and protein G-agarose, and the recovered proteins were analyzed by Western blotting with anti-Cdc7 and anti-ASK antibodies. Lanes 1 and 2 contained whole cell extract. To improve detection of ASK, the samples in lanes 2–4 were treated with λ-protein phosphatase (λ PPase ). The bands corresponding to ASK are indicated with asterisks .

Techniques Used: Construct, Binding Assay, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Migration, Incubation

Residues 625–674 of ASK are required for binding to LEDGF. A , schematic of ASK truncations. Locations of the N, M, and C motifs are indicated. B , C terminus of ASK is required for the interaction with LEDGF. S-tagged Cdc7-ASK or its indicated mutant forms were incubated with S-protein-agarose in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. Lane 14 contains a mock pull-down of LEDGF with S-protein-agarose. Input quantities of proteins ( lanes 1–5 ) or proteins captured on the beads ( lanes 6–14 ) separated by SDS-PAGE were stained with Coomassie Blue ( top ) and analyzed by Western blotting using anti-LEDGF antibody ( bottom ). C , deletion of 50 residues from the C terminus of ASK is sufficient to ablate the interaction with LEDGF. Cdc7-ASK or its mutants were incubated with S-protein-agarose beads in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. LEDGF was incubated with S-protein-agarose alone in lane 14. Lanes 1–5 contained input levels of the indicated proteins. Samples were analyzed as in B .
Figure Legend Snippet: Residues 625–674 of ASK are required for binding to LEDGF. A , schematic of ASK truncations. Locations of the N, M, and C motifs are indicated. B , C terminus of ASK is required for the interaction with LEDGF. S-tagged Cdc7-ASK or its indicated mutant forms were incubated with S-protein-agarose in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. Lane 14 contains a mock pull-down of LEDGF with S-protein-agarose. Input quantities of proteins ( lanes 1–5 ) or proteins captured on the beads ( lanes 6–14 ) separated by SDS-PAGE were stained with Coomassie Blue ( top ) and analyzed by Western blotting using anti-LEDGF antibody ( bottom ). C , deletion of 50 residues from the C terminus of ASK is sufficient to ablate the interaction with LEDGF. Cdc7-ASK or its mutants were incubated with S-protein-agarose beads in the presence ( lanes 6–9 ) or absence ( lanes 10–13 ) of LEDGF. LEDGF was incubated with S-protein-agarose alone in lane 14. Lanes 1–5 contained input levels of the indicated proteins. Samples were analyzed as in B .

Techniques Used: Binding Assay, Mutagenesis, Incubation, SDS Page, Staining, Western Blot

The interaction between LEDGF IBD and the C terminus of ASK relieves autoinhibition of Cdc7-ASK kinase activity. Schematic of the Cdc7-ASK kinase activity in the absence ( A ) or presence ( B ) of LEDGF. Cdc7 is shown as a gray rectangle . ASK domains are shown as black circles ; the smaller circle is the C-terminal regulatory peptide ( RP ). Ovals represent LEDGF PWWP and IBD domains; the MCM2–7 protein complex is shown as hexagons ( P , phosphorylation). Interaction with LEDGF results in full activation of Cdc7-ASK kinase activity.
Figure Legend Snippet: The interaction between LEDGF IBD and the C terminus of ASK relieves autoinhibition of Cdc7-ASK kinase activity. Schematic of the Cdc7-ASK kinase activity in the absence ( A ) or presence ( B ) of LEDGF. Cdc7 is shown as a gray rectangle . ASK domains are shown as black circles ; the smaller circle is the C-terminal regulatory peptide ( RP ). Ovals represent LEDGF PWWP and IBD domains; the MCM2–7 protein complex is shown as hexagons ( P , phosphorylation). Interaction with LEDGF results in full activation of Cdc7-ASK kinase activity.

Techniques Used: Activity Assay, Activation Assay

Intracellular distribution of overexpressed LEDGF, ASK and Cdc7. A , confocal laser-scanning microscopy images of HeLa cells transfected with EGFP-Cdc7, full-length HA-ASK ( top and middle rows of images ), HA-ASK-(1–624) ( bottom row ), HcRed1-LEDGF ( top and bottom rows ), and/or HcRed1-p52 ( middle row ). B , ASK and Cdc7 are enriched in the chromatin-containing Triton-insoluble fraction when co-overexpressed with LEDGF. 293T cells were transfected with expression vectors for FLAG-Cdc7 and HA-ASK ( lanes w1–w4 , s1–s4 , and i1–i4 ), WT LEDGF ( lanes w2 , s2 , and i2 ), LEDGF K401E/K402E/R405E ( EEE ) ( lanes w3 , s3 , and i3 ), LEDGFΔIBD ( lanes w4 , s4 , and i4 ), or empty vector ( lane w0 ). Whole cell extracts ( lanes w0–w4 ) and Triton X-100-soluble ( lanes s1–s4 ), and -insoluble ( lanes i1–i4 ) fractions were analyzed by Western blotting using anti-HA, Cdc7, LEDGF, lamin B, and β-tubulin antibodies.
Figure Legend Snippet: Intracellular distribution of overexpressed LEDGF, ASK and Cdc7. A , confocal laser-scanning microscopy images of HeLa cells transfected with EGFP-Cdc7, full-length HA-ASK ( top and middle rows of images ), HA-ASK-(1–624) ( bottom row ), HcRed1-LEDGF ( top and bottom rows ), and/or HcRed1-p52 ( middle row ). B , ASK and Cdc7 are enriched in the chromatin-containing Triton-insoluble fraction when co-overexpressed with LEDGF. 293T cells were transfected with expression vectors for FLAG-Cdc7 and HA-ASK ( lanes w1–w4 , s1–s4 , and i1–i4 ), WT LEDGF ( lanes w2 , s2 , and i2 ), LEDGF K401E/K402E/R405E ( EEE ) ( lanes w3 , s3 , and i3 ), LEDGFΔIBD ( lanes w4 , s4 , and i4 ), or empty vector ( lane w0 ). Whole cell extracts ( lanes w0–w4 ) and Triton X-100-soluble ( lanes s1–s4 ), and -insoluble ( lanes i1–i4 ) fractions were analyzed by Western blotting using anti-HA, Cdc7, LEDGF, lamin B, and β-tubulin antibodies.

Techniques Used: Confocal Laser Scanning Microscopy, Transfection, Expressing, Plasmid Preparation, Western Blot

5) Product Images from "LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis 1LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis 1 [OPEN]"

Article Title: LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis 1LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis 1 [OPEN]

Journal: Plant Physiology

doi: 10.1104/pp.15.01480

GhLEC1 , GhCKI , and GhTCP15 Affect Callus Proliferation
Figure Legend Snippet: GhLEC1 , GhCKI , and GhTCP15 Affect Callus Proliferation

Techniques Used:

Ectopic expression of GhLEC1 , GhCKI , and GhTCP15 wild type (WT), OE25-6 , OE15-7 , CDi-1 , Lov6-29 , lec1-1 , OETCP6-9 , and tcp14/15 at 21 d. Calluses in red circles are highlighted
Figure Legend Snippet: Ectopic expression of GhLEC1 , GhCKI , and GhTCP15 wild type (WT), OE25-6 , OE15-7 , CDi-1 , Lov6-29 , lec1-1 , OETCP6-9 , and tcp14/15 at 21 d. Calluses in red circles are highlighted

Techniques Used: Expressing

6) Product Images from "Role of Apoptosis Signal-regulating Kinase 1 (ASK1) as an Activator of the GAPDH-Siah1 Stress-Signaling Cascade *"

Article Title: Role of Apoptosis Signal-regulating Kinase 1 (ASK1) as an Activator of the GAPDH-Siah1 Stress-Signaling Cascade *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.596205

Siah1 and GAPDH bind ASK1. A , Siah1-ASK1 and GAPDH-ASK1 binding in mouse brain extracts. Mouse brain extracts were immunoprecipitated with anti-Siah1 or anti-GAPDH antibodies and analyzed by Western blot with anti-ASK1, GAPDH, and Siah1 antibodies. Input
Figure Legend Snippet: Siah1 and GAPDH bind ASK1. A , Siah1-ASK1 and GAPDH-ASK1 binding in mouse brain extracts. Mouse brain extracts were immunoprecipitated with anti-Siah1 or anti-GAPDH antibodies and analyzed by Western blot with anti-ASK1, GAPDH, and Siah1 antibodies. Input

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot

ASK1 phosphorylates Siah1 in cells and in vitro . A , phosphorylation of Siah1 by ASK1 in vitro. In vitro phosphorylation assays were performed by incubation of recombinant GST-Siah1 or GST with constitutively active GST-ASK1 (aa 649–946) in the
Figure Legend Snippet: ASK1 phosphorylates Siah1 in cells and in vitro . A , phosphorylation of Siah1 by ASK1 in vitro. In vitro phosphorylation assays were performed by incubation of recombinant GST-Siah1 or GST with constitutively active GST-ASK1 (aa 649–946) in the

Techniques Used: In Vitro, Incubation, Recombinant

Scheme to represent the mechanisms through GAPDH-Siah1 pathway is activated under stress.
Figure Legend Snippet: Scheme to represent the mechanisms through GAPDH-Siah1 pathway is activated under stress.

Techniques Used:

ASK1 facilitates GAPDH-Siah1 binding in cells. Upper panel , ASK1 augments GAPDH-Siah1 binding in a kinase-dependent manner. Cell lysates of HEK293 cells expressing Myc-tagged wild-type ( WT ) Siah1 together with HA-WT ASK1, kinase-dead (KD) ASK1, or constitutively
Figure Legend Snippet: ASK1 facilitates GAPDH-Siah1 binding in cells. Upper panel , ASK1 augments GAPDH-Siah1 binding in a kinase-dependent manner. Cell lysates of HEK293 cells expressing Myc-tagged wild-type ( WT ) Siah1 together with HA-WT ASK1, kinase-dead (KD) ASK1, or constitutively

Techniques Used: Binding Assay, Expressing

Siah1 and GAPDH form a ternary complex with ASK1 in cells. HEK293 cells expressing FLAG-ASK1, HA-GAPDH, and Myc-Siah1 were treated with 1 m m H 2 O 2 for 30 min to induce activation of ASK1 ( lower panel ). Cell lysates were immunoprecipitated in sequence with
Figure Legend Snippet: Siah1 and GAPDH form a ternary complex with ASK1 in cells. HEK293 cells expressing FLAG-ASK1, HA-GAPDH, and Myc-Siah1 were treated with 1 m m H 2 O 2 for 30 min to induce activation of ASK1 ( lower panel ). Cell lysates were immunoprecipitated in sequence with

Techniques Used: Expressing, Activation Assay, Immunoprecipitation, Sequencing

PUMA expression, a downstream target of p300, was found reduced in M2+M4 mutant of Siah1. HEK293 cells expressing WT-Siah1 and M2+M4 Siah1 mutants were treated with 1 m m H 2 O 2 for 30 min. Cell lysates were analyzed by Western blot with an anti-PUMA antibody.
Figure Legend Snippet: PUMA expression, a downstream target of p300, was found reduced in M2+M4 mutant of Siah1. HEK293 cells expressing WT-Siah1 and M2+M4 Siah1 mutants were treated with 1 m m H 2 O 2 for 30 min. Cell lysates were analyzed by Western blot with an anti-PUMA antibody.

Techniques Used: Expressing, Mutagenesis, Western Blot

GAPDH-Siah1 binding is independent of p38 and JNK signaling. GAPDH-Siah1-ASK1 interactions occur independent of p38 and JNK activation. A , representative figure in which GAPDH-Siah1-ASK1 interactions were assessed in lysates from HEK293 treated with 10
Figure Legend Snippet: GAPDH-Siah1 binding is independent of p38 and JNK signaling. GAPDH-Siah1-ASK1 interactions occur independent of p38 and JNK activation. A , representative figure in which GAPDH-Siah1-ASK1 interactions were assessed in lysates from HEK293 treated with 10

Techniques Used: Binding Assay, Activation Assay

ASK1 directly binds to Siah1 and is modulated by GAPDH in vitro . A , recombinant GST-ASK1 (aa 1–940) or GST were incubated with recombinant Siah1 in vitro and subjected to GST pull-down, followed Western blot with an anti-Siah1 antibody. Input
Figure Legend Snippet: ASK1 directly binds to Siah1 and is modulated by GAPDH in vitro . A , recombinant GST-ASK1 (aa 1–940) or GST were incubated with recombinant Siah1 in vitro and subjected to GST pull-down, followed Western blot with an anti-Siah1 antibody. Input

Techniques Used: In Vitro, Recombinant, Incubation, Western Blot

7) Product Images from "Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *"

Article Title: Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *

Journal:

doi: 10.1074/jbc.M708782200

eEF1A1 enhances SK1 activity in vitro . A , the effect of purified GST-eEF1A1( eEF1A ) or GST control( Ctl ) on the catalytic activity of purified rec-SK1 was determined by in vitro SK assay. GST-eEF1A1 and GST were added to rec-SK1 in 10-fold molar excess.
Figure Legend Snippet: eEF1A1 enhances SK1 activity in vitro . A , the effect of purified GST-eEF1A1( eEF1A ) or GST control( Ctl ) on the catalytic activity of purified rec-SK1 was determined by in vitro SK assay. GST-eEF1A1 and GST were added to rec-SK1 in 10-fold molar excess.

Techniques Used: Activity Assay, In Vitro, Purification, CTL Assay

Phosphorylation of SK1 does not alter binding to eEF1A1. A , GST-SK1 bound to GSH-Sepharose was phosphorylated using ERK2, as assessed through IB with anti-phospho-SK1 antibodies. GST-SK1 and GST-phospho-SK1 (GST-pSK1) were then incubated with lysates
Figure Legend Snippet: Phosphorylation of SK1 does not alter binding to eEF1A1. A , GST-SK1 bound to GSH-Sepharose was phosphorylated using ERK2, as assessed through IB with anti-phospho-SK1 antibodies. GST-SK1 and GST-phospho-SK1 (GST-pSK1) were then incubated with lysates

Techniques Used: Binding Assay, Incubation

eEF1A2 interacts with SK1 and increases its activity. A , HEK-293T cells were co-transfected with FLAG-tagged SK1 and either HA-tagged eEF1A1 or eEF1A2. eEF1A1 or eEF1A2 were immunoprecipitated ( IP ) from the lysates using anti-HA antibodies and the
Figure Legend Snippet: eEF1A2 interacts with SK1 and increases its activity. A , HEK-293T cells were co-transfected with FLAG-tagged SK1 and either HA-tagged eEF1A1 or eEF1A2. eEF1A1 or eEF1A2 were immunoprecipitated ( IP ) from the lysates using anti-HA antibodies and the

Techniques Used: Activity Assay, Transfection, Immunoprecipitation

eEF1A1 interacts with SK1. A , GST-SK1 bound to GSH-Sepharose was incubated with lysates from HEK-293T cells expressing HA-tagged eEF1A1. eEF1A1 pulled down by the GST-SK1 was detected by IB with anti-HA. B , GST-eEF1A1 bound to GSH-Sepharose was incubated
Figure Legend Snippet: eEF1A1 interacts with SK1. A , GST-SK1 bound to GSH-Sepharose was incubated with lysates from HEK-293T cells expressing HA-tagged eEF1A1. eEF1A1 pulled down by the GST-SK1 was detected by IB with anti-HA. B , GST-eEF1A1 bound to GSH-Sepharose was incubated

Techniques Used: Incubation, Expressing

Phosphorylation of eEF1A1 does not alter binding to SK1 or SK2. A , GST-eEF1A1 bound to GSH-Sepharose was incubated with S6K in the presence of γ- 32 P]ATP, and the phosphorylation of GST-eEF1A1 was detected using phosphorimaging ( upper panel
Figure Legend Snippet: Phosphorylation of eEF1A1 does not alter binding to SK1 or SK2. A , GST-eEF1A1 bound to GSH-Sepharose was incubated with S6K in the presence of γ- 32 P]ATP, and the phosphorylation of GST-eEF1A1 was detected using phosphorimaging ( upper panel

Techniques Used: Binding Assay, Incubation

8) Product Images from "Inhibition of the PI3K/AKT pathway potentiates cytotoxicity of EGFR kinase inhibitors in triple-negative breast cancer cells"

Article Title: Inhibition of the PI3K/AKT pathway potentiates cytotoxicity of EGFR kinase inhibitors in triple-negative breast cancer cells

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12046

Combination of PI-103 with gefitinib abolishes AKT and ERK pathways in the susceptible cells. ( A ) Cells were treated with either gefitinib (3 μM), PI-103 (3 μM) alone or a combination of both drugs for 2 or 24 hrs. α-tubulin (for 2-hr treatment) or β-actin (for 24-hr treatment) was used as a loading control. ( B ) Two susceptible cell lines (SUM149PT and MDA-MB-468) were treated with increasing amounts of gefitinib, PI-103 or in combination of both drugs for 24 hrs. β-actin was used as a loading control. (A–B) Western blot analysis was performed with the indicated antibodies. Representative data from two independent experiments are shown.
Figure Legend Snippet: Combination of PI-103 with gefitinib abolishes AKT and ERK pathways in the susceptible cells. ( A ) Cells were treated with either gefitinib (3 μM), PI-103 (3 μM) alone or a combination of both drugs for 2 or 24 hrs. α-tubulin (for 2-hr treatment) or β-actin (for 24-hr treatment) was used as a loading control. ( B ) Two susceptible cell lines (SUM149PT and MDA-MB-468) were treated with increasing amounts of gefitinib, PI-103 or in combination of both drugs for 24 hrs. β-actin was used as a loading control. (A–B) Western blot analysis was performed with the indicated antibodies. Representative data from two independent experiments are shown.

Techniques Used: Multiple Displacement Amplification, Western Blot

EGFR is overexpressed in TNBC cell lines. Cells were harvested the day after subculture and cell lysates were analysed by western blot with the indicated antibodies. α-tubulin was used as a loading control. MSL: mesenchymal stem-like; BL: basal-like.
Figure Legend Snippet: EGFR is overexpressed in TNBC cell lines. Cells were harvested the day after subculture and cell lysates were analysed by western blot with the indicated antibodies. α-tubulin was used as a loading control. MSL: mesenchymal stem-like; BL: basal-like.

Techniques Used: Western Blot

9) Product Images from "Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins"

Article Title: Human cytomegalovirus-encoded viral cyclin-dependent kinase (v-CDK) UL97 phosphorylates and inactivates the retinoblastoma protein-related p107 and p130 proteins

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.773150

UL97 phosphorylates p107 and p130 during HCMV lytic infection. HFFs were serum-starved for 48 h then infected with HCMV or UL97-null virus (Δ97) at an m.o.i. of 1. Whole-cell lysates were prepared at the indicated times postinfection, and equal amounts of protein from each sample were loaded onto a normal gel ( A ) or phosphate affinity (phos-tag) gel ( B ) and subjected to Western blotting analysis with the indicated antibodies. The percentage of phosphorylated p107 (compared with total) quantified by ImageJ is presented below the panel. M, mock infection. C, lysates from HCMV-infected HFFs were treated (+) or not (−) with λ protein phosphatase ( l-PPase ) and analyzed as in B. D, Saos-2 cells were transfected with expression plasmids for HA-tagged p107 (HA-p107) together with either an empty vector ( EV ), one expressing V5-tagged UL97 ( 97 ), cyclin A2 ( A2 )/CDK2 ( K2 ), cyclin E1 ( E1 )/CDK2, or cyclin D2 ( D2 )/CDK4 ( K4 ). Lysates harvested 48 h after transfection were analyzed by Western blotting with the indicated antibodies. E, transfections were performed as in D with HA-tagged p130 ( HA-p130 ), p130ΔCDK4, or p130-PM19A. F and G, lysates of Saos-2 cells transfected with plasmids expressing HA-tagged p107 ( HA-p107 ) or p130 ( HA-p130 ) were immunoprecipitated with anti-p107 or p130 antibodies. Either wild-type ( WT ) or kinase-dead ( KD ) UL97 was incubated with immune-enriched p107 or p130 substrates in the presence of [γ- 32 P]ATP. Kinase reactions were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. They were visualized by both autoradiography and Western blotting with an anti-HA antibody for detection of HA-UL97 and HA-p107 and an anti-p130 antibody for detection of HA-p130. M.W. , molecular weight. H, kinase reaction was conducted as in F except normal ATP and bacterially purified GST-tagged p130 were utilized. Samples were analyzed by Western blotting with the indicated antibodies. Experiments were performed in biological triplicate except those in D and E that were performed in biological duplicates.
Figure Legend Snippet: UL97 phosphorylates p107 and p130 during HCMV lytic infection. HFFs were serum-starved for 48 h then infected with HCMV or UL97-null virus (Δ97) at an m.o.i. of 1. Whole-cell lysates were prepared at the indicated times postinfection, and equal amounts of protein from each sample were loaded onto a normal gel ( A ) or phosphate affinity (phos-tag) gel ( B ) and subjected to Western blotting analysis with the indicated antibodies. The percentage of phosphorylated p107 (compared with total) quantified by ImageJ is presented below the panel. M, mock infection. C, lysates from HCMV-infected HFFs were treated (+) or not (−) with λ protein phosphatase ( l-PPase ) and analyzed as in B. D, Saos-2 cells were transfected with expression plasmids for HA-tagged p107 (HA-p107) together with either an empty vector ( EV ), one expressing V5-tagged UL97 ( 97 ), cyclin A2 ( A2 )/CDK2 ( K2 ), cyclin E1 ( E1 )/CDK2, or cyclin D2 ( D2 )/CDK4 ( K4 ). Lysates harvested 48 h after transfection were analyzed by Western blotting with the indicated antibodies. E, transfections were performed as in D with HA-tagged p130 ( HA-p130 ), p130ΔCDK4, or p130-PM19A. F and G, lysates of Saos-2 cells transfected with plasmids expressing HA-tagged p107 ( HA-p107 ) or p130 ( HA-p130 ) were immunoprecipitated with anti-p107 or p130 antibodies. Either wild-type ( WT ) or kinase-dead ( KD ) UL97 was incubated with immune-enriched p107 or p130 substrates in the presence of [γ- 32 P]ATP. Kinase reactions were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. They were visualized by both autoradiography and Western blotting with an anti-HA antibody for detection of HA-UL97 and HA-p107 and an anti-p130 antibody for detection of HA-p130. M.W. , molecular weight. H, kinase reaction was conducted as in F except normal ATP and bacterially purified GST-tagged p130 were utilized. Samples were analyzed by Western blotting with the indicated antibodies. Experiments were performed in biological triplicate except those in D and E that were performed in biological duplicates.

Techniques Used: Infection, Western Blot, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Incubation, SDS Page, Autoradiography, Molecular Weight, Purification

10) Product Images from "Phosphodiesterase 5 Inhibition Limits Doxorubicin-induced Heart Failure by Attenuating Protein Kinase G Iα Oxidation *"

Article Title: Phosphodiesterase 5 Inhibition Limits Doxorubicin-induced Heart Failure by Attenuating Protein Kinase G Iα Oxidation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.724070

Tadalafil limits doxorubicin-induced PKG Iα oxidation and the associated apoptotic signaling in WT, whereas KI are basally protected. A , PKG Iα disulfide dimerization, RhoA Ser-188 phosphorylation, RhoA, and ROCK activity in WT or KI hearts
Figure Legend Snippet: Tadalafil limits doxorubicin-induced PKG Iα oxidation and the associated apoptotic signaling in WT, whereas KI are basally protected. A , PKG Iα disulfide dimerization, RhoA Ser-188 phosphorylation, RhoA, and ROCK activity in WT or KI hearts

Techniques Used: Activity Assay

Oxidation of PKG Iα to the interprotein disulfide state attenuates cGMP-stimulated phosphorylation of Ser-188 RhoA. A , comparison of phosphorylation of Ser-188 RhoA with that of Ser-27 histone H1 by recombinant interprotein disulfide dimer or
Figure Legend Snippet: Oxidation of PKG Iα to the interprotein disulfide state attenuates cGMP-stimulated phosphorylation of Ser-188 RhoA. A , comparison of phosphorylation of Ser-188 RhoA with that of Ser-27 histone H1 by recombinant interprotein disulfide dimer or

Techniques Used: Recombinant

11) Product Images from ""

Article Title:

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.069948

Rap1a-GTP and Gβγ promote the translocation of Radil at cell-matrix contacts in HT1080 cells. A , shown is localization of GFP ( a and b ), Venus-Radil ( c and d ), or Venus-Radil co-expressed with wild-type HA-Rap1 ( e–g ), HA-Rap1aQ63E ( h–j ), HA-Rap1G12V ( k–m ), or FLAG-Gβ 2 /Gγ1 ( n–p ). HA- and FLAG-tagged Rap1a or Gβ 2 + Gγ (untagged) were transiently transfected in HT1080 cells stably expressing Venus-Radil as indicated. Cells were fixed followed by immunodetection with α-HA ( e–m ) or α-FLAG ( n–p ) monoclonal antibody followed by secondary detection with goat-anti-mouse conjugated to Alexa 594 antibody. Cells were visualized using a Zeiss LSM 510 confocal microscope under 63× oil immersion objective. White arrows depict plasma-membrane co-localization. White boxes indicate magnified sections cropped in Adobe Photoshop CS3. Images shown are representative of 10–15 cells analyzed in three independent experiments. Bars , 50 μm. B , membrane fractions show enrichment of Venus-Radil when Rap1aQ63E and Gβγ are expressed. HT1080 cells stably expressing Venus-Radil were transiently transfected to express the indicated proteins. Cells were lysed in hypotonic buffer and subjected to subcellular fractionation as described under “Experimental Procedures.” Membrane fractions and the inputs were analyzed by Western blot ( WB ) to determine the amount of Venus-Radil present in the membrane pool when HA-Rap1aQ63E or FLAG-Gβ 2 was co-expressed. The blot for the membrane fractions was stripped and re-probed using α-Na-K-ATPase antibody to provide for an internal loading control.
Figure Legend Snippet: Rap1a-GTP and Gβγ promote the translocation of Radil at cell-matrix contacts in HT1080 cells. A , shown is localization of GFP ( a and b ), Venus-Radil ( c and d ), or Venus-Radil co-expressed with wild-type HA-Rap1 ( e–g ), HA-Rap1aQ63E ( h–j ), HA-Rap1G12V ( k–m ), or FLAG-Gβ 2 /Gγ1 ( n–p ). HA- and FLAG-tagged Rap1a or Gβ 2 + Gγ (untagged) were transiently transfected in HT1080 cells stably expressing Venus-Radil as indicated. Cells were fixed followed by immunodetection with α-HA ( e–m ) or α-FLAG ( n–p ) monoclonal antibody followed by secondary detection with goat-anti-mouse conjugated to Alexa 594 antibody. Cells were visualized using a Zeiss LSM 510 confocal microscope under 63× oil immersion objective. White arrows depict plasma-membrane co-localization. White boxes indicate magnified sections cropped in Adobe Photoshop CS3. Images shown are representative of 10–15 cells analyzed in three independent experiments. Bars , 50 μm. B , membrane fractions show enrichment of Venus-Radil when Rap1aQ63E and Gβγ are expressed. HT1080 cells stably expressing Venus-Radil were transiently transfected to express the indicated proteins. Cells were lysed in hypotonic buffer and subjected to subcellular fractionation as described under “Experimental Procedures.” Membrane fractions and the inputs were analyzed by Western blot ( WB ) to determine the amount of Venus-Radil present in the membrane pool when HA-Rap1aQ63E or FLAG-Gβ 2 was co-expressed. The blot for the membrane fractions was stripped and re-probed using α-Na-K-ATPase antibody to provide for an internal loading control.

Techniques Used: Translocation Assay, Transfection, Stable Transfection, Expressing, Immunodetection, Microscopy, Fractionation, Western Blot

Interaction between Gβγ, Rap1a, and Radil. A , Radil physically connects Gβγ and Rap1a. HEK293T cells were transfected with different plasmid combinations coding for Strep-HA-Gβ 2 , Gγ 2 , HA-Rap1a, and Venus-Radil. Strep-HA-Gβ 2 -containing complexes were affinity-purified with streptavidin-Sepharose beads, and the association with Rap1a and Radil was monitored by Western blot ( WB ) with α-HA and α-GFP antibodies, respectively. The efficiency of Gβ 2 purification and expression was also followed by Western blot using HA antibodies. A fraction of the lysates for each samples was probed with α-HA or α-GFP antibodies to assess protein expression. The association of Rap1a with Gβ 2 was enhanced when Radil was overexpressed (compare lanes 2 and 3) ( n = 3). *, nonspecific band. B , formation of the Gβγ·Rap1a·Radil complex requires active Rap1a. HEK293T cells were transfected with expression plasmids for Strep-HA-Radil and HA-Rap1a only ( lane 2 ) or together with a vector coding for FLAG-Rap1GAP ( lane 3 ). Strep-HA-Radil was purified using streptavidin-Sepharose beads, and its association with Rap1 and Gβ was monitored by a Western blot using α-HA and α-Gβ antibodies, respectively. The expression of Rap1GAP was followed using α-FLAG antibodies. Although HA-Rap1a and Gβ bound to Strep-HA-Radil ( lane 2 ), these interactions were compromised in the presence of overexpressed FLAG-Rap1GAP ( lane 3 ) ( n = 4). C , expression of a constitutively active Rap1a mutant (Rap1aQ63E) promotes the interaction of Gβ with Radil. Strep-HA-Radil expressing stable cells were transfected or not with HA-Rap1aQ63E. 48 h after transfection, Radil was affinity-purified with streptavidin-Sepharose beads for 1.5 h, and the eluates were analyzed by Western blot using the indicated antibodies. Untransfected HEK293T cells were used as the negative control for streptavidin purification. AP , affinity purification. M r , molecular weights. K , ×1000.
Figure Legend Snippet: Interaction between Gβγ, Rap1a, and Radil. A , Radil physically connects Gβγ and Rap1a. HEK293T cells were transfected with different plasmid combinations coding for Strep-HA-Gβ 2 , Gγ 2 , HA-Rap1a, and Venus-Radil. Strep-HA-Gβ 2 -containing complexes were affinity-purified with streptavidin-Sepharose beads, and the association with Rap1a and Radil was monitored by Western blot ( WB ) with α-HA and α-GFP antibodies, respectively. The efficiency of Gβ 2 purification and expression was also followed by Western blot using HA antibodies. A fraction of the lysates for each samples was probed with α-HA or α-GFP antibodies to assess protein expression. The association of Rap1a with Gβ 2 was enhanced when Radil was overexpressed (compare lanes 2 and 3) ( n = 3). *, nonspecific band. B , formation of the Gβγ·Rap1a·Radil complex requires active Rap1a. HEK293T cells were transfected with expression plasmids for Strep-HA-Radil and HA-Rap1a only ( lane 2 ) or together with a vector coding for FLAG-Rap1GAP ( lane 3 ). Strep-HA-Radil was purified using streptavidin-Sepharose beads, and its association with Rap1 and Gβ was monitored by a Western blot using α-HA and α-Gβ antibodies, respectively. The expression of Rap1GAP was followed using α-FLAG antibodies. Although HA-Rap1a and Gβ bound to Strep-HA-Radil ( lane 2 ), these interactions were compromised in the presence of overexpressed FLAG-Rap1GAP ( lane 3 ) ( n = 4). C , expression of a constitutively active Rap1a mutant (Rap1aQ63E) promotes the interaction of Gβ with Radil. Strep-HA-Radil expressing stable cells were transfected or not with HA-Rap1aQ63E. 48 h after transfection, Radil was affinity-purified with streptavidin-Sepharose beads for 1.5 h, and the eluates were analyzed by Western blot using the indicated antibodies. Untransfected HEK293T cells were used as the negative control for streptavidin purification. AP , affinity purification. M r , molecular weights. K , ×1000.

Techniques Used: Transfection, Plasmid Preparation, Affinity Purification, Western Blot, Purification, Expressing, Mutagenesis, Negative Control

Identification of Radil as a novel interactor of Gβγ subunits of heterotrimeric G proteins. A , shown is the protein-protein interaction network of Gβ 2 , Gγ 2 , and Radil. Single-headed arrows represent interactions found in Gβ 2 ( red ), Gγ 2 ( blue ), and Radil (purple) pulldown experiments, color coded according to the color of the bait. Dark double-sided arrows represent proteins reciprocally identified using the other as bait. Gβ 2 ( n = 3), Gγ 2 ( n = 2), and Radil pulldown assays ( n = 2) were performed in HEK293T and HT1080 cells. Analysis of the tandem affinity-purified Gβ 2 or Gγ 2 protein complexes using mass spectrometry reveals several known Gβγ interactors. The small GTP-binding protein Rap1a and the newly characterized protein Radil were also identified in the Gβ 2 complexes. The reciprocal analysis of Radil protein complexes confirmed Radil as a Gβγ-associated protein and also revealed that it binds small G proteins of the Ras family. B , streptavidin affinity purification ( AP ) of Strep-HA-Radil ( left panel ) and Strep-HA-Gγ 2 ( right panel ) and immunodetection of co-purified endogenous Gβ, Rap1a, or Radil as indicated validates the mass spectrometry results. WB , Western blot. C , left panel , HEK293T cells were transiently transfected with expression vectors coding for FLAG-GFP, FLAG-Radil, or the closely related FLAG-AF6, and proteins were immunoprecipitated ( IP )using α-FLAG M2-conjugated agarose beads followed by a Western blot with α-FLAG ( top panel ) or α-Gβ ( bottom panel ) antibodies ( n = 3). Gβ co-immunoprecipitates with FLAG-Radil but not with FLAG-GFP or FLAG-AF6. Right panel , a schematic representation of Radil and AF6 proteins shows the similarity between the two proteins containing RA, DIL, and PDZ domains.
Figure Legend Snippet: Identification of Radil as a novel interactor of Gβγ subunits of heterotrimeric G proteins. A , shown is the protein-protein interaction network of Gβ 2 , Gγ 2 , and Radil. Single-headed arrows represent interactions found in Gβ 2 ( red ), Gγ 2 ( blue ), and Radil (purple) pulldown experiments, color coded according to the color of the bait. Dark double-sided arrows represent proteins reciprocally identified using the other as bait. Gβ 2 ( n = 3), Gγ 2 ( n = 2), and Radil pulldown assays ( n = 2) were performed in HEK293T and HT1080 cells. Analysis of the tandem affinity-purified Gβ 2 or Gγ 2 protein complexes using mass spectrometry reveals several known Gβγ interactors. The small GTP-binding protein Rap1a and the newly characterized protein Radil were also identified in the Gβ 2 complexes. The reciprocal analysis of Radil protein complexes confirmed Radil as a Gβγ-associated protein and also revealed that it binds small G proteins of the Ras family. B , streptavidin affinity purification ( AP ) of Strep-HA-Radil ( left panel ) and Strep-HA-Gγ 2 ( right panel ) and immunodetection of co-purified endogenous Gβ, Rap1a, or Radil as indicated validates the mass spectrometry results. WB , Western blot. C , left panel , HEK293T cells were transiently transfected with expression vectors coding for FLAG-GFP, FLAG-Radil, or the closely related FLAG-AF6, and proteins were immunoprecipitated ( IP )using α-FLAG M2-conjugated agarose beads followed by a Western blot with α-FLAG ( top panel ) or α-Gβ ( bottom panel ) antibodies ( n = 3). Gβ co-immunoprecipitates with FLAG-Radil but not with FLAG-GFP or FLAG-AF6. Right panel , a schematic representation of Radil and AF6 proteins shows the similarity between the two proteins containing RA, DIL, and PDZ domains.

Techniques Used: Affinity Purification, Mass Spectrometry, Binding Assay, Immunodetection, Purification, Western Blot, Transfection, Expressing, Immunoprecipitation

12) Product Images from "NH4Cl Treatment Prevents Tissue Calcification in Klotho Deficiency"

Article Title: NH4Cl Treatment Prevents Tissue Calcification in Klotho Deficiency

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2014030230

Effect of NH4 Cl Treatment on Phenotype, Body Weight, and Survival of kl/kl Mice
Figure Legend Snippet: Effect of NH4 Cl Treatment on Phenotype, Body Weight, and Survival of kl/kl Mice

Techniques Used: Mouse Assay

13) Product Images from "Palmitoylation of Desmoglein 2 Is a Regulator of Assembly Dynamics and Protein Turnover *"

Article Title: Palmitoylation of Desmoglein 2 Is a Regulator of Assembly Dynamics and Protein Turnover *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.739458

Palmitoylation-deficient Dsg2 localizes to a perinuclear subcellular compartment. A–L , A431 cells stably expressing wild-type Dsg2/GFP ( A–C ), Dsg2/GFP 7mut ( D–F ), Dsg2/GFP 5mut ( G–I ), and Dsg2/GFP CACS ( J–L ) were immunostained using anti-desmoplakin antibody, and co-localization of GFP with desmoplakin is shown. The localization of Dsg2/GFP fusion proteins was determined in at least three independent cultures grown on individual coverslips.  Scale bar  = 10 μm.  M , whole cell lysates were prepared from A431 cells expressing wild-type Dsg2/GFP and Dsg2/GFP CACS. Lysates were subjected to immunoblot ( IB ) analysis. Immunoblot analysis was performed using cell lysates prepared from three different cultures. These data demonstrate equal expression of the GFP fusion proteins.  N , A431 cells expressing Dsg2/GFP and Dsg2/GFP CACS were separated into Triton X-100-insoluble ( P ) and Triton X-100-soluble ( S ) fractions and subjected to immunoblot analysis using anti-GFP.  O , quantitation of soluble and insoluble fractions shown in  N . The lysates were prepared from four independent cultures, and immunoblot analysis band intensities were collected using LiCor Odyssey infrared scanning. Student's  t  test was performed to determine differences in solubility (*,  p
Figure Legend Snippet: Palmitoylation-deficient Dsg2 localizes to a perinuclear subcellular compartment. A–L , A431 cells stably expressing wild-type Dsg2/GFP ( A–C ), Dsg2/GFP 7mut ( D–F ), Dsg2/GFP 5mut ( G–I ), and Dsg2/GFP CACS ( J–L ) were immunostained using anti-desmoplakin antibody, and co-localization of GFP with desmoplakin is shown. The localization of Dsg2/GFP fusion proteins was determined in at least three independent cultures grown on individual coverslips. Scale bar = 10 μm. M , whole cell lysates were prepared from A431 cells expressing wild-type Dsg2/GFP and Dsg2/GFP CACS. Lysates were subjected to immunoblot ( IB ) analysis. Immunoblot analysis was performed using cell lysates prepared from three different cultures. These data demonstrate equal expression of the GFP fusion proteins. N , A431 cells expressing Dsg2/GFP and Dsg2/GFP CACS were separated into Triton X-100-insoluble ( P ) and Triton X-100-soluble ( S ) fractions and subjected to immunoblot analysis using anti-GFP. O , quantitation of soluble and insoluble fractions shown in N . The lysates were prepared from four independent cultures, and immunoblot analysis band intensities were collected using LiCor Odyssey infrared scanning. Student's t test was performed to determine differences in solubility (*, p

Techniques Used: Stable Transfection, Expressing, Quantitation Assay, Solubility

14) Product Images from "Study on site‐specific expression of bone formation and resorption factors in human dental follicles. Study on site‐specific expression of bone formation and resorption factors in human dental follicles"

Article Title: Study on site‐specific expression of bone formation and resorption factors in human dental follicles. Study on site‐specific expression of bone formation and resorption factors in human dental follicles

Journal: European Journal of Oral Sciences

doi: 10.1111/eos.12568

Effects of forskolin (FSK) on tumour necrosis factor (ligand) superfamily, member 11 ( RANKL ) and tumour necrosis factor receptor superfamily, member 11b ( OPG ) gene expression levels in cultured human dental follicle cells (HDFCs). The HDFCs from four patients were precultured in MEM Alpha medium ( α ‐MEM) with or without the presence of osteogenic induction medium for 14 d and thereafter cultured in α ‐MEM with FSK (10 −6 M) for the stated time periods. The results are presented as the average of two different experiments performed in duplicate. The expression levels of RANKL (A) and OPG (B), measured by RT‐qPCR, are adjusted by standardization based on the expression levels of glucuronidase, beta ( GUSB ) and are presented as fold increases relative to the control over time (0, 24, and 48 h). The RANKL/OPG ratios are presented as mean ± SEM ( n = 5). (C) Ratios of RANKL/OPG gene expression are presented as ▵▵Ct values.
Figure Legend Snippet: Effects of forskolin (FSK) on tumour necrosis factor (ligand) superfamily, member 11 ( RANKL ) and tumour necrosis factor receptor superfamily, member 11b ( OPG ) gene expression levels in cultured human dental follicle cells (HDFCs). The HDFCs from four patients were precultured in MEM Alpha medium ( α ‐MEM) with or without the presence of osteogenic induction medium for 14 d and thereafter cultured in α ‐MEM with FSK (10 −6 M) for the stated time periods. The results are presented as the average of two different experiments performed in duplicate. The expression levels of RANKL (A) and OPG (B), measured by RT‐qPCR, are adjusted by standardization based on the expression levels of glucuronidase, beta ( GUSB ) and are presented as fold increases relative to the control over time (0, 24, and 48 h). The RANKL/OPG ratios are presented as mean ± SEM ( n = 5). (C) Ratios of RANKL/OPG gene expression are presented as ▵▵Ct values.

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

15) Product Images from "Aurora B Interacts with NIR-p53, Leading to p53 Phosphorylation in Its DNA-binding Domain and Subsequent Functional Suppression *"

Article Title: Aurora B Interacts with NIR-p53, Leading to p53 Phosphorylation in Its DNA-binding Domain and Subsequent Functional Suppression *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.174755

Identification of Aurora B as a novel NIR-interacting protein. A , the elution products of tandem affinity purification from 293T cells stably expressing S/FLAG/SBP ( SFB )-tagged NIR were separated by SDS-PAGE and visualized by Coomassie Blue staining. The SFB-NIR band is shown on the  right. B , association between endogenous NIR and Aurora B. Whole cell lysates from U2OS cells were immunoprecipitated ( IP ) with antibody specific for NIR or control IgG and immunoblotted with antibodies against NIR or Aurora B ( AurB ) as indicated.  C , plasmid encoding Myc-tagged Aurora B was co-transfected with empty vector or plasmid encoding S/FLAG-tagged NIR. Cell lysates were then subjected to immunoprecipitation with S-Sepharose and blotted with antibodies against Myc ( AurB ) or FLAG ( NIR ). Input lysates were probed with anti-Myc antibody.  D , plasmid encoding Myc-tagged NIR was co-transfected with or without plasmid encoding S/FLAG-tagged Aurora B ( S/F-AurB ). Immunoprecipitation and blotting were performed as described in  C. E , U2OS cells were transfected with plasmid encoding FLAG-tagged Aurora B. After 24 h, cells were stained with anti-FLAG and NIR antibodies, counterstained with DAPI, and observed under a fluorescence microscope.
Figure Legend Snippet: Identification of Aurora B as a novel NIR-interacting protein. A , the elution products of tandem affinity purification from 293T cells stably expressing S/FLAG/SBP ( SFB )-tagged NIR were separated by SDS-PAGE and visualized by Coomassie Blue staining. The SFB-NIR band is shown on the right. B , association between endogenous NIR and Aurora B. Whole cell lysates from U2OS cells were immunoprecipitated ( IP ) with antibody specific for NIR or control IgG and immunoblotted with antibodies against NIR or Aurora B ( AurB ) as indicated. C , plasmid encoding Myc-tagged Aurora B was co-transfected with empty vector or plasmid encoding S/FLAG-tagged NIR. Cell lysates were then subjected to immunoprecipitation with S-Sepharose and blotted with antibodies against Myc ( AurB ) or FLAG ( NIR ). Input lysates were probed with anti-Myc antibody. D , plasmid encoding Myc-tagged NIR was co-transfected with or without plasmid encoding S/FLAG-tagged Aurora B ( S/F-AurB ). Immunoprecipitation and blotting were performed as described in C. E , U2OS cells were transfected with plasmid encoding FLAG-tagged Aurora B. After 24 h, cells were stained with anti-FLAG and NIR antibodies, counterstained with DAPI, and observed under a fluorescence microscope.

Techniques Used: Affinity Purification, Stable Transfection, Expressing, SDS Page, Staining, Immunoprecipitation, Plasmid Preparation, Transfection, Fluorescence, Microscopy

16) Product Images from "Requirement of TCF7L2 for TGF-?-dependent Transcriptional Activation of the TMEPAI Gene *"

Article Title: Requirement of TCF7L2 for TGF-?-dependent Transcriptional Activation of the TMEPAI Gene *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.132209

TGF-β-dependent interaction of TCF7L2 with TTE. A , depiction of Smad3 mutants. MH , mad homology region. B , interaction of Smad3 with TCF7L2 via its MH2 domain. Immunoprecipitations were carried out using anti-FLAG M5 antibody, and coimmunoprecipitated
Figure Legend Snippet: TGF-β-dependent interaction of TCF7L2 with TTE. A , depiction of Smad3 mutants. MH , mad homology region. B , interaction of Smad3 with TCF7L2 via its MH2 domain. Immunoprecipitations were carried out using anti-FLAG M5 antibody, and coimmunoprecipitated

Techniques Used:

17) Product Images from "RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3 *"

Article Title: RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.644633

Anti-PHLPP antibodies abrogate fragment N2-mediated inhibition of Akt Ser-473 phosphorylation in oocytes expressing FGFR. A , anti-PHLPP and anti-Sin1 antibodies were injected into oocytes expressing FGFR(MDA). They were then stimulated or not with 5 n
Figure Legend Snippet: Anti-PHLPP antibodies abrogate fragment N2-mediated inhibition of Akt Ser-473 phosphorylation in oocytes expressing FGFR. A , anti-PHLPP and anti-Sin1 antibodies were injected into oocytes expressing FGFR(MDA). They were then stimulated or not with 5 n

Techniques Used: Inhibition, Expressing, Injection

The SH2-SH3-SH2 RasGAP fragment does not inhibit phosphorylation of Akt Ser-473 in insulin-stimulated oocytes. Naive oocytes were microinjected with the SH2-SH3-SH2 construct of RasGAP alone and together with the indicated combinations of anti-PHLPP and
Figure Legend Snippet: The SH2-SH3-SH2 RasGAP fragment does not inhibit phosphorylation of Akt Ser-473 in insulin-stimulated oocytes. Naive oocytes were microinjected with the SH2-SH3-SH2 construct of RasGAP alone and together with the indicated combinations of anti-PHLPP and

Techniques Used: Construct

Schematic representation of the impact of full-length RasGAP and its caspase-3-generated fragments on FGFR1 signaling. Top , binding of full-length RasGAP to the FGF1 receptor is required for Akt to be incorporated in the FGFR signaling complex. In this
Figure Legend Snippet: Schematic representation of the impact of full-length RasGAP and its caspase-3-generated fragments on FGFR1 signaling. Top , binding of full-length RasGAP to the FGF1 receptor is required for Akt to be incorporated in the FGFR signaling complex. In this

Techniques Used: Generated, Binding Assay

Inhibition of Akt recruitment to the plasma membrane and expression of individual RasGAP SH domains block FGF-mediated ERK2 phosphorylation. Oocytes expressing FGFR(MDA) for 48 h were treated as described in the legend to . They were then microinjected
Figure Legend Snippet: Inhibition of Akt recruitment to the plasma membrane and expression of individual RasGAP SH domains block FGF-mediated ERK2 phosphorylation. Oocytes expressing FGFR(MDA) for 48 h were treated as described in the legend to . They were then microinjected

Techniques Used: Inhibition, Expressing, Blocking Assay

18) Product Images from "Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment"

Article Title: Dendritic Cell-derived Extracellular Vesicles mediate Mesenchymal Stem/Stromal Cell recruitment

Journal: Scientific Reports

doi: 10.1038/s41598-017-01809-x

Stimulation of MSC with DC-derived EV is not cytotoxic and do not affect significantly their proliferation nor differentiation capacity. ( a ) MSC were incubated alone, with DC-derived EV (10 μg of protein), total conditioned media or 100 K ultracentrifugation supernatant (UC control) for 14 days. At days 1, 7 and 14, MSC were incubated with resazurin and conversion to resorufin was determined by fluorescence measurement (RFU = relative fluorescence units). Relative fluorescence at each timepoint normalized to day 0 is depicted. ( b ) MSC were incubated alone (Control), with 50 μg protein from EV, total conditioned media (CM), or 100 K ultracentrifugation supernatant (UC control) for 24 h, before staining for the Ki67(-AlexaFluor 647; pink) proliferation marker and DAPI for nuclei (blue). Scale bar: 100 μm. ( b ) Stained cells were imaged by fluorescence microscopy in 20 random fields, the total number and the number of Ki67 +  cells were counted, and are represented as a ratio to non-stimulated control condition (dashed line) (n = 4 different DC donors). All bar graphs represent average ± SD. n = 3 different DC donors. ( d ) Fold-change gene expression by RT-qPCR (average ± SD) of osteogenic markers (ALP and RUNX2) and a chondrogenic marker (SOX9) of MSC stimulated with DC-derived EV, osteogenic media (osteo: supplemented with Dexamethasone 10 −7  M, β-glycerophosphate 10 −2  M and ascorbic acid 5 × 10 −5  M), conditioned media (CM), or protein extracted from complete media (including cytokines) that was not in contact with cells (media), comparatively to MSC in basal conditions (dashed line; n = 2 different DC donors). ( e ) ALP activity staining (pink/red), associated with early osteogenic differentiation, of MSC cultured in the presence of the indicated stimuli for 14 days. Although ALP activity staining is similar in stimulated conditions, only osteogenic media lead to morphological changes of MSC characteristic their osteogenic commitment. Scale bar: 1 mm.
Figure Legend Snippet: Stimulation of MSC with DC-derived EV is not cytotoxic and do not affect significantly their proliferation nor differentiation capacity. ( a ) MSC were incubated alone, with DC-derived EV (10 μg of protein), total conditioned media or 100 K ultracentrifugation supernatant (UC control) for 14 days. At days 1, 7 and 14, MSC were incubated with resazurin and conversion to resorufin was determined by fluorescence measurement (RFU = relative fluorescence units). Relative fluorescence at each timepoint normalized to day 0 is depicted. ( b ) MSC were incubated alone (Control), with 50 μg protein from EV, total conditioned media (CM), or 100 K ultracentrifugation supernatant (UC control) for 24 h, before staining for the Ki67(-AlexaFluor 647; pink) proliferation marker and DAPI for nuclei (blue). Scale bar: 100 μm. ( b ) Stained cells were imaged by fluorescence microscopy in 20 random fields, the total number and the number of Ki67 + cells were counted, and are represented as a ratio to non-stimulated control condition (dashed line) (n = 4 different DC donors). All bar graphs represent average ± SD. n = 3 different DC donors. ( d ) Fold-change gene expression by RT-qPCR (average ± SD) of osteogenic markers (ALP and RUNX2) and a chondrogenic marker (SOX9) of MSC stimulated with DC-derived EV, osteogenic media (osteo: supplemented with Dexamethasone 10 −7  M, β-glycerophosphate 10 −2 M and ascorbic acid 5 × 10 −5  M), conditioned media (CM), or protein extracted from complete media (including cytokines) that was not in contact with cells (media), comparatively to MSC in basal conditions (dashed line; n = 2 different DC donors). ( e ) ALP activity staining (pink/red), associated with early osteogenic differentiation, of MSC cultured in the presence of the indicated stimuli for 14 days. Although ALP activity staining is similar in stimulated conditions, only osteogenic media lead to morphological changes of MSC characteristic their osteogenic commitment. Scale bar: 1 mm.

Techniques Used: Derivative Assay, Incubation, Fluorescence, Staining, Marker, Microscopy, Expressing, Quantitative RT-PCR, ALP Assay, Activity Assay, Cell Culture

19) Product Images from "Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *"

Article Title: Phosphorylation of Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) at Threonine 72 in Spindle Assembly *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.591545

Localization of Thr(P) 72 TPX2 in HeLa and 293 cells. Mitotic ( A and B ) and interphase ( C ) HeLa cells were stained with Abs directed against Thr(P) 72 TPX2, TPX2, and tubulin or with the Thr(P) 72 Abs pre-absorbed with blocking peptide at different ratios. A , in mitotic cells, TPX2 phosphorylated at Thr 72 is localized in the cytosol and does not strictly associate with the mitotic spindle. B , HeLa cells stained with pan-TPX2 and Thr(P)72 TPX2 Abs preincubated with Thr(P)72 blocking peptides. C , during interphase, Thr(P) 72 TPX2 is localized in the nucleus. Note that the expression levels of Thr(P) 72 are much lower in interphase cells than in mitotic cells. Note that only the Thr(P) 72 signal was blocked. D , representative photographs of mitotic 293 cells transfected with GFP-TPX2 WT (WT) and GFP-TPX2 T72A (T72A). Scatter plots show the GFP signal at microtubules relative to total GFP signal. GFP-TPX2 T72A is significantly enriched on microtubules when compared with GFP-TPX2 WT (GFP-TPX2 WT (0.26 ± 0.01, n = 29) versus GFP-TPX2 T72A (0.39 ± 0.02, n = 22), group (mean ± S.E.); ***, p
Figure Legend Snippet: Localization of Thr(P) 72 TPX2 in HeLa and 293 cells. Mitotic ( A and B ) and interphase ( C ) HeLa cells were stained with Abs directed against Thr(P) 72 TPX2, TPX2, and tubulin or with the Thr(P) 72 Abs pre-absorbed with blocking peptide at different ratios. A , in mitotic cells, TPX2 phosphorylated at Thr 72 is localized in the cytosol and does not strictly associate with the mitotic spindle. B , HeLa cells stained with pan-TPX2 and Thr(P)72 TPX2 Abs preincubated with Thr(P)72 blocking peptides. C , during interphase, Thr(P) 72 TPX2 is localized in the nucleus. Note that the expression levels of Thr(P) 72 are much lower in interphase cells than in mitotic cells. Note that only the Thr(P) 72 signal was blocked. D , representative photographs of mitotic 293 cells transfected with GFP-TPX2 WT (WT) and GFP-TPX2 T72A (T72A). Scatter plots show the GFP signal at microtubules relative to total GFP signal. GFP-TPX2 T72A is significantly enriched on microtubules when compared with GFP-TPX2 WT (GFP-TPX2 WT (0.26 ± 0.01, n = 29) versus GFP-TPX2 T72A (0.39 ± 0.02, n = 22), group (mean ± S.E.); ***, p

Techniques Used: Staining, Blocking Assay, Expressing, Transfection

Overactivation of Aurora A and increased spindle length, a measure of Eg5 activity, in TPX2 T72A-expressing cells. A–C show 293 mitotic cells (prometaphase/metaphase) previously transfected with GFP-TPX2 WT (WT) or GFP-TPX2 T72A (T72A) expression vectors. A , representative photographs of WT- and T72A-transfected cells stained for Thr(P) 288 , a phosphoresidue indicative of the activity of Aurora kinase A. Dotted circles identify the poles. Scatter plots show the P-Aurora signal at centrosomes relative to total GFP signal. GFP-TPX2 T72A induces higher Aurora A activity than GFP-TPX2 WT (GFP-TPX2 WT (0.07 ± 0.01, n = 13) versus GFP-TPX2 T72A (0.14 ± 0.03, n = 18); *, p
Figure Legend Snippet: Overactivation of Aurora A and increased spindle length, a measure of Eg5 activity, in TPX2 T72A-expressing cells. A–C show 293 mitotic cells (prometaphase/metaphase) previously transfected with GFP-TPX2 WT (WT) or GFP-TPX2 T72A (T72A) expression vectors. A , representative photographs of WT- and T72A-transfected cells stained for Thr(P) 288 , a phosphoresidue indicative of the activity of Aurora kinase A. Dotted circles identify the poles. Scatter plots show the P-Aurora signal at centrosomes relative to total GFP signal. GFP-TPX2 T72A induces higher Aurora A activity than GFP-TPX2 WT (GFP-TPX2 WT (0.07 ± 0.01, n = 13) versus GFP-TPX2 T72A (0.14 ± 0.03, n = 18); *, p

Techniques Used: Activity Assay, Expressing, Transfection, Staining

Effects of GFP-TPX2 T72A on the polarity of mitotic spindles in HeLa cells with or without endogenous TPX2. A , representative photographs of mitotic HeLa cells at prometaphase and metaphase with monopolar, bipolar, and multipolar mitotic spindle poles. Scale bar , 10 μm. B , Western blots showing the levels of endogenous TPX2, GFP-TPX2 WT, and GFP-TPX2 T72A in cells with intact levels of TPX2. C, bar graphs showing the number of cells with different mono-, bi-, or multipolar mitotic spindles in each group. Cells with mitotic spindles were fixed and stained with Cy3-conjugated tubulin for MT visualization. GFP-TPX2 T72A expression results in a significant increase in the percentage of cells with multipolar spindles in the presence of endogenous TPX2. ANOVA comparing the three groups shows high significance with p
Figure Legend Snippet: Effects of GFP-TPX2 T72A on the polarity of mitotic spindles in HeLa cells with or without endogenous TPX2. A , representative photographs of mitotic HeLa cells at prometaphase and metaphase with monopolar, bipolar, and multipolar mitotic spindle poles. Scale bar , 10 μm. B , Western blots showing the levels of endogenous TPX2, GFP-TPX2 WT, and GFP-TPX2 T72A in cells with intact levels of TPX2. C, bar graphs showing the number of cells with different mono-, bi-, or multipolar mitotic spindles in each group. Cells with mitotic spindles were fixed and stained with Cy3-conjugated tubulin for MT visualization. GFP-TPX2 T72A expression results in a significant increase in the percentage of cells with multipolar spindles in the presence of endogenous TPX2. ANOVA comparing the three groups shows high significance with p

Techniques Used: Western Blot, Staining, Expressing

20) Product Images from "Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells"

Article Title: Tissue Source and Cell Expansion Condition Influence Phenotypic Changes of Adipose-Derived Stem Cells

Journal: Stem Cells International

doi: 10.1155/2017/7108458

Light micrographs of Alizarin red S staining. ASCs bought from a commercial vendor (RB) were compared to ASCs derived from abdominoplasty (HAP) or burned human skin (BH) cultured in α MEM with 10% FBS and 10% hPL and differentiated under osteogenic conditions with identical supplements for 14 days, then stained to assess mineralization. Representative images of undifferentiated RB (a), HAP cells (b, c), and BH cells (d, e) from control media conditions and differentiated RB (f), HAP cells (g, h), and BH (i, j) are given. White scale bar represents 100 μ m.
Figure Legend Snippet: Light micrographs of Alizarin red S staining. ASCs bought from a commercial vendor (RB) were compared to ASCs derived from abdominoplasty (HAP) or burned human skin (BH) cultured in α MEM with 10% FBS and 10% hPL and differentiated under osteogenic conditions with identical supplements for 14 days, then stained to assess mineralization. Representative images of undifferentiated RB (a), HAP cells (b, c), and BH cells (d, e) from control media conditions and differentiated RB (f), HAP cells (g, h), and BH (i, j) are given. White scale bar represents 100 μ m.

Techniques Used: Staining, Derivative Assay, Cell Culture

21) Product Images from "Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification *"

Article Title: Osterix Regulates Calcification and Degradation of Chondrogenic Matrices through Matrix Metalloproteinase 13 (MMP13) Expression in Association with Transcription Factor Runx2 during Endochondral Ossification *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.337063

Osterix and Runx2 required for up-regulation of MMP13. A , up-regulation of MMP13 by Osterix was determined by real-time PCR. Limb bud cells isolated from Osterix knock-out mice were infected with control or Osterix adenovirus, and RNA was isolated from the cells. Values represent means ± S.D. B , ATDC5 cells were transfected with the MMP13 gene promoter luciferase construct and then infected with control ( Cont ) or Osterix adenovirus. Luciferase activity in the cell lysates was measured. Values represent means ± S.D. C , limb bud cells of wild-type or Osterix knock-out mice (Δ/Δ) were infected with control, Runx2, and/or Osterix adenovirus, and RNA was subjected to real-time PCR analysis. Values represent means ± S.D. D , co-immunoprecipitation experiment using nuclear extracts containing 293 cells transfected with FLAG-Runx2 and/or Myc-Osterix. Co-immunoprecipitated Myc-Osterix with FLAG-Runx2 is shown by the red arrow. IP , immunoprecipitation; WB , Western blotting. E , 293 cells were transfected with DsRed-tagged-Runx2 and Venus-tagged-Osterix and then monitored under confocal microscopy. F , ATDC5 cells infected control ( Cont ) or Myc-Osterix adenovirus were subjected to a ChIP assay. Immunoprecipitated chromatin samples with anti-Myc antibody and input samples were determined by real-time PCR analysis using a specific Taqman probe against the ∼500 bp upstream region of the MMP13 gene promoter. G , limb bud cells of wild-type or Osterix knock-out mice (Δ/Δ) were subjected to micromass culture. The cells were infected with control, Osterix, or both MMP13 and Cre adenovirus and were subsequently cultured in the presence or absence of BMP2 for 7 days; cells were then stained with alcian blue ( top panel ) or alizarin red ( lower panel ).
Figure Legend Snippet: Osterix and Runx2 required for up-regulation of MMP13. A , up-regulation of MMP13 by Osterix was determined by real-time PCR. Limb bud cells isolated from Osterix knock-out mice were infected with control or Osterix adenovirus, and RNA was isolated from the cells. Values represent means ± S.D. B , ATDC5 cells were transfected with the MMP13 gene promoter luciferase construct and then infected with control ( Cont ) or Osterix adenovirus. Luciferase activity in the cell lysates was measured. Values represent means ± S.D. C , limb bud cells of wild-type or Osterix knock-out mice (Δ/Δ) were infected with control, Runx2, and/or Osterix adenovirus, and RNA was subjected to real-time PCR analysis. Values represent means ± S.D. D , co-immunoprecipitation experiment using nuclear extracts containing 293 cells transfected with FLAG-Runx2 and/or Myc-Osterix. Co-immunoprecipitated Myc-Osterix with FLAG-Runx2 is shown by the red arrow. IP , immunoprecipitation; WB , Western blotting. E , 293 cells were transfected with DsRed-tagged-Runx2 and Venus-tagged-Osterix and then monitored under confocal microscopy. F , ATDC5 cells infected control ( Cont ) or Myc-Osterix adenovirus were subjected to a ChIP assay. Immunoprecipitated chromatin samples with anti-Myc antibody and input samples were determined by real-time PCR analysis using a specific Taqman probe against the ∼500 bp upstream region of the MMP13 gene promoter. G , limb bud cells of wild-type or Osterix knock-out mice (Δ/Δ) were subjected to micromass culture. The cells were infected with control, Osterix, or both MMP13 and Cre adenovirus and were subsequently cultured in the presence or absence of BMP2 for 7 days; cells were then stained with alcian blue ( top panel ) or alizarin red ( lower panel ).

Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Knock-Out, Mouse Assay, Infection, Transfection, Luciferase, Construct, Activity Assay, Immunoprecipitation, Western Blot, Confocal Microscopy, Chromatin Immunoprecipitation, Cell Culture, Staining

22) Product Images from "Role of Apoptosis Signal-regulating Kinase 1 (ASK1) as an Activator of the GAPDH-Siah1 Stress-Signaling Cascade *"

Article Title: Role of Apoptosis Signal-regulating Kinase 1 (ASK1) as an Activator of the GAPDH-Siah1 Stress-Signaling Cascade *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.596205

Siah1 and GAPDH bind ASK1. A , Siah1-ASK1 and GAPDH-ASK1 binding in mouse brain extracts. Mouse brain extracts were immunoprecipitated with anti-Siah1 or anti-GAPDH antibodies and analyzed by Western blot with anti-ASK1, GAPDH, and Siah1 antibodies. Input
Figure Legend Snippet: Siah1 and GAPDH bind ASK1. A , Siah1-ASK1 and GAPDH-ASK1 binding in mouse brain extracts. Mouse brain extracts were immunoprecipitated with anti-Siah1 or anti-GAPDH antibodies and analyzed by Western blot with anti-ASK1, GAPDH, and Siah1 antibodies. Input

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot

ASK1 phosphorylates Siah1 in cells and in vitro . A , phosphorylation of Siah1 by ASK1 in vitro. In vitro phosphorylation assays were performed by incubation of recombinant GST-Siah1 or GST with constitutively active GST-ASK1 (aa 649–946) in the
Figure Legend Snippet: ASK1 phosphorylates Siah1 in cells and in vitro . A , phosphorylation of Siah1 by ASK1 in vitro. In vitro phosphorylation assays were performed by incubation of recombinant GST-Siah1 or GST with constitutively active GST-ASK1 (aa 649–946) in the

Techniques Used: In Vitro, Incubation, Recombinant

ASK1 facilitates GAPDH-Siah1 binding in cells. Upper panel , ASK1 augments GAPDH-Siah1 binding in a kinase-dependent manner. Cell lysates of HEK293 cells expressing Myc-tagged wild-type ( WT ) Siah1 together with HA-WT ASK1, kinase-dead (KD) ASK1, or constitutively
Figure Legend Snippet: ASK1 facilitates GAPDH-Siah1 binding in cells. Upper panel , ASK1 augments GAPDH-Siah1 binding in a kinase-dependent manner. Cell lysates of HEK293 cells expressing Myc-tagged wild-type ( WT ) Siah1 together with HA-WT ASK1, kinase-dead (KD) ASK1, or constitutively

Techniques Used: Binding Assay, Expressing

Siah1 and GAPDH form a ternary complex with ASK1 in cells. HEK293 cells expressing FLAG-ASK1, HA-GAPDH, and Myc-Siah1 were treated with 1 m m H 2 O 2 for 30 min to induce activation of ASK1 ( lower panel ). Cell lysates were immunoprecipitated in sequence with
Figure Legend Snippet: Siah1 and GAPDH form a ternary complex with ASK1 in cells. HEK293 cells expressing FLAG-ASK1, HA-GAPDH, and Myc-Siah1 were treated with 1 m m H 2 O 2 for 30 min to induce activation of ASK1 ( lower panel ). Cell lysates were immunoprecipitated in sequence with

Techniques Used: Expressing, Activation Assay, Immunoprecipitation, Sequencing

ASK1 augments nuclear translocation of GAPDH and p300 acetylation in cells. A , ASK1 augments nuclear translocation of GAPDH in a kinase-dependent manner. Effects of ASK1 kinase activity on nuclear translocation of GAPDH were examined in HEK293 cells expressing
Figure Legend Snippet: ASK1 augments nuclear translocation of GAPDH and p300 acetylation in cells. A , ASK1 augments nuclear translocation of GAPDH in a kinase-dependent manner. Effects of ASK1 kinase activity on nuclear translocation of GAPDH were examined in HEK293 cells expressing

Techniques Used: Translocation Assay, Activity Assay, Expressing

GAPDH-Siah1 binding is independent of p38 and JNK signaling. GAPDH-Siah1-ASK1 interactions occur independent of p38 and JNK activation. A , representative figure in which GAPDH-Siah1-ASK1 interactions were assessed in lysates from HEK293 treated with 10
Figure Legend Snippet: GAPDH-Siah1 binding is independent of p38 and JNK signaling. GAPDH-Siah1-ASK1 interactions occur independent of p38 and JNK activation. A , representative figure in which GAPDH-Siah1-ASK1 interactions were assessed in lysates from HEK293 treated with 10

Techniques Used: Binding Assay, Activation Assay

ASK1 directly binds to Siah1 and is modulated by GAPDH in vitro . A , recombinant GST-ASK1 (aa 1–940) or GST were incubated with recombinant Siah1 in vitro and subjected to GST pull-down, followed Western blot with an anti-Siah1 antibody. Input
Figure Legend Snippet: ASK1 directly binds to Siah1 and is modulated by GAPDH in vitro . A , recombinant GST-ASK1 (aa 1–940) or GST were incubated with recombinant Siah1 in vitro and subjected to GST pull-down, followed Western blot with an anti-Siah1 antibody. Input

Techniques Used: In Vitro, Recombinant, Incubation, Western Blot

23) Product Images from "Degradation of Tiam1 by Casein Kinase 1 and the SCFβTrCP Ubiquitin Ligase Controls the Duration of mTOR-S6K Signaling *"

Article Title: Degradation of Tiam1 by Casein Kinase 1 and the SCFβTrCP Ubiquitin Ligase Controls the Duration of mTOR-S6K Signaling *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.575571

Tiam1 interacts with and is ubiquitylated by the SCF βTrCP ubiquitin ligase. A , peptide coverage of Tiam1 in the mass spectrometry analysis of βTrCP2 immunopurification. Amino acid sequences of detected Tiam1 peptides are highlighted in yellow. B , peptide coverage of βTrCP2 (also known as FBXW11) in the mass spectrometry analysis of Tiam1 immunopurification. Amino acid sequences of detected βTrCP2 peptides are highlighted in yellow. C , the indicated FLAG-tagged F-box proteins containing WD40 repeats (FBXWs) or an empty vector ( EV ) were expressed in HEK293T cells. Forty-eight hours after transfection, cells were treated for 5 h with the proteasome inhibitors MG132, then harvested, and lysed. Whole cell extracts ( WCE ) were immunoprecipitated ( IP ) with anti-FLAG antibody and immunoblotted with antibodies specific for the indicated proteins. D , HEK293T cells were transfected with GFP-tagged wild type Tiam1 or an empty vector. Cells were collected and lysed. Tiam1 was immunoprecipitated from whole cell extracts, and immunocomplexes were analyzed by immunoblotting with antibodies specific for the indicated proteins. E , arginine 447 in the WD40 repeat of βTrCP2 is required for the interaction with Tiam1. HEK293T cells were transfected as indicated and analyzed as in C. F , Tiam1, Skp1, Cul1, and Rbx1 were expressed in HEK293T in the absence or presence of βTrCP1 or an inactive βTrCP1-ΔF-box mutant. After immunopurification with anti-FLAG resin, an in vitro ubiquitylation assay of Tiam1 was performed. Samples were analyzed by immunoblotting with an anti-Tiam1 antibody. The bracket indicates a ladder of bands corresponding to polyubiquitylated Tiam1.
Figure Legend Snippet: Tiam1 interacts with and is ubiquitylated by the SCF βTrCP ubiquitin ligase. A , peptide coverage of Tiam1 in the mass spectrometry analysis of βTrCP2 immunopurification. Amino acid sequences of detected Tiam1 peptides are highlighted in yellow. B , peptide coverage of βTrCP2 (also known as FBXW11) in the mass spectrometry analysis of Tiam1 immunopurification. Amino acid sequences of detected βTrCP2 peptides are highlighted in yellow. C , the indicated FLAG-tagged F-box proteins containing WD40 repeats (FBXWs) or an empty vector ( EV ) were expressed in HEK293T cells. Forty-eight hours after transfection, cells were treated for 5 h with the proteasome inhibitors MG132, then harvested, and lysed. Whole cell extracts ( WCE ) were immunoprecipitated ( IP ) with anti-FLAG antibody and immunoblotted with antibodies specific for the indicated proteins. D , HEK293T cells were transfected with GFP-tagged wild type Tiam1 or an empty vector. Cells were collected and lysed. Tiam1 was immunoprecipitated from whole cell extracts, and immunocomplexes were analyzed by immunoblotting with antibodies specific for the indicated proteins. E , arginine 447 in the WD40 repeat of βTrCP2 is required for the interaction with Tiam1. HEK293T cells were transfected as indicated and analyzed as in C. F , Tiam1, Skp1, Cul1, and Rbx1 were expressed in HEK293T in the absence or presence of βTrCP1 or an inactive βTrCP1-ΔF-box mutant. After immunopurification with anti-FLAG resin, an in vitro ubiquitylation assay of Tiam1 was performed. Samples were analyzed by immunoblotting with an anti-Tiam1 antibody. The bracket indicates a ladder of bands corresponding to polyubiquitylated Tiam1.

Techniques Used: Mass Spectrometry, Immu-Puri, Plasmid Preparation, Transfection, Immunoprecipitation, Mutagenesis, In Vitro, Ubiquitin Assay

24) Product Images from "miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis"

Article Title: miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis

Journal: Oncotarget

doi:

microRNA levels are altered during osteogenesis Osteoblast differentiation was induced in MC3T3 cells with dexamethasone, β-glycerophosphate and ascorbic acid. A. ALP staining detected ALP activity in cells grown with osteogenic differentiation but not with basal media (without osteogenic differentiation supplements); Alizarin Red S staining detected presence of calcium deposits (mineralization) in cells grown in osteogenic differentiation media but not in basal media (10X; scale: 100 μm). B. ALP, RUNX2, OSX and OPN mRNA levels were measured by quantitative real-time PCR. GAPDH was used as reference control. Expression levels at day 1 (D1), day 3 (D3), day 7 (D7) and day 14 (D14) of differentiation (dif) were normalized to expression levels of cells grown in basal conditions for the same time points (mean±SD, N = 4; * P
Figure Legend Snippet: microRNA levels are altered during osteogenesis Osteoblast differentiation was induced in MC3T3 cells with dexamethasone, β-glycerophosphate and ascorbic acid. A. ALP staining detected ALP activity in cells grown with osteogenic differentiation but not with basal media (without osteogenic differentiation supplements); Alizarin Red S staining detected presence of calcium deposits (mineralization) in cells grown in osteogenic differentiation media but not in basal media (10X; scale: 100 μm). B. ALP, RUNX2, OSX and OPN mRNA levels were measured by quantitative real-time PCR. GAPDH was used as reference control. Expression levels at day 1 (D1), day 3 (D3), day 7 (D7) and day 14 (D14) of differentiation (dif) were normalized to expression levels of cells grown in basal conditions for the same time points (mean±SD, N = 4; * P

Techniques Used: ALP Assay, Staining, Activity Assay, Real-time Polymerase Chain Reaction, Expressing

25) Product Images from "Anabolic and Antiresorptive Modulation of Bone Homeostasis by the Epigenetic Modulator Sulforaphane, a Naturally Occurring Isothiocyanate *"

Article Title: Anabolic and Antiresorptive Modulation of Bone Homeostasis by the Epigenetic Modulator Sulforaphane, a Naturally Occurring Isothiocyanate *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.678235

DMSO and SFN show structural similarities and analogous biological effects. DMSO and SFN contain a polar sulfoxide functional group. SFN carries an additional butane group with a terminal isothiocyanate group ( A ). DMSO and SFN increase matrix mineralization in MC3T3-E1 cells at 14 days of differentiation as revealed by Alizarin Red staining ( B ).
Figure Legend Snippet: DMSO and SFN show structural similarities and analogous biological effects. DMSO and SFN contain a polar sulfoxide functional group. SFN carries an additional butane group with a terminal isothiocyanate group ( A ). DMSO and SFN increase matrix mineralization in MC3T3-E1 cells at 14 days of differentiation as revealed by Alizarin Red staining ( B ).

Techniques Used: Functional Assay, Staining

26) Product Images from "The Transcription Factor Protein Sox11 Enhances Early Osteoblast Differentiation by Facilitating Proliferation and the Survival of Mesenchymal and Osteoblast Progenitors *"

Article Title: The Transcription Factor Protein Sox11 Enhances Early Osteoblast Differentiation by Facilitating Proliferation and the Survival of Mesenchymal and Osteoblast Progenitors *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.413377

Endogenous expression of Sox11 in different cell lines, bone tissues and during osteoblast differentiation of MC3T3-E1 cells. A , RT-PCR analysis of Sox11 expression in MC3T3-E1, MLOY4, primary calvaria, ATDC5 cells and in adult mouse calvaria and long
Figure Legend Snippet: Endogenous expression of Sox11 in different cell lines, bone tissues and during osteoblast differentiation of MC3T3-E1 cells. A , RT-PCR analysis of Sox11 expression in MC3T3-E1, MLOY4, primary calvaria, ATDC5 cells and in adult mouse calvaria and long

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

The effect of Sox11 knockdown on cell number, proliferation, cell viability, and apoptosis in MC3T3-E1 and primary mouse calvaria cells. A , after Sox11 knockdown, the total cell number of MC3T3-E1 and primary mouse calvaria cells ( a b ) were
Figure Legend Snippet: The effect of Sox11 knockdown on cell number, proliferation, cell viability, and apoptosis in MC3T3-E1 and primary mouse calvaria cells. A , after Sox11 knockdown, the total cell number of MC3T3-E1 and primary mouse calvaria cells ( a b ) were

Techniques Used:

27) Product Images from "Winner for Outstanding Research in the Ph.D. Category for the 2013 Society for Biomaterials Meeting and Exposition, April 10-13, 2013, Boston, Massachusetts"

Article Title: Winner for Outstanding Research in the Ph.D. Category for the 2013 Society for Biomaterials Meeting and Exposition, April 10-13, 2013, Boston, Massachusetts

Journal: Journal of biomedical materials research. Part A

doi: 10.1002/jbm.a.34611

Cell viability of MSC or ASC in COL/FIB or COL/FIB/HA microbeads at days 1 and 7. Scale bar = 100 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure Legend Snippet: Cell viability of MSC or ASC in COL/FIB or COL/FIB/HA microbeads at days 1 and 7. Scale bar = 100 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Techniques Used:

Morphology and size distribution of acellular microbeads. Coomasie staining allowed for visualization of microbeads under light microscopy (4×, 10×). Images of the microarchitecture of microbeads obtained through confocal reflectance (60×).
Figure Legend Snippet: Morphology and size distribution of acellular microbeads. Coomasie staining allowed for visualization of microbeads under light microscopy (4×, 10×). Images of the microarchitecture of microbeads obtained through confocal reflectance (60×).

Techniques Used: Staining, Light Microscopy

Confocal reflectance imaging of MSC or ASC in COL/FIB or COL/FIB/HA microbeads at days 1 and 7. Scale bar = 50 μm.
Figure Legend Snippet: Confocal reflectance imaging of MSC or ASC in COL/FIB or COL/FIB/HA microbeads at days 1 and 7. Scale bar = 50 μm.

Techniques Used: Imaging

Schematic of microbead fabrication process. COL/FIB and COL/FIB/HA microbeads were formed through a water-in-oil emulsification process which resulted in spherical three-dimensional cell-seeded hydrogel microenvironments. [Color figure can be viewed in
Figure Legend Snippet: Schematic of microbead fabrication process. COL/FIB and COL/FIB/HA microbeads were formed through a water-in-oil emulsification process which resulted in spherical three-dimensional cell-seeded hydrogel microenvironments. [Color figure can be viewed in

Techniques Used:

28) Product Images from "Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *"

Article Title: Eukaryotic Elongation Factor 1A Interacts with Sphingosine Kinase and Directly Enhances Its Catalytic Activity *

Journal:

doi: 10.1074/jbc.M708782200

eEF1A1 interacts with and activates SK2 in vitro . A , purified recombinant His-tagged SK2 (rec-SK2) was incubated with GST-eEF1A1 or GST alone control bound to GSH-Sepharose. SK2 associated with GST-eEF1A1 or GST control was detected by IB with anti-HIS
Figure Legend Snippet: eEF1A1 interacts with and activates SK2 in vitro . A , purified recombinant His-tagged SK2 (rec-SK2) was incubated with GST-eEF1A1 or GST alone control bound to GSH-Sepharose. SK2 associated with GST-eEF1A1 or GST control was detected by IB with anti-HIS

Techniques Used: In Vitro, Purification, Recombinant, Incubation

Phosphorylation of SK1 does not alter binding to eEF1A1. A , GST-SK1 bound to GSH-Sepharose was phosphorylated using ERK2, as assessed through IB with anti-phospho-SK1 antibodies. GST-SK1 and GST-phospho-SK1 (GST-pSK1) were then incubated with lysates
Figure Legend Snippet: Phosphorylation of SK1 does not alter binding to eEF1A1. A , GST-SK1 bound to GSH-Sepharose was phosphorylated using ERK2, as assessed through IB with anti-phospho-SK1 antibodies. GST-SK1 and GST-phospho-SK1 (GST-pSK1) were then incubated with lysates

Techniques Used: Binding Assay, Incubation

eEF1A1 interacts with SK1. A , GST-SK1 bound to GSH-Sepharose was incubated with lysates from HEK-293T cells expressing HA-tagged eEF1A1. eEF1A1 pulled down by the GST-SK1 was detected by IB with anti-HA. B , GST-eEF1A1 bound to GSH-Sepharose was incubated
Figure Legend Snippet: eEF1A1 interacts with SK1. A , GST-SK1 bound to GSH-Sepharose was incubated with lysates from HEK-293T cells expressing HA-tagged eEF1A1. eEF1A1 pulled down by the GST-SK1 was detected by IB with anti-HA. B , GST-eEF1A1 bound to GSH-Sepharose was incubated

Techniques Used: Incubation, Expressing

Phosphorylation of eEF1A1 does not alter binding to SK1 or SK2. A , GST-eEF1A1 bound to GSH-Sepharose was incubated with S6K in the presence of γ- 32 P]ATP, and the phosphorylation of GST-eEF1A1 was detected using phosphorimaging ( upper panel
Figure Legend Snippet: Phosphorylation of eEF1A1 does not alter binding to SK1 or SK2. A , GST-eEF1A1 bound to GSH-Sepharose was incubated with S6K in the presence of γ- 32 P]ATP, and the phosphorylation of GST-eEF1A1 was detected using phosphorimaging ( upper panel

Techniques Used: Binding Assay, Incubation

29) Product Images from "Dual Functions of the Trans-2-Enoyl-CoA Reductase TER in the Sphingosine 1-Phosphate Metabolic Pathway and in Fatty Acid Elongation *"

Article Title: Dual Functions of the Trans-2-Enoyl-CoA Reductase TER in the Sphingosine 1-Phosphate Metabolic Pathway and in Fatty Acid Elongation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.571869

The pathways of sphingolipid degradation and synthesis. A , sphingolipid degradation pathway and involved enzymes. Complex sphingolipids are degraded to SPH by lysosomal degrading enzymes. After SPH is phosphorylated, the resulting S1P is metabolized to
Figure Legend Snippet: The pathways of sphingolipid degradation and synthesis. A , sphingolipid degradation pathway and involved enzymes. Complex sphingolipids are degraded to SPH by lysosomal degrading enzymes. After SPH is phosphorylated, the resulting S1P is metabolized to

Techniques Used:

Blockage of the S1P metabolic pathway in tsc13 Δ cells is recovered by expression of mammalian TER. A , ABY83 ( tsc13 Δ cells bearing the pTW6 plasmid encoding TSC13 (p TSC13 ) and URA3 ; ABY83/pTW6) were transfected with the pAKNF313 ( HIS3 vector,
Figure Legend Snippet: Blockage of the S1P metabolic pathway in tsc13 Δ cells is recovered by expression of mammalian TER. A , ABY83 ( tsc13 Δ cells bearing the pTW6 plasmid encoding TSC13 (p TSC13 ) and URA3 ; ABY83/pTW6) were transfected with the pAKNF313 ( HIS3 vector,

Techniques Used: Expressing, Plasmid Preparation, Transfection

The S1P metabolic pathway is impaired in membrane fractions prepared from TER knockdown HeLa cells. A , reactions and cofactors in the S1P metabolic pathway as well as the expected products of the S1P metabolites by methyl esterification and KIO 4 /KMnO
Figure Legend Snippet: The S1P metabolic pathway is impaired in membrane fractions prepared from TER knockdown HeLa cells. A , reactions and cofactors in the S1P metabolic pathway as well as the expected products of the S1P metabolites by methyl esterification and KIO 4 /KMnO

Techniques Used:

Yeast Tsc13 and mammalian TER are involved in the S1P metabolic pathway. A , metabolic pathways of SPH, DHS, and palmitic acid to sphingolipids and glycerophospholipids. Most of the enzymes are common to metabolisms of SPH and DHS (reactions 1–4).
Figure Legend Snippet: Yeast Tsc13 and mammalian TER are involved in the S1P metabolic pathway. A , metabolic pathways of SPH, DHS, and palmitic acid to sphingolipids and glycerophospholipids. Most of the enzymes are common to metabolisms of SPH and DHS (reactions 1–4).

Techniques Used:

30) Product Images from "Nanofibers Regulate Single Bone Marrow Stem Cell Osteogenesis via FAK/RhoA/YAP1 Pathway"

Article Title: Nanofibers Regulate Single Bone Marrow Stem Cell Osteogenesis via FAK/RhoA/YAP1 Pathway

Journal: ACS applied materials & interfaces

doi: 10.1021/acsami.8b11449

Morphology and differentiation of a single BMSC on the nanofibers were modulated by a RhoA/ROCK pathway. (a) ALP staining of a single BMSC with the addition of ROCK inhibitor Y-27632 on the NF-MP and FF-MP microislands, separately. (b) Cell area of a single BMSC with the addition of Y-27632 on the NF-MP and FF-MP microislands, separately. (c, d) The effect of Y-27632 on a single BMSC osteogenic differentiation on the NF-MP and FF-MP microislands. Scale bar: 20 μ m.
Figure Legend Snippet: Morphology and differentiation of a single BMSC on the nanofibers were modulated by a RhoA/ROCK pathway. (a) ALP staining of a single BMSC with the addition of ROCK inhibitor Y-27632 on the NF-MP and FF-MP microislands, separately. (b) Cell area of a single BMSC with the addition of Y-27632 on the NF-MP and FF-MP microislands, separately. (c, d) The effect of Y-27632 on a single BMSC osteogenic differentiation on the NF-MP and FF-MP microislands. Scale bar: 20 μ m.

Techniques Used: ALP Assay, Staining

31) Product Images from "Palmitoylation of Desmoglein 2 Is a Regulator of Assembly Dynamics and Protein Turnover *"

Article Title: Palmitoylation of Desmoglein 2 Is a Regulator of Assembly Dynamics and Protein Turnover *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.739458

Palmitoylation-deficient Dsg2 localizes to a perinuclear subcellular compartment. A–L , A431 cells stably expressing wild-type Dsg2/GFP ( A–C ), Dsg2/GFP 7mut ( D–F ), Dsg2/GFP 5mut ( G–I ), and Dsg2/GFP CACS ( J–L ) were immunostained using anti-desmoplakin antibody, and co-localization of GFP with desmoplakin is shown. The localization of Dsg2/GFP fusion proteins was determined in at least three independent cultures grown on individual coverslips.  Scale bar  = 10 μm.  M , whole cell lysates were prepared from A431 cells expressing wild-type Dsg2/GFP and Dsg2/GFP CACS. Lysates were subjected to immunoblot ( IB ) analysis. Immunoblot analysis was performed using cell lysates prepared from three different cultures. These data demonstrate equal expression of the GFP fusion proteins.  N , A431 cells expressing Dsg2/GFP and Dsg2/GFP CACS were separated into Triton X-100-insoluble ( P ) and Triton X-100-soluble ( S ) fractions and subjected to immunoblot analysis using anti-GFP.  O , quantitation of soluble and insoluble fractions shown in  N . The lysates were prepared from four independent cultures, and immunoblot analysis band intensities were collected using LiCor Odyssey infrared scanning. Student's  t  test was performed to determine differences in solubility (*,  p
Figure Legend Snippet: Palmitoylation-deficient Dsg2 localizes to a perinuclear subcellular compartment. A–L , A431 cells stably expressing wild-type Dsg2/GFP ( A–C ), Dsg2/GFP 7mut ( D–F ), Dsg2/GFP 5mut ( G–I ), and Dsg2/GFP CACS ( J–L ) were immunostained using anti-desmoplakin antibody, and co-localization of GFP with desmoplakin is shown. The localization of Dsg2/GFP fusion proteins was determined in at least three independent cultures grown on individual coverslips. Scale bar = 10 μm. M , whole cell lysates were prepared from A431 cells expressing wild-type Dsg2/GFP and Dsg2/GFP CACS. Lysates were subjected to immunoblot ( IB ) analysis. Immunoblot analysis was performed using cell lysates prepared from three different cultures. These data demonstrate equal expression of the GFP fusion proteins. N , A431 cells expressing Dsg2/GFP and Dsg2/GFP CACS were separated into Triton X-100-insoluble ( P ) and Triton X-100-soluble ( S ) fractions and subjected to immunoblot analysis using anti-GFP. O , quantitation of soluble and insoluble fractions shown in N . The lysates were prepared from four independent cultures, and immunoblot analysis band intensities were collected using LiCor Odyssey infrared scanning. Student's t test was performed to determine differences in solubility (*, p

Techniques Used: Stable Transfection, Expressing, Quantitation Assay, Solubility

32) Product Images from "Differential Regulation of Mitogen- and Stress-activated Protein Kinase-1 and -2 (MSK1 and MSK2) by CK2 following UV Radiation *"

Article Title: Differential Regulation of Mitogen- and Stress-activated Protein Kinase-1 and -2 (MSK1 and MSK2) by CK2 following UV Radiation *

Journal:

doi: 10.1074/jbc.M109.083808

p65-Ser 276 phosphorylation is induced following UV-C radiation in a MSK2-dependent manner. A , MDA-MB-231 cells were serum-starved for 24 h; pretreated with vehicle (DMSO), DMAT (10 μ m ), SB203580 (10 μ m ), or H89 (10 μ m ) in serum-free
Figure Legend Snippet: p65-Ser 276 phosphorylation is induced following UV-C radiation in a MSK2-dependent manner. A , MDA-MB-231 cells were serum-starved for 24 h; pretreated with vehicle (DMSO), DMAT (10 μ m ), SB203580 (10 μ m ), or H89 (10 μ m ) in serum-free

Techniques Used: Multiple Displacement Amplification

33) Product Images from "In Vivo and in Vitro Studies of a Functional Peroxisome Proliferator-activated Receptor ? Response Element in the Mousepdx-1 Promoter *"

Article Title: In Vivo and in Vitro Studies of a Functional Peroxisome Proliferator-activated Receptor ? Response Element in the Mousepdx-1 Promoter *

Journal:

doi: 10.1074/jbc.M801813200

Quantitative PCR analysis ( A ), PDX-1 immunoblot ( B ), and glucose-stimulated insulin secretion ( C ), in isolated islets from 8-week-old male floxed control (Cre - ) and PANC PPARγ -/- mice (Cre + ). A, quantitative PCR analysis of various genes from
Figure Legend Snippet: Quantitative PCR analysis ( A ), PDX-1 immunoblot ( B ), and glucose-stimulated insulin secretion ( C ), in isolated islets from 8-week-old male floxed control (Cre - ) and PANC PPARγ -/- mice (Cre + ). A, quantitative PCR analysis of various genes from

Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Mouse Assay

PDX-1 immunoblot post 72 h of incubation with troglitazone or the diluent DMSO in isolated islets from 8-week-old male floxed control (Cre - ) and PANC PPARγ -/- mice (Cre + ). Isolated islets were cultured 72 h in medium with 10 μ m troglitazone
Figure Legend Snippet: PDX-1 immunoblot post 72 h of incubation with troglitazone or the diluent DMSO in isolated islets from 8-week-old male floxed control (Cre - ) and PANC PPARγ -/- mice (Cre + ). Isolated islets were cultured 72 h in medium with 10 μ m troglitazone

Techniques Used: Incubation, Isolation, Mouse Assay, Cell Culture

Chromatin immunoprecipitation assay of BTC6 cells. 500–600-bp chromatin preparations of BTC6 cells were prepared as described under “Experimental Procedures.” A, they were immunoprecipitated using mouse monoclonal PPARγ
Figure Legend Snippet: Chromatin immunoprecipitation assay of BTC6 cells. 500–600-bp chromatin preparations of BTC6 cells were prepared as described under “Experimental Procedures.” A, they were immunoprecipitated using mouse monoclonal PPARγ

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation

In vitro translated PPARγ and RXR-α are binding partners for acyl-CoA oxidase, mouse pdx-1 , and human pdx-1 × PPRE probes. In vitro translated RXR-α and PPARγ proteins were used in the DNA binding reaction in
Figure Legend Snippet: In vitro translated PPARγ and RXR-α are binding partners for acyl-CoA oxidase, mouse pdx-1 , and human pdx-1 × PPRE probes. In vitro translated RXR-α and PPARγ proteins were used in the DNA binding reaction in

Techniques Used: In Vitro, Binding Assay

Tissue panel for PPARγ immunoblot ( A ), pancreas histology ( B ), pancreas morphometrics ( C ), and intraperitoneal glucose tolerance test results ( D ) in 8-week-old male floxed control (Cre - ) and PANC PPARγ -/- mice (Cre + ). A, representative
Figure Legend Snippet: Tissue panel for PPARγ immunoblot ( A ), pancreas histology ( B ), pancreas morphometrics ( C ), and intraperitoneal glucose tolerance test results ( D ) in 8-week-old male floxed control (Cre - ) and PANC PPARγ -/- mice (Cre + ). A, representative

Techniques Used: Mouse Assay

34) Product Images from "Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor"

Article Title: Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor

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

doi:

Coelution of GST-I-1(T35A) kinase and NCLK from various chromatographic columns. Bovine brain extract was chromatographed through a DEAE-Sephacel column. The flow-through fraction containing GST-I-1(T35A) kinase activity was subjected to SP-Sepharose ( A ), hydroxylapatite ( B ), and Superose 12 gel filtration ( C ) chromatographies sequentially. Aliquots from indicated fractions were withdrawn and assayed for GST-I-1(T35A) kinase and NCLK activities. Gel filtration was carried out with the Pharmacia FPLC system at a flow rate of 1 ml/min. The size of each fraction in A , B , and C was 5, 5, and 1 ml, respectively.
Figure Legend Snippet: Coelution of GST-I-1(T35A) kinase and NCLK from various chromatographic columns. Bovine brain extract was chromatographed through a DEAE-Sephacel column. The flow-through fraction containing GST-I-1(T35A) kinase activity was subjected to SP-Sepharose ( A ), hydroxylapatite ( B ), and Superose 12 gel filtration ( C ) chromatographies sequentially. Aliquots from indicated fractions were withdrawn and assayed for GST-I-1(T35A) kinase and NCLK activities. Gel filtration was carried out with the Pharmacia FPLC system at a flow rate of 1 ml/min. The size of each fraction in A , B , and C was 5, 5, and 1 ml, respectively.

Techniques Used: Flow Cytometry, Activity Assay, Filtration, Fast Protein Liquid Chromatography

35) Product Images from "Cell Cycle-dependent Regulation of Structure of Endoplasmic Reticulum and Inositol 1,4,5-Trisphosphate-induced Ca2+ Release in Mouse Oocytes and Embryos"

Article Title: Cell Cycle-dependent Regulation of Structure of Endoplasmic Reticulum and Inositol 1,4,5-Trisphosphate-induced Ca2+ Release in Mouse Oocytes and Embryos

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-07-0431

H1 kinase activity after fertilization, and treatment with MG132 or roscovitine. The presence of ER clusters in relation to cdk1-cyclin B activity, as measured by histone H1 kinase activity, requires manipulation of cdk1-cyclin B activity. The H1 kinase activity in MII oocytes has been set to 100% against which other treatments are compared. Fertilized one-cells at the Pb2 stage and Pn stage have reduced levels of H1 kinase activity, consistent with previous observations. Fertilized embryos treated with MG132 (50 μM) maintain an elevated level of H1 kinase activity. The cdk inhibitor roscovitine (75 μM) inhibits histone H1 kinase activity after incubation for 1 and 2 h. Data are from two experiments with two replicates on each day.
Figure Legend Snippet: H1 kinase activity after fertilization, and treatment with MG132 or roscovitine. The presence of ER clusters in relation to cdk1-cyclin B activity, as measured by histone H1 kinase activity, requires manipulation of cdk1-cyclin B activity. The H1 kinase activity in MII oocytes has been set to 100% against which other treatments are compared. Fertilized one-cells at the Pb2 stage and Pn stage have reduced levels of H1 kinase activity, consistent with previous observations. Fertilized embryos treated with MG132 (50 μM) maintain an elevated level of H1 kinase activity. The cdk inhibitor roscovitine (75 μM) inhibits histone H1 kinase activity after incubation for 1 and 2 h. Data are from two experiments with two replicates on each day.

Techniques Used: Activity Assay, Incubation

36) Product Images from "CD45-mediated control of TCR tuning in naïve and memory CD8+ T cells"

Article Title: CD45-mediated control of TCR tuning in naïve and memory CD8+ T cells

Journal: Nature Communications

doi: 10.1038/ncomms13373

TCR sensitivity of naïve versus memory CD8 + T cells. ( a ) Proliferation of CFSE-labelled CD44 lo naïve and CD44 hi memory-phenotype (MP) B6 CD8 + T cells after incubation with Cx-αCD3 mAb. ( b ) ERK phosphorylation in CD44 lo and CD44 hi B6 CD8 + T cells after incubation with S-αCD3 or Cx-αCD3 mAb. ( c , d ) Phosphorylation of ERK and ZAP70 ( c ) and densitometric levels (relative to β-actin; mean±s.d.) ( d ) in CD44 lo and CD44 hi B6 CD8 + T cells after incubation with S-αCD3 mAb. ( e ) Phosphorylation of ERK, ZAP-70, and PLCγ in CD44 lo CD5 lo and CD5 hi and CD44 hi B6 CD8 + T cells after incubation with S-αCD3 mAb. ( f , g ) ERK phosphorylation in CD44 lo and CD44 hi B6 CD8 + T cells after incubation with S- or Cx-αCD3±Cx-αCD28 ( f ) and S-αCD3±S- or Cx-αCD28 mAbs ( g ). ( h ) Phosphorylation of ERK, ZAP-70, and PLCγ in CD44 lo and CD44 hi CD8 + T cells after incubation with Cx-αCD3 and -αCD28 mAbs. ( i ) Flow cytometry for Ca 2+ flux in CD44 lo and CD44 hi (top) or CD44 lo CD5 lo and CD5 hi and CD44 hi CD8 + T cells (bottom) gated from Indo-1-loaded total B6 LN cells after incubation with the indicated stimuli. ( j ) Phosphorylation of ERK and AKT in CD44 lo and CD44 hi B6 CD8 + T cells at 2 and 24 h after incubation with Cx-αCD3 mAb±αIL-2 mAb blockade. Data are representative of three ( a , b , e – j ) and at least four independent experiments ( c , d ). Unpaired Student's t -test was used for the statistical analysis. * P
Figure Legend Snippet: TCR sensitivity of naïve versus memory CD8 + T cells. ( a ) Proliferation of CFSE-labelled CD44 lo naïve and CD44 hi memory-phenotype (MP) B6 CD8 + T cells after incubation with Cx-αCD3 mAb. ( b ) ERK phosphorylation in CD44 lo and CD44 hi B6 CD8 + T cells after incubation with S-αCD3 or Cx-αCD3 mAb. ( c , d ) Phosphorylation of ERK and ZAP70 ( c ) and densitometric levels (relative to β-actin; mean±s.d.) ( d ) in CD44 lo and CD44 hi B6 CD8 + T cells after incubation with S-αCD3 mAb. ( e ) Phosphorylation of ERK, ZAP-70, and PLCγ in CD44 lo CD5 lo and CD5 hi and CD44 hi B6 CD8 + T cells after incubation with S-αCD3 mAb. ( f , g ) ERK phosphorylation in CD44 lo and CD44 hi B6 CD8 + T cells after incubation with S- or Cx-αCD3±Cx-αCD28 ( f ) and S-αCD3±S- or Cx-αCD28 mAbs ( g ). ( h ) Phosphorylation of ERK, ZAP-70, and PLCγ in CD44 lo and CD44 hi CD8 + T cells after incubation with Cx-αCD3 and -αCD28 mAbs. ( i ) Flow cytometry for Ca 2+ flux in CD44 lo and CD44 hi (top) or CD44 lo CD5 lo and CD5 hi and CD44 hi CD8 + T cells (bottom) gated from Indo-1-loaded total B6 LN cells after incubation with the indicated stimuli. ( j ) Phosphorylation of ERK and AKT in CD44 lo and CD44 hi B6 CD8 + T cells at 2 and 24 h after incubation with Cx-αCD3 mAb±αIL-2 mAb blockade. Data are representative of three ( a , b , e – j ) and at least four independent experiments ( c , d ). Unpaired Student's t -test was used for the statistical analysis. * P

Techniques Used: Incubation, Flow Cytometry, Cytometry

Expression of TCR signalling proteins in CD8 + T cell subsets. ( a ) Expression levels of TCR signalling complex proteins, LCK, ZAP-70, SLP-76, PLCγ, LAT and PKCθ in CD44 lo B6 naïve CD5 lo and CD5 hi and CD44 hi MP CD8 + T cells, shown as fold change of densitometric levels (relative to β-actin; mean±s.d.). ( b,c ) Expression levels of LAT, PKCθ and CBL-B in 2C naïve, Ag MEM and HP MEM CD8 + T cells as in Fig. 3a ( b ) and CBL-B levels in B6 CD44 lo CD5 lo and CD5 hi and CD44 hi CD8 + T cells ( c ; mean±s.d.). ( d ) ERK phosphorylation in CD44 lo and CD44 hi CD8 + T cells from cblb +/+ , cblb +/− or cblb −/− mice after incubation with S-αCD3 mAb. ( e ) Expression (Western blot and densitometry) of SHP-1 and SHP-2 in CD44 lo B6 naïve CD5 lo and CD5 hi and CD44 hi MP CD8 + T cells (mean±s.d.). ( f ) Expression of PTPN22 (LYP) and PTPN2 (TCPTP) in CD44 lo B6 naïve CD5 lo and CD5 hi and CD44 hi MP CD8 + T cells. ( g ) ERK phosphorylation in CD44 lo naïve CD5 lo and CD5 hi cells and CD44 hi MP CD8 + T cells from ptpn2 +/+ ( Ptpn2 fl/fl ) and ptpn2 −/− ( Lck-cre.Ptpn2 fl/fl ) mice after incubation with S-αCD3 mAb. ( h ) ERK phosphorylation in CD44 lo and CD44 hi CD8 + T cells from shp1 +/+ and shp1 +/− mice (derived from SHP-1-deficient me v /me v mice) after incubation with S-αCD3 mAb. Data are representative of three ( a – e ) and two independent experiments ( f – g ). Unpaired Student's t -test was used for the statistical analysis. * P
Figure Legend Snippet: Expression of TCR signalling proteins in CD8 + T cell subsets. ( a ) Expression levels of TCR signalling complex proteins, LCK, ZAP-70, SLP-76, PLCγ, LAT and PKCθ in CD44 lo B6 naïve CD5 lo and CD5 hi and CD44 hi MP CD8 + T cells, shown as fold change of densitometric levels (relative to β-actin; mean±s.d.). ( b,c ) Expression levels of LAT, PKCθ and CBL-B in 2C naïve, Ag MEM and HP MEM CD8 + T cells as in Fig. 3a ( b ) and CBL-B levels in B6 CD44 lo CD5 lo and CD5 hi and CD44 hi CD8 + T cells ( c ; mean±s.d.). ( d ) ERK phosphorylation in CD44 lo and CD44 hi CD8 + T cells from cblb +/+ , cblb +/− or cblb −/− mice after incubation with S-αCD3 mAb. ( e ) Expression (Western blot and densitometry) of SHP-1 and SHP-2 in CD44 lo B6 naïve CD5 lo and CD5 hi and CD44 hi MP CD8 + T cells (mean±s.d.). ( f ) Expression of PTPN22 (LYP) and PTPN2 (TCPTP) in CD44 lo B6 naïve CD5 lo and CD5 hi and CD44 hi MP CD8 + T cells. ( g ) ERK phosphorylation in CD44 lo naïve CD5 lo and CD5 hi cells and CD44 hi MP CD8 + T cells from ptpn2 +/+ ( Ptpn2 fl/fl ) and ptpn2 −/− ( Lck-cre.Ptpn2 fl/fl ) mice after incubation with S-αCD3 mAb. ( h ) ERK phosphorylation in CD44 lo and CD44 hi CD8 + T cells from shp1 +/+ and shp1 +/− mice (derived from SHP-1-deficient me v /me v mice) after incubation with S-αCD3 mAb. Data are representative of three ( a – e ) and two independent experiments ( f – g ). Unpaired Student's t -test was used for the statistical analysis. * P

Techniques Used: Expressing, Mouse Assay, Incubation, Western Blot, Derivative Assay

TCR sensitivity of CD5 lo versus CD5 hi naïve CD8 + T cells. ( a ) Levels of tyrosine-phosphorylated CD3ζ chain (p-CD3ζ; 21 kDa) in freshly isolated CD5 lo and CD5 hi B6 naïve (CD44 lo ) CD8 + T cells (relative to β-actin; mean±s.d.). ( b ) Proliferation of CFSE-labelled CD5 lo and CD5 hi B6 naïve CD8 + T cells after incubation with IL-2, IL-7, or cross-linked anti-CD3 (Cx-αCD3) mAb. ( c – e ) ERK phosphorylation ( c ) and densitometric levels (relative to total ERK; d or β-actin; e ) of phosphorylated ERK ( d , e ), ZAP-70 ( e ), PLCγ ( e ), and AKT ( e ) in CD5 lo and CD5 hi B6 naïve CD8 + T cells after incubation with soluble anti-CD3 (S-αCD3) mAb (graphs in d and e show mean±s.d.). ( f , g ) Phosphorylation of ERK and ZAP70 ( f ) and levels of phosphorylated ZAP-70, PLCγ, ERK and AKT (relative to β-actin; mean±s.d.) ( g ) in CD5 lo and CD5 hi B6 naïve CD8 + T cells after incubation with Cx-αCD3 mAb. ( h ) Phosphorylation of ERK and STAT5 in CD5 lo and CD5 hi B6 naïve CD8 + T cells after S-αCD3 mAb incubation with or without IL-2. ( i ) CD5 levels by flow cytometry (top left) and S-αCD3 mAb-induced ERK phosphorylation by Western blot (top right) in naïve CD8 + T cells from HY, 2C and OT-I TCR Tg mice (top and bottom) or CD5 lo and CD5 hi naïve CD8 + T cells from B6 and HY mice (bottom); note that CD5 hi HY cells are TCR-clonotype negative. Data are representative of at least four ( a , c - g ) and three independent experiments ( b , h , i ). Unpaired Student's t -test was used for the statistical analysis. * P
Figure Legend Snippet: TCR sensitivity of CD5 lo versus CD5 hi naïve CD8 + T cells. ( a ) Levels of tyrosine-phosphorylated CD3ζ chain (p-CD3ζ; 21 kDa) in freshly isolated CD5 lo and CD5 hi B6 naïve (CD44 lo ) CD8 + T cells (relative to β-actin; mean±s.d.). ( b ) Proliferation of CFSE-labelled CD5 lo and CD5 hi B6 naïve CD8 + T cells after incubation with IL-2, IL-7, or cross-linked anti-CD3 (Cx-αCD3) mAb. ( c – e ) ERK phosphorylation ( c ) and densitometric levels (relative to total ERK; d or β-actin; e ) of phosphorylated ERK ( d , e ), ZAP-70 ( e ), PLCγ ( e ), and AKT ( e ) in CD5 lo and CD5 hi B6 naïve CD8 + T cells after incubation with soluble anti-CD3 (S-αCD3) mAb (graphs in d and e show mean±s.d.). ( f , g ) Phosphorylation of ERK and ZAP70 ( f ) and levels of phosphorylated ZAP-70, PLCγ, ERK and AKT (relative to β-actin; mean±s.d.) ( g ) in CD5 lo and CD5 hi B6 naïve CD8 + T cells after incubation with Cx-αCD3 mAb. ( h ) Phosphorylation of ERK and STAT5 in CD5 lo and CD5 hi B6 naïve CD8 + T cells after S-αCD3 mAb incubation with or without IL-2. ( i ) CD5 levels by flow cytometry (top left) and S-αCD3 mAb-induced ERK phosphorylation by Western blot (top right) in naïve CD8 + T cells from HY, 2C and OT-I TCR Tg mice (top and bottom) or CD5 lo and CD5 hi naïve CD8 + T cells from B6 and HY mice (bottom); note that CD5 hi HY cells are TCR-clonotype negative. Data are representative of at least four ( a , c - g ) and three independent experiments ( b , h , i ). Unpaired Student's t -test was used for the statistical analysis. * P

Techniques Used: Isolation, Incubation, Flow Cytometry, Cytometry, Western Blot, Mouse Assay

37) Product Images from "Dissociating β-Amyloid from α7 Nicotinic Acetylcholine Receptor by a Novel Therapeutic Agent, S 24795, Normalizes α7 Nicotinic Acetylcholine and NMDA Receptor Function in Alzheimer's Disease Brain"

Article Title: Dissociating β-Amyloid from α7 Nicotinic Acetylcholine Receptor by a Novel Therapeutic Agent, S 24795, Normalizes α7 Nicotinic Acetylcholine and NMDA Receptor Function in Alzheimer's Disease Brain

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.6088-08.2009

Aβ 15-20 is the primary Aβ 42 binding domain to α7nAChRs and the action site of S 24795 on dissociating Aβ 42 from Aβ 42 –α7nAChR complexes. A , Top, A representative blot depicting the level of Aβ 42 –α7nAChR complexes in the anti-Aβ 42 /-actin immunoprecipitates of lysates preparing from synaptosomes of postmortem FCX of control subjects incubated with Aβ 42 and Aβ 15-20 , Aβ 1-11 , Aβ 25-35 , or Aβ 37-43 . The levels of Aβ 42 -associated α7nAChRs and β-actin (immunoprecipitation and loading control) were detected in the anti-Aβ 42 /-actin immunoprecipitates by Western blotting with antibodies to α7nAChR and β-actin and quantified by densitometric scanning (bottom). Data are means ± SEM of the percentage inhibition by comparing optical intensity of the α7nAChR band of Aβ 42 plus the indicated Aβ peptides to Aβ 42 alone group derived from four independent determinations from an individual postmortem control brain. There were no discernible changes in β-actin level by any of the in vitro treatments. * p
Figure Legend Snippet: Aβ 15-20 is the primary Aβ 42 binding domain to α7nAChRs and the action site of S 24795 on dissociating Aβ 42 from Aβ 42 –α7nAChR complexes. A , Top, A representative blot depicting the level of Aβ 42 –α7nAChR complexes in the anti-Aβ 42 /-actin immunoprecipitates of lysates preparing from synaptosomes of postmortem FCX of control subjects incubated with Aβ 42 and Aβ 15-20 , Aβ 1-11 , Aβ 25-35 , or Aβ 37-43 . The levels of Aβ 42 -associated α7nAChRs and β-actin (immunoprecipitation and loading control) were detected in the anti-Aβ 42 /-actin immunoprecipitates by Western blotting with antibodies to α7nAChR and β-actin and quantified by densitometric scanning (bottom). Data are means ± SEM of the percentage inhibition by comparing optical intensity of the α7nAChR band of Aβ 42 plus the indicated Aβ peptides to Aβ 42 alone group derived from four independent determinations from an individual postmortem control brain. There were no discernible changes in β-actin level by any of the in vitro treatments. * p

Techniques Used: Binding Assay, Incubation, Immunoprecipitation, Western Blot, Inhibition, Derivative Assay, In Vitro

Hypothetical pathways through which S 24795 normalizes Aβ 42 ), soluble Aβ 42 or Aβ 42 ) followed immediately by rapid desensitizing and restricting Ca 2+ influx through α7nAChRs and downstream NMDAR activity and NMDAR signaling. In addition, Aβ 42 ). Similar to partial pharmacological blockade of NMDARs, Aβ 42 ) that further dampens excitatory neurotransmission. Treatment with S 24795 removes Aβ 42 monomers and/or oligomers from Aβ 42 –α7nAChR complexes. This relieves Aβ 42 -inhibited α7nAChR activity leading to an improved NMDAR–PSD-95 linkage and consequently the NMDAR signaling and activity. The augmented NMDAR signal and activation favor LTP induction and healthier dendritic spines resulting in more normal excitatory neurotransmission and cognitive processing. The red triangles indicate the effect of Aβ 42 , and the blue lines and triangles reflect the impact of S 24795.
Figure Legend Snippet: Hypothetical pathways through which S 24795 normalizes Aβ 42 ), soluble Aβ 42 or Aβ 42 ) followed immediately by rapid desensitizing and restricting Ca 2+ influx through α7nAChRs and downstream NMDAR activity and NMDAR signaling. In addition, Aβ 42 ). Similar to partial pharmacological blockade of NMDARs, Aβ 42 ) that further dampens excitatory neurotransmission. Treatment with S 24795 removes Aβ 42 monomers and/or oligomers from Aβ 42 –α7nAChR complexes. This relieves Aβ 42 -inhibited α7nAChR activity leading to an improved NMDAR–PSD-95 linkage and consequently the NMDAR signaling and activity. The augmented NMDAR signal and activation favor LTP induction and healthier dendritic spines resulting in more normal excitatory neurotransmission and cognitive processing. The red triangles indicate the effect of Aβ 42 , and the blue lines and triangles reflect the impact of S 24795.

Techniques Used: Activity Assay, Activation Assay

38) Product Images from "Characterization of Porcine Aortic Valvular Interstitial Cell 'Calcified' Nodules"

Article Title: Characterization of Porcine Aortic Valvular Interstitial Cell 'Calcified' Nodules

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048154

Transmission Electron Micrographs of cultured PAVICs. PAVICs were grown in A - CTL medium and B - OST+TGF-β1 medium (VIC – valvular interstitial cell, ECM – extracellular matrix) (scale = 2 µm).
Figure Legend Snippet: Transmission Electron Micrographs of cultured PAVICs. PAVICs were grown in A - CTL medium and B - OST+TGF-β1 medium (VIC – valvular interstitial cell, ECM – extracellular matrix) (scale = 2 µm).

Techniques Used: Transmission Assay, Cell Culture, CTL Assay

Raman spectroscopy of PAVICs and MOBs in culture compared through univariate and multivariate statistical analysis. A - Raman spectra comparing a representative MOBs mineralized nodule spectrum (black), PAVICs grown in CTL media mean spectrum (dark grey), and PAVICs cultured in OST+TGF-β1 medium mean spectrum (shaded box over entire spectrum). Vertical shaded areas highlight the 960 cm −1 apatite peak and the 1070 cm −1 carbonate peak spectral range. B - Higher magnification of the PAVICs mean spectrum grown in CTL (dark grey) and in OST+TGF-β1 medium (light grey). The red spectrum is the difference between the two PAVICs mean spectra. Shaded red bands highlight spectral ranges which discriminate between groups. C - Scatter plot showing group separation between PAVICs grown in CTL medium (red triangles) vs. PAVICs grown in OST+TGF-β1 medium (green circles). D - Latent variables loadings used in the interval partial least squares discriminant analysis (iPLS-DA) model, bands highlighted are spectral ranges which contributed to the distinction between groups.
Figure Legend Snippet: Raman spectroscopy of PAVICs and MOBs in culture compared through univariate and multivariate statistical analysis. A - Raman spectra comparing a representative MOBs mineralized nodule spectrum (black), PAVICs grown in CTL media mean spectrum (dark grey), and PAVICs cultured in OST+TGF-β1 medium mean spectrum (shaded box over entire spectrum). Vertical shaded areas highlight the 960 cm −1 apatite peak and the 1070 cm −1 carbonate peak spectral range. B - Higher magnification of the PAVICs mean spectrum grown in CTL (dark grey) and in OST+TGF-β1 medium (light grey). The red spectrum is the difference between the two PAVICs mean spectra. Shaded red bands highlight spectral ranges which discriminate between groups. C - Scatter plot showing group separation between PAVICs grown in CTL medium (red triangles) vs. PAVICs grown in OST+TGF-β1 medium (green circles). D - Latent variables loadings used in the interval partial least squares discriminant analysis (iPLS-DA) model, bands highlighted are spectral ranges which contributed to the distinction between groups.

Techniques Used: Raman Spectroscopy, CTL Assay, Cell Culture

Micrographs showing the morphology and staining of PAVICs and MOBs in culture. A,D,G - Phase contrast images of cultured PAVICs grown in CTL medium for 21 days, PAVICs grown in OST+TGF-β1 medium for 21 days, and MOBs grown in OST medium for 21 days respectively (scale = 100 µm). B,E,H - Alizarin Red S staining negative for PAVICs grown in CTL medium for 21 days, positive for PAVICs grown in OST+TGF-β1 medium for 21 days, and positive for MOBs grown in OST medium respectively (scale = 100 µm). C,F,I - SEM images of cultured PAVICs grown in CTL medium for 21 days, PAVICs grown in OST+TGF-β1 medium for 21 days, and MOBs grown in OST medium for 21 days respectively (scale = 50 µm).
Figure Legend Snippet: Micrographs showing the morphology and staining of PAVICs and MOBs in culture. A,D,G - Phase contrast images of cultured PAVICs grown in CTL medium for 21 days, PAVICs grown in OST+TGF-β1 medium for 21 days, and MOBs grown in OST medium for 21 days respectively (scale = 100 µm). B,E,H - Alizarin Red S staining negative for PAVICs grown in CTL medium for 21 days, positive for PAVICs grown in OST+TGF-β1 medium for 21 days, and positive for MOBs grown in OST medium respectively (scale = 100 µm). C,F,I - SEM images of cultured PAVICs grown in CTL medium for 21 days, PAVICs grown in OST+TGF-β1 medium for 21 days, and MOBs grown in OST medium for 21 days respectively (scale = 50 µm).

Techniques Used: Staining, Cell Culture, CTL Assay

39) Product Images from "In vitro and in vivo biocompatibility and osteogenesis of graphene-reinforced nanohydroxyapatite polyamide66 ternary biocomposite as orthopedic implant material"

Article Title: In vitro and in vivo biocompatibility and osteogenesis of graphene-reinforced nanohydroxyapatite polyamide66 ternary biocomposite as orthopedic implant material

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S105794

Confocal laser scanning microscope images of C3H10T1/2 cells cultured on nHA/PA66 ( A – C ) and G/nHA/PA66 ( D – F ) discs (scale bar =100 μm, magnification 200×). Abbreviations: G/nHA/PA66, graphene/nanohydroxyapatite/polyamide66; nHA/PA66, nanohydroxyapatite/polyamide66.
Figure Legend Snippet: Confocal laser scanning microscope images of C3H10T1/2 cells cultured on nHA/PA66 ( A – C ) and G/nHA/PA66 ( D – F ) discs (scale bar =100 μm, magnification 200×). Abbreviations: G/nHA/PA66, graphene/nanohydroxyapatite/polyamide66; nHA/PA66, nanohydroxyapatite/polyamide66.

Techniques Used: Laser-Scanning Microscopy, Cell Culture

qRT-PCR analysis of the mRNA level of ALP and OC in C3H10T1/2 cells cultured for 7 ( A ) and 14 ( B ) days. Note: * P
Figure Legend Snippet: qRT-PCR analysis of the mRNA level of ALP and OC in C3H10T1/2 cells cultured for 7 ( A ) and 14 ( B ) days. Note: * P

Techniques Used: Quantitative RT-PCR, ALP Assay, Cell Culture

40) Product Images from "Generation of Osteosarcomas from a Combination of Rb Silencing and c‐Myc Overexpression in Human Mesenchymal Stem Cells"

Article Title: Generation of Osteosarcomas from a Combination of Rb Silencing and c‐Myc Overexpression in Human Mesenchymal Stem Cells

Journal: Stem Cells Translational Medicine

doi: 10.5966/sctm.2015-0226

Combination of retinoblastoma (Rb) knockdown and c‐Myc overexpression caused phenotypic changes. (A): Flow cytometric analysis for cell surface markers in parental MSCs and MSCs with SiRb and c‐Myc overexpression (SiRb‐OeMyc). (B left ): Cells were induced for osteogenesis in osteogenic induction medium for 2 weeks, followed by ARS staining. (B right ): Stained dye was extracted, and OD values were measured. (C left ): Cells were induced for adipogenesis in adipogenic induction medium for 2 weeks, followed by Oil Red O staining. (C right ): Stained dye was extracted, and OD values were measured. Bars = 100 μm. The gene expression levels of RUNX2 at 7 days of osteogenic induction and PPARγ2 at 7 days of adipogenic induction were analyzed by quantitative reverse‐transcriptase polymerase chain reaction assay. (D): Compared with parental MSCs, SiRb‐OeMyc cells significantly increased in RUNX2 mRNA level (left), whereas they decreased in PPARγ2 mRNA level (right). ∗, p
Figure Legend Snippet: Combination of retinoblastoma (Rb) knockdown and c‐Myc overexpression caused phenotypic changes. (A): Flow cytometric analysis for cell surface markers in parental MSCs and MSCs with SiRb and c‐Myc overexpression (SiRb‐OeMyc). (B left ): Cells were induced for osteogenesis in osteogenic induction medium for 2 weeks, followed by ARS staining. (B right ): Stained dye was extracted, and OD values were measured. (C left ): Cells were induced for adipogenesis in adipogenic induction medium for 2 weeks, followed by Oil Red O staining. (C right ): Stained dye was extracted, and OD values were measured. Bars = 100 μm. The gene expression levels of RUNX2 at 7 days of osteogenic induction and PPARγ2 at 7 days of adipogenic induction were analyzed by quantitative reverse‐transcriptase polymerase chain reaction assay. (D): Compared with parental MSCs, SiRb‐OeMyc cells significantly increased in RUNX2 mRNA level (left), whereas they decreased in PPARγ2 mRNA level (right). ∗, p

Techniques Used: Over Expression, Flow Cytometry, Staining, Expressing, Polymerase Chain Reaction

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Incubation:

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Blocking Assay:

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