Structured Review

Millipore pfmsrp 2
In vitro protein binding assay of Pf MSP-1 with PfMSRP-1 and <t>PfMSRP-2.</t> (A) (Top) Coomassie gel (lane 1, GST-PfMSP-1; lane 2, His-PfMSRP-2) and Western blot analysis of fractions from a binding reaction using a mouse anti-His antibody (lane 1′, GST-PfMSP-1; lane 2′, His-PfMSRP-2; lane S, supernatant; lanes W1 to W3, washes 1 to 3; lane P, eluted fraction). (Bottom) Coomassie gel (lane 1, GST alone; lane 2, His-PfMSRP-2) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-His antibody. (B) (Top) Coomassie gel (lane 1, GST-PfMSRP-1; lane 2, His-PfMSP-1) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-GST antibody. (Bottom) Coomassie gel (lane 1, GST alone; lane 2, His-PfMSP-1) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-GST antibody.
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Images

1) Product Images from "A Multigene Family That Interacts with the Amino Terminus of Plasmodium MSP-1 Identified Using the Yeast Two-Hybrid System"

Article Title: A Multigene Family That Interacts with the Amino Terminus of Plasmodium MSP-1 Identified Using the Yeast Two-Hybrid System

Journal: Eukaryotic Cell

doi: 10.1128/EC.1.6.915-925.2002

In vitro protein binding assay of Pf MSP-1 with PfMSRP-1 and PfMSRP-2. (A) (Top) Coomassie gel (lane 1, GST-PfMSP-1; lane 2, His-PfMSRP-2) and Western blot analysis of fractions from a binding reaction using a mouse anti-His antibody (lane 1′, GST-PfMSP-1; lane 2′, His-PfMSRP-2; lane S, supernatant; lanes W1 to W3, washes 1 to 3; lane P, eluted fraction). (Bottom) Coomassie gel (lane 1, GST alone; lane 2, His-PfMSRP-2) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-His antibody. (B) (Top) Coomassie gel (lane 1, GST-PfMSRP-1; lane 2, His-PfMSP-1) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-GST antibody. (Bottom) Coomassie gel (lane 1, GST alone; lane 2, His-PfMSP-1) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-GST antibody.
Figure Legend Snippet: In vitro protein binding assay of Pf MSP-1 with PfMSRP-1 and PfMSRP-2. (A) (Top) Coomassie gel (lane 1, GST-PfMSP-1; lane 2, His-PfMSRP-2) and Western blot analysis of fractions from a binding reaction using a mouse anti-His antibody (lane 1′, GST-PfMSP-1; lane 2′, His-PfMSRP-2; lane S, supernatant; lanes W1 to W3, washes 1 to 3; lane P, eluted fraction). (Bottom) Coomassie gel (lane 1, GST alone; lane 2, His-PfMSRP-2) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-His antibody. (B) (Top) Coomassie gel (lane 1, GST-PfMSRP-1; lane 2, His-PfMSP-1) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-GST antibody. (Bottom) Coomassie gel (lane 1, GST alone; lane 2, His-PfMSP-1) and Western blot analysis of fractions (designated as explained above) from a binding reaction using a mouse anti-GST antibody.

Techniques Used: In Vitro, Protein Binding, Western Blot, Binding Assay

Colocalization of PfMSRP-1 and PfMSRP-2 with P. falciparum MSP-1 as demonstrated by immunofluorescence of thin blood smears of asynchronous cultures of P. falciparum 3D7 prepared as described in Materials and Methods. (a) Slides were incubated with a mouse anti-PfMSRP-1 serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (b and f) Slides were incubated with a guinea pig anti-Pf83a serum and a rhodamine-labeled donkey anti-guinea pig IgG secondary antibody. (c) Overlay of panels a and b. (d) Bright-field image of panels a, b, and c. (e) Slides were incubated with a mouse anti-PfMSRP-2 serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (g) Overlay of panels e and f. (h) Bright-field image of panels e, f, and g. (i) Slides were incubated with a preimmune guinea pig serum and a rhodamine-labeled donkey anti-guinea pig IgG secondary antibody. (j) Bright-field image of panel i. (k) Slides were incubated with normal mouse serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (l) Bright field image of panel k.
Figure Legend Snippet: Colocalization of PfMSRP-1 and PfMSRP-2 with P. falciparum MSP-1 as demonstrated by immunofluorescence of thin blood smears of asynchronous cultures of P. falciparum 3D7 prepared as described in Materials and Methods. (a) Slides were incubated with a mouse anti-PfMSRP-1 serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (b and f) Slides were incubated with a guinea pig anti-Pf83a serum and a rhodamine-labeled donkey anti-guinea pig IgG secondary antibody. (c) Overlay of panels a and b. (d) Bright-field image of panels a, b, and c. (e) Slides were incubated with a mouse anti-PfMSRP-2 serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (g) Overlay of panels e and f. (h) Bright-field image of panels e, f, and g. (i) Slides were incubated with a preimmune guinea pig serum and a rhodamine-labeled donkey anti-guinea pig IgG secondary antibody. (j) Bright-field image of panel i. (k) Slides were incubated with normal mouse serum and a fluorescein isothiocyanate-labeled rabbit anti-mouse IgG secondary antibody. (l) Bright field image of panel k.

Techniques Used: Immunofluorescence, Incubation, Labeling

(A) Northern blot analysis of total RNA isolated from asynchronous cultures of P. falciparum strain 3D7. Blots were probed using PfMSRP-1 (lane 1), PfMSRP-2 (lane 2), and PfMSRP-3 (lane 3). (B) RNA slot blots containing total RNA isolated from synchronized cultures of P. falciparum strain 3D7 as described in Materials and Methods. The filters were probed with PfMSRP-1, PfMSRP-2, PfMSRP-3, and a probe corresponding to the 5′ region of MSP-1. The blot was also probed with a primer specific for the P. falciparum small ribosomal subunit to normalize loading. Lanes 1, 15.0 μg of RNA; lanes 2, 10.0 μg of RNA; lanes 3, 5.0 μg of RNA; lanes 4, 1.0 μg of RNA.
Figure Legend Snippet: (A) Northern blot analysis of total RNA isolated from asynchronous cultures of P. falciparum strain 3D7. Blots were probed using PfMSRP-1 (lane 1), PfMSRP-2 (lane 2), and PfMSRP-3 (lane 3). (B) RNA slot blots containing total RNA isolated from synchronized cultures of P. falciparum strain 3D7 as described in Materials and Methods. The filters were probed with PfMSRP-1, PfMSRP-2, PfMSRP-3, and a probe corresponding to the 5′ region of MSP-1. The blot was also probed with a primer specific for the P. falciparum small ribosomal subunit to normalize loading. Lanes 1, 15.0 μg of RNA; lanes 2, 10.0 μg of RNA; lanes 3, 5.0 μg of RNA; lanes 4, 1.0 μg of RNA.

Techniques Used: Northern Blot, Isolation

Lack of cross-reactivity of antisera directed against recombinant PfMSRP-1 and PfMSRP-2. A 50.0-ng portion of PfMSRP-1 is run on each blot in lanes 1 to 4, and 50.0 ng of PfMSRP-2 is run on each of the blots in lanes 5 to 8. Lane 1, polyclonal mouse anti-GST IgG antibody as a positive control; lanes 2 and 7, mouse anti-PfMSRP-1; lanes 3 and 6, mouse anti-PfMSRP-2; lane 4, Coomassie stain of recombinant PfMSRP-1 used in the studies; lane 5, monoclonal mouse anti-His antibody; lane 8, Coomassie stain of recombinant PfMSRP-2 used in the studies. PfMSRP-1 and PfMSRP-2 did not show any reactivity when incubated with preimmune mouse serum or with the goat anti-mouse horseradish peroxidase-conjugated secondary antibody alone. The same molecular weight marker is run in lanes 1 and 5.
Figure Legend Snippet: Lack of cross-reactivity of antisera directed against recombinant PfMSRP-1 and PfMSRP-2. A 50.0-ng portion of PfMSRP-1 is run on each blot in lanes 1 to 4, and 50.0 ng of PfMSRP-2 is run on each of the blots in lanes 5 to 8. Lane 1, polyclonal mouse anti-GST IgG antibody as a positive control; lanes 2 and 7, mouse anti-PfMSRP-1; lanes 3 and 6, mouse anti-PfMSRP-2; lane 4, Coomassie stain of recombinant PfMSRP-1 used in the studies; lane 5, monoclonal mouse anti-His antibody; lane 8, Coomassie stain of recombinant PfMSRP-2 used in the studies. PfMSRP-1 and PfMSRP-2 did not show any reactivity when incubated with preimmune mouse serum or with the goat anti-mouse horseradish peroxidase-conjugated secondary antibody alone. The same molecular weight marker is run in lanes 1 and 5.

Techniques Used: Recombinant, Positive Control, Staining, Incubation, Molecular Weight, Marker

2) Product Images from "The p65 (RelA) Subunit of NF-?B Interacts with the Histone Deacetylase (HDAC) Corepressors HDAC1 and HDAC2 To Negatively Regulate Gene Expression"

Article Title: The p65 (RelA) Subunit of NF-?B Interacts with the Histone Deacetylase (HDAC) Corepressors HDAC1 and HDAC2 To Negatively Regulate Gene Expression

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.20.7065-7077.2001

HDAC1 interacts directly with p65 in in vitro binding assays. (A) Diagram of p65 full-length and deletion mutants used for in vitro interaction. (B) Lanes 1 to 6, 1/10 of the indicated input in vitro transcription and translation products used for in vitro binding assays. The products of the in vitro transcription and translation reactions were fractionated by SDS-polyacrylamide gel electrophoresis and the bands were visualized by autoradiography. (C) After the in vitro transcription and translation reactions were performed, 10 μl (of a 50-μl reaction) of the indicated products was mixed with GST-HDAC1 (upper panel) or GST (lower panel) bound to glutathione-agarose beads. After incubation for 1 h at 4°C, the beads were washed extensively and interacting proteins were visualized as for panel A.
Figure Legend Snippet: HDAC1 interacts directly with p65 in in vitro binding assays. (A) Diagram of p65 full-length and deletion mutants used for in vitro interaction. (B) Lanes 1 to 6, 1/10 of the indicated input in vitro transcription and translation products used for in vitro binding assays. The products of the in vitro transcription and translation reactions were fractionated by SDS-polyacrylamide gel electrophoresis and the bands were visualized by autoradiography. (C) After the in vitro transcription and translation reactions were performed, 10 μl (of a 50-μl reaction) of the indicated products was mixed with GST-HDAC1 (upper panel) or GST (lower panel) bound to glutathione-agarose beads. After incubation for 1 h at 4°C, the beads were washed extensively and interacting proteins were visualized as for panel A.

Techniques Used: In Vitro, Binding Assay, Polyacrylamide Gel Electrophoresis, Autoradiography, Incubation

3) Product Images from "Identification of Amino Acid Residues of ERH Required for Its Recruitment to Nuclear Speckles and Replication Foci in HeLa Cells"

Article Title: Identification of Amino Acid Residues of ERH Required for Its Recruitment to Nuclear Speckles and Replication Foci in HeLa Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074885

GST pull-down assay with substituted forms of human ERH. Indicated FLAG-tagged ERH forms incubated with either GST-tagged fragment L7 of human PDIP46/SKAR (GST-PDIP46/SKAR[L7]) or GST-tagged fragment B of human Ciz1 (GST-Ciz1[B]) and detected by western blotting with anti-FLAG antibody followed by enhanced chemiluminescence reaction. PDIP46/SKAR does not interact with ERH H3A Q9A or ERH H3A Q9A E37A T51A, and Ciz1 does not interact with ERH E37A T51A or ERH H3A Q9A E37A T51A.
Figure Legend Snippet: GST pull-down assay with substituted forms of human ERH. Indicated FLAG-tagged ERH forms incubated with either GST-tagged fragment L7 of human PDIP46/SKAR (GST-PDIP46/SKAR[L7]) or GST-tagged fragment B of human Ciz1 (GST-Ciz1[B]) and detected by western blotting with anti-FLAG antibody followed by enhanced chemiluminescence reaction. PDIP46/SKAR does not interact with ERH H3A Q9A or ERH H3A Q9A E37A T51A, and Ciz1 does not interact with ERH E37A T51A or ERH H3A Q9A E37A T51A.

Techniques Used: Pull Down Assay, Incubation, Western Blot

Recruitment of substituted forms of human ERH to nuclear speckles and replication foci in HeLa cells visualized by confocal microscopy. EGFP-tagged substituted forms of ERH expressed alone (top) or coexpressed with mCherry-tagged human PDIP46/SKAR (middle) or mCherry-tagged human Ciz1 (bottom). A . ERH T18A S24A localizes to the nucleus and is recruited both to nuclear speckles and to replication foci similarly to wild-type ERH. B . ERH H3A Q9A is present not only in the nucleus but also in the cytoplasm, shows diminished recruitment to nuclear speckles but still accumulates in replication foci. C . ERH E37A T51A localizes partly to the cytoplasm, is recruited to nuclear speckles, and shows very week accumulation in replication foci. D . ERH H3A Q9A E37A T51A is also present in the cytoplasm and recruited neither to nuclear speckles nor to replication foci.
Figure Legend Snippet: Recruitment of substituted forms of human ERH to nuclear speckles and replication foci in HeLa cells visualized by confocal microscopy. EGFP-tagged substituted forms of ERH expressed alone (top) or coexpressed with mCherry-tagged human PDIP46/SKAR (middle) or mCherry-tagged human Ciz1 (bottom). A . ERH T18A S24A localizes to the nucleus and is recruited both to nuclear speckles and to replication foci similarly to wild-type ERH. B . ERH H3A Q9A is present not only in the nucleus but also in the cytoplasm, shows diminished recruitment to nuclear speckles but still accumulates in replication foci. C . ERH E37A T51A localizes partly to the cytoplasm, is recruited to nuclear speckles, and shows very week accumulation in replication foci. D . ERH H3A Q9A E37A T51A is also present in the cytoplasm and recruited neither to nuclear speckles nor to replication foci.

Techniques Used: Confocal Microscopy

4) Product Images from "The Chemokine Fractalkine Can Activate Integrins without CX3CR1 through Direct Binding to a Ligand-Binding Site Distinct from the Classical RGD-Binding Site"

Article Title: The Chemokine Fractalkine Can Activate Integrins without CX3CR1 through Direct Binding to a Ligand-Binding Site Distinct from the Classical RGD-Binding Site

Journal: PLoS ONE

doi: 10.1371/journal.pone.0096372

FKN-CD activates α5β1 integrin in a CX3CR1-independent manner through the binding to site 2. a. Activation of α5β1 by FKN-CD in K562 cells (CX3CR1-negative). The binding of FITC-labeled FN8-11 (specific ligand to α5β1) was measured as described in the methods. Data are shown as means ± SEM of MFI of three independent experiments. b. K562 cells adhesion to FN8-11. Cell adhesion to immobilized FN8-11 was measured as described in the methods. Data are shown as means ± SEM of three independent experiments. c. Effect of S2-β3 on FKN-CD induced integrin activation in K562 cells. Cells were incubated with FITC-labeled FN8-11 in the presence of FKN-CD or the mixtures of FKN-CD and S2-β3. FKN-CD (20 µg/ml) was preincubated with S2-β3 (300 µg/ml) in PBS for 30 min at room temperature. Binding of FITC-labeled FN8-11 to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. d. Activation of α5β1 by FKN-CD in CHO cells (CX3CR1-negative) in a CX3CR1-independent manner. The binding of FITC-labeled FN8-11 (specific ligand to α5β1) was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. e. Activation of α5β1 by FKN-CD in CHO cells at low FKN-CD concentrations. Experiments were performed as described in (d) except that FKN-CD and K36E/R37E were used at 0.1 and 1 µg/ml. Data are shown as means ± SEM of MFI of three independent experiments. f. Effect of S2-β3 peptide on FKN-CD induced integrin activation in CHO cells. Experiments were performed as descibed in c) except that CHO cells were used. Data are shown as means ± SEM of MFI of three independent experiments.
Figure Legend Snippet: FKN-CD activates α5β1 integrin in a CX3CR1-independent manner through the binding to site 2. a. Activation of α5β1 by FKN-CD in K562 cells (CX3CR1-negative). The binding of FITC-labeled FN8-11 (specific ligand to α5β1) was measured as described in the methods. Data are shown as means ± SEM of MFI of three independent experiments. b. K562 cells adhesion to FN8-11. Cell adhesion to immobilized FN8-11 was measured as described in the methods. Data are shown as means ± SEM of three independent experiments. c. Effect of S2-β3 on FKN-CD induced integrin activation in K562 cells. Cells were incubated with FITC-labeled FN8-11 in the presence of FKN-CD or the mixtures of FKN-CD and S2-β3. FKN-CD (20 µg/ml) was preincubated with S2-β3 (300 µg/ml) in PBS for 30 min at room temperature. Binding of FITC-labeled FN8-11 to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. d. Activation of α5β1 by FKN-CD in CHO cells (CX3CR1-negative) in a CX3CR1-independent manner. The binding of FITC-labeled FN8-11 (specific ligand to α5β1) was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. e. Activation of α5β1 by FKN-CD in CHO cells at low FKN-CD concentrations. Experiments were performed as described in (d) except that FKN-CD and K36E/R37E were used at 0.1 and 1 µg/ml. Data are shown as means ± SEM of MFI of three independent experiments. f. Effect of S2-β3 peptide on FKN-CD induced integrin activation in CHO cells. Experiments were performed as descibed in c) except that CHO cells were used. Data are shown as means ± SEM of MFI of three independent experiments.

Techniques Used: Binding Assay, Activation Assay, Labeling, Incubation, Flow Cytometry, Cytometry

A peptide derived from the predicted site 2 of αvβ3 (S2-β3) binds to FKN-CD and suppresses CX3CR1-independent FKN-CD-induced αvβ3 activation. a. Binding of S2-β3 peptide to immobilized FKN-CD. The binding of the peptide to immobilized FKN-CD was measured by ELISA. Data are shown as means ± SEM of three independent experiments. b. Pull-down assays. FKN-CD (with 6His tag) was incubated with S2-β3 or S2-β1 peptide (GST fusion protein) and the complexes were analyzed by Western blotting. c. Binding of site 2 peptides from different integrin β subunits (S2-β1, β2, β3, and β4) to immobilized FKN-CD. The binding of peptides to immobilized FKN-CD was measured as described in (a). Data are shown as means ± SEM of three independent experiments. d. Binding of S2-β3 peptide to FKN-CD. The binding of the peptide to immobilized FKN-CD, γC399tr, FN-H120, FN-8-11 (5 µM) was measured as described in (a). Data are shown as means ± SEM of three independent experiments. e. Effect of S2-β3 peptide on FKN-CD induced integrin activation in αvβ3-K562 cells. Cells were incubated with FITC-labeled γC399tr in the presence of FKN-CD or the mixture of FKN-CD and S2-β3 peptide. FKN-CD (20 µg/ml) were preincubated with S2-β3 (300 µg/ml) in PBS for 30 min at room temperature. Binding of FITC-labeled γC399tr to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. f. Effect of S2-β3 peptide on FKN-CD induced integrin activation in β3-CHO cells. The binding of γC399tr to cells was measured as described in e). Data are shown as means ± SEM of MFI of three independent experiments.
Figure Legend Snippet: A peptide derived from the predicted site 2 of αvβ3 (S2-β3) binds to FKN-CD and suppresses CX3CR1-independent FKN-CD-induced αvβ3 activation. a. Binding of S2-β3 peptide to immobilized FKN-CD. The binding of the peptide to immobilized FKN-CD was measured by ELISA. Data are shown as means ± SEM of three independent experiments. b. Pull-down assays. FKN-CD (with 6His tag) was incubated with S2-β3 or S2-β1 peptide (GST fusion protein) and the complexes were analyzed by Western blotting. c. Binding of site 2 peptides from different integrin β subunits (S2-β1, β2, β3, and β4) to immobilized FKN-CD. The binding of peptides to immobilized FKN-CD was measured as described in (a). Data are shown as means ± SEM of three independent experiments. d. Binding of S2-β3 peptide to FKN-CD. The binding of the peptide to immobilized FKN-CD, γC399tr, FN-H120, FN-8-11 (5 µM) was measured as described in (a). Data are shown as means ± SEM of three independent experiments. e. Effect of S2-β3 peptide on FKN-CD induced integrin activation in αvβ3-K562 cells. Cells were incubated with FITC-labeled γC399tr in the presence of FKN-CD or the mixture of FKN-CD and S2-β3 peptide. FKN-CD (20 µg/ml) were preincubated with S2-β3 (300 µg/ml) in PBS for 30 min at room temperature. Binding of FITC-labeled γC399tr to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. f. Effect of S2-β3 peptide on FKN-CD induced integrin activation in β3-CHO cells. The binding of γC399tr to cells was measured as described in e). Data are shown as means ± SEM of MFI of three independent experiments.

Techniques Used: Derivative Assay, Activation Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Labeling, Flow Cytometry, Cytometry

FKN-CD activates α4β1 integrin in a CX3CR1-independent manner through the binding to site 2. a. Activation of α4β1 by FKN-CD in α4-K562 cells (CX3CR1-negative). The binding of FITC-labeled H120 (specific ligand to α4β1) to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. b. Adhesion of α4-K562 cell to VCAM-1. Cell adhesion to immobilized VCAM-1 was measured as described in the methods. Data are shown as means ± SEM of three independent experiments. c. Effect of S2-β3 on FKN-CD induced α4β1 activation. α4-K562 cells were incubated with FITC-labeled H120 in the presence of FKN-CD or the mixtures of FKN-CD and S2-β3. FKN-CD (20 µg/ml) was preincubated with S2-β3 (300 µg/ml) in PBS for 30 min at room temperature. Binding of FITC-labeled H120 to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. d. Activation of α4β1 by FKN-CD in α4-CHO cells (CX3CR1-negative) in a CX3CR1-independent manner. The binding of FITC-labeled H120 (specific ligand to α4β1) was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. e. Activation of α4β1 by FKN-CD in α4-CHO cells at low FKN-CD concentrations. Experiments were performed as described in (d) except that FKN-CD and K36E/R37E were used at 0.1 and 1 µg/ml. Data are shown as means ± SEM of MFI of three independent experiments. f. Effect of S2-β3 peptide on FKN-CD induced integrin activation in α4-CHO cells. Experiments were performed as descibed in c) except that α4-CHO cells were used. Data are shown as means ± SEM of MFI of three independent experiments.
Figure Legend Snippet: FKN-CD activates α4β1 integrin in a CX3CR1-independent manner through the binding to site 2. a. Activation of α4β1 by FKN-CD in α4-K562 cells (CX3CR1-negative). The binding of FITC-labeled H120 (specific ligand to α4β1) to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. b. Adhesion of α4-K562 cell to VCAM-1. Cell adhesion to immobilized VCAM-1 was measured as described in the methods. Data are shown as means ± SEM of three independent experiments. c. Effect of S2-β3 on FKN-CD induced α4β1 activation. α4-K562 cells were incubated with FITC-labeled H120 in the presence of FKN-CD or the mixtures of FKN-CD and S2-β3. FKN-CD (20 µg/ml) was preincubated with S2-β3 (300 µg/ml) in PBS for 30 min at room temperature. Binding of FITC-labeled H120 to cells was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. d. Activation of α4β1 by FKN-CD in α4-CHO cells (CX3CR1-negative) in a CX3CR1-independent manner. The binding of FITC-labeled H120 (specific ligand to α4β1) was measured by flow cytometry. Data are shown as means ± SEM of MFI of three independent experiments. e. Activation of α4β1 by FKN-CD in α4-CHO cells at low FKN-CD concentrations. Experiments were performed as described in (d) except that FKN-CD and K36E/R37E were used at 0.1 and 1 µg/ml. Data are shown as means ± SEM of MFI of three independent experiments. f. Effect of S2-β3 peptide on FKN-CD induced integrin activation in α4-CHO cells. Experiments were performed as descibed in c) except that α4-CHO cells were used. Data are shown as means ± SEM of MFI of three independent experiments.

Techniques Used: Binding Assay, Activation Assay, Labeling, Flow Cytometry, Cytometry, Incubation

5) Product Images from "Distinct molecular forms of ?-catenin are targeted to adhesive or transcriptional complexes"

Article Title: Distinct molecular forms of ?-catenin are targeted to adhesive or transcriptional complexes

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200402153

Cadherin phosphorylation reverses β-catenin binding selectivity during Wnt signaling. (A) Phosphorylation of cad-GST increases β-catenin binding to cadherin compared with TCF. A cytosolic fraction from L cells transfected with Wnt3a were incubated with equimolar amounts of cad-GST, TCF-GST, and CK2-P-cad-GST-glutathione–coupled beads for 1 h at 4°C (see Fig. S1 for characterization of GST fusion proteins). The resulting anti–β-catenin and anti-GST immunoblots are shown. (B) Fraction of β-catenin that binds cadherin is a subset of fraction of β-catenin that binds TCF. Cytosolic fraction of Wnt cells was sequentially affinity precipitated with cad-GST (lanes 1–3) or TCF-GST (lanes 6–8) proteins. After cad-GST depletion (lanes 1–3), half of the cad-GST non-binding fraction (NB/2) was precipitated with TCF-GST (lane 4); the other half was precipitated with TCA to show amount remaining (lane 5, far right). After TCF-GST depletion (lanes 6–8), half of the TCF-GST non-binding fraction (NB/2, lane 9) was precipitated with cad-GST, whereas the other half was precipitated with TCA to show amount remaining (lane 10, far right). Lanes 5 and 10 reveal a fraction of β-catenin that binds neither TCF nor cadherin. This fraction is likely due to β-catenin already complexed with partners such as ICAT ( Gottardi and Gumbiner, 2004 ). (C) Phosphorylated cadherin-GST and TCF-GST bind the same pool of β-catenin in Wnt-activated cells. Cytosolic fraction was precipitated with cad-GST (top blot), TCF-GST (bottom left) or P-cadherin-GST (bottom right) fusion proteins. After cad-GST depletion (lanes 2–4 and 7–9), there is a fraction of β-catenin that binds TCF-GST (lane 5) and P-cadherin-GST (lane 10). Note that after TCF-GST depletion (lanes 13–15), there is no β-catenin remaining to bind P-cadherin-GST (lane 16). After P-cadherin-GST depletion (lanes 18–20), there is no β-catenin remaining to bind TCF-GST (lane 21). Reciprocal depletions suggest that P-cadherin-GST and TCF-GST bind the same form of β-catenin.
Figure Legend Snippet: Cadherin phosphorylation reverses β-catenin binding selectivity during Wnt signaling. (A) Phosphorylation of cad-GST increases β-catenin binding to cadherin compared with TCF. A cytosolic fraction from L cells transfected with Wnt3a were incubated with equimolar amounts of cad-GST, TCF-GST, and CK2-P-cad-GST-glutathione–coupled beads for 1 h at 4°C (see Fig. S1 for characterization of GST fusion proteins). The resulting anti–β-catenin and anti-GST immunoblots are shown. (B) Fraction of β-catenin that binds cadherin is a subset of fraction of β-catenin that binds TCF. Cytosolic fraction of Wnt cells was sequentially affinity precipitated with cad-GST (lanes 1–3) or TCF-GST (lanes 6–8) proteins. After cad-GST depletion (lanes 1–3), half of the cad-GST non-binding fraction (NB/2) was precipitated with TCF-GST (lane 4); the other half was precipitated with TCA to show amount remaining (lane 5, far right). After TCF-GST depletion (lanes 6–8), half of the TCF-GST non-binding fraction (NB/2, lane 9) was precipitated with cad-GST, whereas the other half was precipitated with TCA to show amount remaining (lane 10, far right). Lanes 5 and 10 reveal a fraction of β-catenin that binds neither TCF nor cadherin. This fraction is likely due to β-catenin already complexed with partners such as ICAT ( Gottardi and Gumbiner, 2004 ). (C) Phosphorylated cadherin-GST and TCF-GST bind the same pool of β-catenin in Wnt-activated cells. Cytosolic fraction was precipitated with cad-GST (top blot), TCF-GST (bottom left) or P-cadherin-GST (bottom right) fusion proteins. After cad-GST depletion (lanes 2–4 and 7–9), there is a fraction of β-catenin that binds TCF-GST (lane 5) and P-cadherin-GST (lane 10). Note that after TCF-GST depletion (lanes 13–15), there is no β-catenin remaining to bind P-cadherin-GST (lane 16). After P-cadherin-GST depletion (lanes 18–20), there is no β-catenin remaining to bind TCF-GST (lane 21). Reciprocal depletions suggest that P-cadherin-GST and TCF-GST bind the same form of β-catenin.

Techniques Used: Binding Assay, Transfection, Incubation, Western Blot

The COOH terminus of β-catenin restricts binding to cadherin. COOH terminus of β-catenin competes cadherin, but not TCF binding. (A) Schematic shows where α-catenin, cadherin, and TCF interact with β-catenin ( Huber et al., 1997 ; Graham et al., 2000 ; Pokutta and Weis, 2000 ; Huber and Weis, 2001 ). (B) The COOH terminus of β-catenin binds the arm repeat region of β-catenin in yeast-two hybrid ( Cox et al., 1999 ) and recombinant protein assays ( Piedra et al., 2001 ). (C) COOH-terminal region of β-catenin competes β-catenin binding to cad-GST, but not to TCF-GST fusion protein . Recombinant β-catenin (1.5 μg) purified from baculovirus ( Suh and Gumbiner, 2003 ) was incubated with cadherin-GST (2 μg) or TCF-GST (2.4 μg) coupled agarose beads in the presence of increasing amounts of β-catenin COOH-terminal peptide (amino acids 695–781). Affinity precipitates were analyzed by SDS-PAGE and Western blotting with an antibody to β-catenin. (D) Cadherin-GST preferentially depletes the fraction of β-catenin recognized by a COOH-terminal mAb (M5.2) . A cytosolic fraction from Rat1/Wnt cells was affinity precipitated (×3) with cadherin-GST (lanes 1–3). The cad-GST nonbinding pool (lanes 4 and 5) was divided in two and immunoprecipitated with either an mAb that recognizes a COOH-terminal β-catenin epitope (βC-mAb (M5.2), lane 4) or an NH 2 -terminal β-catenin epitope (βN-mAb (1.1), lane 5). As a control, these antibodies were used to immunoprecipitate β-catenin from the total starting material (not previously depleted with cad-GST; lanes 6 and 7).
Figure Legend Snippet: The COOH terminus of β-catenin restricts binding to cadherin. COOH terminus of β-catenin competes cadherin, but not TCF binding. (A) Schematic shows where α-catenin, cadherin, and TCF interact with β-catenin ( Huber et al., 1997 ; Graham et al., 2000 ; Pokutta and Weis, 2000 ; Huber and Weis, 2001 ). (B) The COOH terminus of β-catenin binds the arm repeat region of β-catenin in yeast-two hybrid ( Cox et al., 1999 ) and recombinant protein assays ( Piedra et al., 2001 ). (C) COOH-terminal region of β-catenin competes β-catenin binding to cad-GST, but not to TCF-GST fusion protein . Recombinant β-catenin (1.5 μg) purified from baculovirus ( Suh and Gumbiner, 2003 ) was incubated with cadherin-GST (2 μg) or TCF-GST (2.4 μg) coupled agarose beads in the presence of increasing amounts of β-catenin COOH-terminal peptide (amino acids 695–781). Affinity precipitates were analyzed by SDS-PAGE and Western blotting with an antibody to β-catenin. (D) Cadherin-GST preferentially depletes the fraction of β-catenin recognized by a COOH-terminal mAb (M5.2) . A cytosolic fraction from Rat1/Wnt cells was affinity precipitated (×3) with cadherin-GST (lanes 1–3). The cad-GST nonbinding pool (lanes 4 and 5) was divided in two and immunoprecipitated with either an mAb that recognizes a COOH-terminal β-catenin epitope (βC-mAb (M5.2), lane 4) or an NH 2 -terminal β-catenin epitope (βN-mAb (1.1), lane 5). As a control, these antibodies were used to immunoprecipitate β-catenin from the total starting material (not previously depleted with cad-GST; lanes 6 and 7).

Techniques Used: Binding Assay, Recombinant, Purification, Incubation, SDS Page, Western Blot, Immunoprecipitation

β-Catenin not phosphorylated at NH 2 -terminal GSK-3β sites binds to cadherin. (A) Cytosolic fraction from HEK293 cells ± Wnt3a was affinity precipitated with cad-GST and TCF-GST, and blotted with pAbs to β-catenin (top blot) or NH 2 -terminal unphosphorylated–β-catenin (amino acids 27–37, bottom blot). (B) NH 2 -terminal unphospho–β-catenin localizes to sites of cell–cell contact in Wnt-expressing cells. Rat1 fibroblasts ± Wnt were fixed and processed for immunofluorescence using standard protocols. Images were captured with the Axioplan 2 microscope and AxioVision2.0 software (Carl Zeiss MicroImaging, Inc.). Note that membrane staining of the unphospho-β-catenin (Cy3) is more readily detected under methanol, rather than PFA fixation conditions, perhaps accounting for the apparent differences observed between our study and Staal et al. (2002) .
Figure Legend Snippet: β-Catenin not phosphorylated at NH 2 -terminal GSK-3β sites binds to cadherin. (A) Cytosolic fraction from HEK293 cells ± Wnt3a was affinity precipitated with cad-GST and TCF-GST, and blotted with pAbs to β-catenin (top blot) or NH 2 -terminal unphosphorylated–β-catenin (amino acids 27–37, bottom blot). (B) NH 2 -terminal unphospho–β-catenin localizes to sites of cell–cell contact in Wnt-expressing cells. Rat1 fibroblasts ± Wnt were fixed and processed for immunofluorescence using standard protocols. Images were captured with the Axioplan 2 microscope and AxioVision2.0 software (Carl Zeiss MicroImaging, Inc.). Note that membrane staining of the unphospho-β-catenin (Cy3) is more readily detected under methanol, rather than PFA fixation conditions, perhaps accounting for the apparent differences observed between our study and Staal et al. (2002) .

Techniques Used: Expressing, Immunofluorescence, Microscopy, Software, Staining

The α-catenin–free, monomeric form of β-catenin exhibits preferential binding to TCF compared with cadherin in Wnt cells. (A) Rat1 cells were labeled to steady-state with [ 35 S]methionine/cysteine, and a cytosolic fraction was prepared from each condition (−Wnt, +Wnt, 10 mM LiCl, 12 h) and immunoprecipitated with the designated antibodies or affinity precipitated with GST proteins. Note that immunoprecipitation of endogenous E-cadherin (from the 100,000 g membrane pellet, lanes 5, 10, and 16) and TCF (lane 11) are also shown. Non-specific bands were not seen with a GST control (not depicted). Overnight incubation with LiCl (10 mM) allows the α-catenin–free pool of β-catenin to bind cad-GST, TCF-GST, and the endogenous E-cadherin (lanes 14–16), equivalently. (B). COOH-terminal epitopes of β-catenin are masked in the α-catenin–free fraction of β-catenin. Equivalent amounts of an S100 fraction from [ 35 S]methionine/cysteine steady-state–labeled Rat1+Wnt cells were immunoprecipitated with the following antibodies: anti–β-catenin NH 2 -terminal mAb (1.1.1; lane 1), anti–β-catenin COOH-terminal mAb (M5.2; lane 2), anti–α-catenin mAb (lane 4), and a nonimmune control (lane 3). (Lanes 5–7) PDZ protein, mLin7, preferentially binds to β-catenin–α-catenin dimer: metabolically labeled Rat1+Wnt lysates were affinity precipitated with (lane 5) anti–β-catenin pAb, (lane 6) control GST, and (lane 7) mLin7-GST.
Figure Legend Snippet: The α-catenin–free, monomeric form of β-catenin exhibits preferential binding to TCF compared with cadherin in Wnt cells. (A) Rat1 cells were labeled to steady-state with [ 35 S]methionine/cysteine, and a cytosolic fraction was prepared from each condition (−Wnt, +Wnt, 10 mM LiCl, 12 h) and immunoprecipitated with the designated antibodies or affinity precipitated with GST proteins. Note that immunoprecipitation of endogenous E-cadherin (from the 100,000 g membrane pellet, lanes 5, 10, and 16) and TCF (lane 11) are also shown. Non-specific bands were not seen with a GST control (not depicted). Overnight incubation with LiCl (10 mM) allows the α-catenin–free pool of β-catenin to bind cad-GST, TCF-GST, and the endogenous E-cadherin (lanes 14–16), equivalently. (B). COOH-terminal epitopes of β-catenin are masked in the α-catenin–free fraction of β-catenin. Equivalent amounts of an S100 fraction from [ 35 S]methionine/cysteine steady-state–labeled Rat1+Wnt cells were immunoprecipitated with the following antibodies: anti–β-catenin NH 2 -terminal mAb (1.1.1; lane 1), anti–β-catenin COOH-terminal mAb (M5.2; lane 2), anti–α-catenin mAb (lane 4), and a nonimmune control (lane 3). (Lanes 5–7) PDZ protein, mLin7, preferentially binds to β-catenin–α-catenin dimer: metabolically labeled Rat1+Wnt lysates were affinity precipitated with (lane 5) anti–β-catenin pAb, (lane 6) control GST, and (lane 7) mLin7-GST.

Techniques Used: Binding Assay, Labeling, Immunoprecipitation, Incubation, Metabolic Labelling

Differential binding activity of recombinant β-catenin as revealed by deletion analysis. (A) Schematic representation of β-catenin constructs. WT-myc- Xenopus β-catenin and GSK3β mutant (S/T > A residues 33, 37, 41, and 45) β-catenin were described previously by Guger and Gumbiner (2000) . WT-human β-catenin-flag, ΔC695-flag and ΔN89-flag constructs were described in Kolligs et al. (1999) . The myc-tagged, Xenopus β-catenin construct encoding only the arm repeat region of β-catenin was described previously ( Funayama et al., 1995 ). (B) Recombinant β-catenin binding to cad-GST versus TCF-GST proteins. HEK293T cells were transfected with decreasing amounts of β-catenin plasmid and incubated in the presence (+) of Wnt3a conditioned media (CM). Cytosolic fractions were affinity precipitated and immunoblotted with anti-myc, -flag, or β-catenin antibodies. Input amounts of wild-type β-catenin, −ΔC695, and arm 12 constructs were the same in accordance with similar expression levels (not depicted).
Figure Legend Snippet: Differential binding activity of recombinant β-catenin as revealed by deletion analysis. (A) Schematic representation of β-catenin constructs. WT-myc- Xenopus β-catenin and GSK3β mutant (S/T > A residues 33, 37, 41, and 45) β-catenin were described previously by Guger and Gumbiner (2000) . WT-human β-catenin-flag, ΔC695-flag and ΔN89-flag constructs were described in Kolligs et al. (1999) . The myc-tagged, Xenopus β-catenin construct encoding only the arm repeat region of β-catenin was described previously ( Funayama et al., 1995 ). (B) Recombinant β-catenin binding to cad-GST versus TCF-GST proteins. HEK293T cells were transfected with decreasing amounts of β-catenin plasmid and incubated in the presence (+) of Wnt3a conditioned media (CM). Cytosolic fractions were affinity precipitated and immunoblotted with anti-myc, -flag, or β-catenin antibodies. Input amounts of wild-type β-catenin, −ΔC695, and arm 12 constructs were the same in accordance with similar expression levels (not depicted).

Techniques Used: Binding Assay, Activity Assay, Recombinant, Construct, Mutagenesis, Transfection, Plasmid Preparation, Incubation, Expressing

Larger molecular size, α-catenin–containing fractions of β-catenin show preferential binding to cad-GST. (A) A cytosolic fraction from stage 12 Xenopus embryos was applied to a Sephacryl 300 gel filtration column, and fractions 28–39 were divided in two: one half of each sample was TCA-precipitated (top blot), whereas the other half was precipitated with cad-GST (middle blot). The top blot was reprobed with an antibody to α-catenin and is shown below. (B) Same as A except that starting material is an S100 fraction from Rat1/Wnt cells. Arrows refer to elution volumes of standard proteins with known molecular weight: (a) catalase (Mr = 232,000); (b) BSA (Mr = 66,000), purified mouse IgG (150 kD) eluted in fractions 31–33.
Figure Legend Snippet: Larger molecular size, α-catenin–containing fractions of β-catenin show preferential binding to cad-GST. (A) A cytosolic fraction from stage 12 Xenopus embryos was applied to a Sephacryl 300 gel filtration column, and fractions 28–39 were divided in two: one half of each sample was TCA-precipitated (top blot), whereas the other half was precipitated with cad-GST (middle blot). The top blot was reprobed with an antibody to α-catenin and is shown below. (B) Same as A except that starting material is an S100 fraction from Rat1/Wnt cells. Arrows refer to elution volumes of standard proteins with known molecular weight: (a) catalase (Mr = 232,000); (b) BSA (Mr = 66,000), purified mouse IgG (150 kD) eluted in fractions 31–33.

Techniques Used: Binding Assay, Filtration, Molecular Weight, Purification

Wnt signaling generates a form of β-catenin that binds preferentially to TCF-GST compared with cadherin-GST. (A) Detergent-free supernatants were prepared from C57MG and Rat1 cells stably expressing Wnt-1, and HEK293T cells incubated overnight ± Wnt3a-conditioned media (CM). Samples were affinity precipitated using equimolar amounts of cad-GST or TCF-GST fusion proteins. GST gives no binding and is not depicted. A fivefold excess of parental cell lysates was required to detect a signal in lanes 5 and 6. Cytosolic β-catenin from C57MG parentals binds cad-GST and TCF-GST proteins equivalently, like the Rat1 and HEK293 controls (not depicted). The blot was probed with a pAb to β-catenin. (B) Preferential binding of β-catenin to TCF-GST over cadherin-GST is not observed with purified, recombinant β-catenin. Recombinant, purified Xenopus β-catenin ( Suh and Gumbiner, 2003 ) and β-catenin from a C57MG/Wnt cytosolic fraction were affinity precipitated with cad-GST and TCF-GST proteins, and blotted with an antibody to β-catenin.
Figure Legend Snippet: Wnt signaling generates a form of β-catenin that binds preferentially to TCF-GST compared with cadherin-GST. (A) Detergent-free supernatants were prepared from C57MG and Rat1 cells stably expressing Wnt-1, and HEK293T cells incubated overnight ± Wnt3a-conditioned media (CM). Samples were affinity precipitated using equimolar amounts of cad-GST or TCF-GST fusion proteins. GST gives no binding and is not depicted. A fivefold excess of parental cell lysates was required to detect a signal in lanes 5 and 6. Cytosolic β-catenin from C57MG parentals binds cad-GST and TCF-GST proteins equivalently, like the Rat1 and HEK293 controls (not depicted). The blot was probed with a pAb to β-catenin. (B) Preferential binding of β-catenin to TCF-GST over cadherin-GST is not observed with purified, recombinant β-catenin. Recombinant, purified Xenopus β-catenin ( Suh and Gumbiner, 2003 ) and β-catenin from a C57MG/Wnt cytosolic fraction were affinity precipitated with cad-GST and TCF-GST proteins, and blotted with an antibody to β-catenin.

Techniques Used: Stable Transfection, Expressing, Incubation, Binding Assay, Purification, Recombinant

6) Product Images from "Distinct molecular forms of ?-catenin are targeted to adhesive or transcriptional complexes"

Article Title: Distinct molecular forms of ?-catenin are targeted to adhesive or transcriptional complexes

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200402153

Cadherin phosphorylation reverses β-catenin binding selectivity during Wnt signaling. (A) Phosphorylation of cad-GST increases β-catenin binding to cadherin compared with TCF. A cytosolic fraction from L cells transfected with Wnt3a were incubated with equimolar amounts of cad-GST, TCF-GST, and CK2-P-cad-GST-glutathione–coupled beads for 1 h at 4°C (see Fig. S1 for characterization of GST fusion proteins). The resulting anti–β-catenin and anti-GST immunoblots are shown. (B) Fraction of β-catenin that binds cadherin is a subset of fraction of β-catenin that binds TCF. Cytosolic fraction of Wnt cells was sequentially affinity precipitated with cad-GST (lanes 1–3) or TCF-GST (lanes 6–8) proteins. After cad-GST depletion (lanes 1–3), half of the cad-GST non-binding fraction (NB/2) was precipitated with TCF-GST (lane 4); the other half was precipitated with TCA to show amount remaining (lane 5, far right). After TCF-GST depletion (lanes 6–8), half of the TCF-GST non-binding fraction (NB/2, lane 9) was precipitated with cad-GST, whereas the other half was precipitated with TCA to show amount remaining (lane 10, far right). Lanes 5 and 10 reveal a fraction of β-catenin that binds neither TCF nor cadherin. This fraction is likely due to β-catenin already complexed with partners such as ICAT ( Gottardi and Gumbiner, 2004 ). (C) Phosphorylated cadherin-GST and TCF-GST bind the same pool of β-catenin in Wnt-activated cells. Cytosolic fraction was precipitated with cad-GST (top blot), TCF-GST (bottom left) or P-cadherin-GST (bottom right) fusion proteins. After cad-GST depletion (lanes 2–4 and 7–9), there is a fraction of β-catenin that binds TCF-GST (lane 5) and P-cadherin-GST (lane 10). Note that after TCF-GST depletion (lanes 13–15), there is no β-catenin remaining to bind P-cadherin-GST (lane 16). After P-cadherin-GST depletion (lanes 18–20), there is no β-catenin remaining to bind TCF-GST (lane 21). Reciprocal depletions suggest that P-cadherin-GST and TCF-GST bind the same form of β-catenin.
Figure Legend Snippet: Cadherin phosphorylation reverses β-catenin binding selectivity during Wnt signaling. (A) Phosphorylation of cad-GST increases β-catenin binding to cadherin compared with TCF. A cytosolic fraction from L cells transfected with Wnt3a were incubated with equimolar amounts of cad-GST, TCF-GST, and CK2-P-cad-GST-glutathione–coupled beads for 1 h at 4°C (see Fig. S1 for characterization of GST fusion proteins). The resulting anti–β-catenin and anti-GST immunoblots are shown. (B) Fraction of β-catenin that binds cadherin is a subset of fraction of β-catenin that binds TCF. Cytosolic fraction of Wnt cells was sequentially affinity precipitated with cad-GST (lanes 1–3) or TCF-GST (lanes 6–8) proteins. After cad-GST depletion (lanes 1–3), half of the cad-GST non-binding fraction (NB/2) was precipitated with TCF-GST (lane 4); the other half was precipitated with TCA to show amount remaining (lane 5, far right). After TCF-GST depletion (lanes 6–8), half of the TCF-GST non-binding fraction (NB/2, lane 9) was precipitated with cad-GST, whereas the other half was precipitated with TCA to show amount remaining (lane 10, far right). Lanes 5 and 10 reveal a fraction of β-catenin that binds neither TCF nor cadherin. This fraction is likely due to β-catenin already complexed with partners such as ICAT ( Gottardi and Gumbiner, 2004 ). (C) Phosphorylated cadherin-GST and TCF-GST bind the same pool of β-catenin in Wnt-activated cells. Cytosolic fraction was precipitated with cad-GST (top blot), TCF-GST (bottom left) or P-cadherin-GST (bottom right) fusion proteins. After cad-GST depletion (lanes 2–4 and 7–9), there is a fraction of β-catenin that binds TCF-GST (lane 5) and P-cadherin-GST (lane 10). Note that after TCF-GST depletion (lanes 13–15), there is no β-catenin remaining to bind P-cadherin-GST (lane 16). After P-cadherin-GST depletion (lanes 18–20), there is no β-catenin remaining to bind TCF-GST (lane 21). Reciprocal depletions suggest that P-cadherin-GST and TCF-GST bind the same form of β-catenin.

Techniques Used: Binding Assay, Transfection, Incubation, Western Blot

The COOH terminus of β-catenin restricts binding to cadherin. COOH terminus of β-catenin competes cadherin, but not TCF binding. (A) Schematic shows where α-catenin, cadherin, and TCF interact with β-catenin ( Huber et al., 1997 ; Graham et al., 2000 ; Pokutta and Weis, 2000 ; Huber and Weis, 2001 ). (B) The COOH terminus of β-catenin binds the arm repeat region of β-catenin in yeast-two hybrid ( Cox et al., 1999 ) and recombinant protein assays ( Piedra et al., 2001 ). (C) COOH-terminal region of β-catenin competes β-catenin binding to cad-GST, but not to TCF-GST fusion protein . Recombinant β-catenin (1.5 μg) purified from baculovirus ( Suh and Gumbiner, 2003 ) was incubated with cadherin-GST (2 μg) or TCF-GST (2.4 μg) coupled agarose beads in the presence of increasing amounts of β-catenin COOH-terminal peptide (amino acids 695–781). Affinity precipitates were analyzed by SDS-PAGE and Western blotting with an antibody to β-catenin. (D) Cadherin-GST preferentially depletes the fraction of β-catenin recognized by a COOH-terminal mAb (M5.2) . A cytosolic fraction from Rat1/Wnt cells was affinity precipitated (×3) with cadherin-GST (lanes 1–3). The cad-GST nonbinding pool (lanes 4 and 5) was divided in two and immunoprecipitated with either an mAb that recognizes a COOH-terminal β-catenin epitope (βC-mAb (M5.2), lane 4) or an NH 2 -terminal β-catenin epitope (βN-mAb (1.1), lane 5). As a control, these antibodies were used to immunoprecipitate β-catenin from the total starting material (not previously depleted with cad-GST; lanes 6 and 7).
Figure Legend Snippet: The COOH terminus of β-catenin restricts binding to cadherin. COOH terminus of β-catenin competes cadherin, but not TCF binding. (A) Schematic shows where α-catenin, cadherin, and TCF interact with β-catenin ( Huber et al., 1997 ; Graham et al., 2000 ; Pokutta and Weis, 2000 ; Huber and Weis, 2001 ). (B) The COOH terminus of β-catenin binds the arm repeat region of β-catenin in yeast-two hybrid ( Cox et al., 1999 ) and recombinant protein assays ( Piedra et al., 2001 ). (C) COOH-terminal region of β-catenin competes β-catenin binding to cad-GST, but not to TCF-GST fusion protein . Recombinant β-catenin (1.5 μg) purified from baculovirus ( Suh and Gumbiner, 2003 ) was incubated with cadherin-GST (2 μg) or TCF-GST (2.4 μg) coupled agarose beads in the presence of increasing amounts of β-catenin COOH-terminal peptide (amino acids 695–781). Affinity precipitates were analyzed by SDS-PAGE and Western blotting with an antibody to β-catenin. (D) Cadherin-GST preferentially depletes the fraction of β-catenin recognized by a COOH-terminal mAb (M5.2) . A cytosolic fraction from Rat1/Wnt cells was affinity precipitated (×3) with cadherin-GST (lanes 1–3). The cad-GST nonbinding pool (lanes 4 and 5) was divided in two and immunoprecipitated with either an mAb that recognizes a COOH-terminal β-catenin epitope (βC-mAb (M5.2), lane 4) or an NH 2 -terminal β-catenin epitope (βN-mAb (1.1), lane 5). As a control, these antibodies were used to immunoprecipitate β-catenin from the total starting material (not previously depleted with cad-GST; lanes 6 and 7).

Techniques Used: Binding Assay, Recombinant, Purification, Incubation, SDS Page, Western Blot, Immunoprecipitation

β-Catenin not phosphorylated at NH 2 -terminal GSK-3β sites binds to cadherin. (A) Cytosolic fraction from HEK293 cells ± Wnt3a was affinity precipitated with cad-GST and TCF-GST, and blotted with pAbs to β-catenin (top blot) or NH 2 -terminal unphosphorylated–β-catenin (amino acids 27–37, bottom blot). (B) NH 2 -terminal unphospho–β-catenin localizes to sites of cell–cell contact in Wnt-expressing cells. Rat1 fibroblasts ± Wnt were fixed and processed for immunofluorescence using standard protocols. Images were captured with the Axioplan 2 microscope and AxioVision2.0 software (Carl Zeiss MicroImaging, Inc.). Note that membrane staining of the unphospho-β-catenin (Cy3) is more readily detected under methanol, rather than PFA fixation conditions, perhaps accounting for the apparent differences observed between our study and Staal et al. (2002) .
Figure Legend Snippet: β-Catenin not phosphorylated at NH 2 -terminal GSK-3β sites binds to cadherin. (A) Cytosolic fraction from HEK293 cells ± Wnt3a was affinity precipitated with cad-GST and TCF-GST, and blotted with pAbs to β-catenin (top blot) or NH 2 -terminal unphosphorylated–β-catenin (amino acids 27–37, bottom blot). (B) NH 2 -terminal unphospho–β-catenin localizes to sites of cell–cell contact in Wnt-expressing cells. Rat1 fibroblasts ± Wnt were fixed and processed for immunofluorescence using standard protocols. Images were captured with the Axioplan 2 microscope and AxioVision2.0 software (Carl Zeiss MicroImaging, Inc.). Note that membrane staining of the unphospho-β-catenin (Cy3) is more readily detected under methanol, rather than PFA fixation conditions, perhaps accounting for the apparent differences observed between our study and Staal et al. (2002) .

Techniques Used: Expressing, Immunofluorescence, Microscopy, Software, Staining

The α-catenin–free, monomeric form of β-catenin exhibits preferential binding to TCF compared with cadherin in Wnt cells. (A) Rat1 cells were labeled to steady-state with [ 35 S]methionine/cysteine, and a cytosolic fraction was prepared from each condition (−Wnt, +Wnt, 10 mM LiCl, 12 h) and immunoprecipitated with the designated antibodies or affinity precipitated with GST proteins. Note that immunoprecipitation of endogenous E-cadherin (from the 100,000 g membrane pellet, lanes 5, 10, and 16) and TCF (lane 11) are also shown. Non-specific bands were not seen with a GST control (not depicted). Overnight incubation with LiCl (10 mM) allows the α-catenin–free pool of β-catenin to bind cad-GST, TCF-GST, and the endogenous E-cadherin (lanes 14–16), equivalently. (B). COOH-terminal epitopes of β-catenin are masked in the α-catenin–free fraction of β-catenin. Equivalent amounts of an S100 fraction from [ 35 S]methionine/cysteine steady-state–labeled Rat1+Wnt cells were immunoprecipitated with the following antibodies: anti–β-catenin NH 2 -terminal mAb (1.1.1; lane 1), anti–β-catenin COOH-terminal mAb (M5.2; lane 2), anti–α-catenin mAb (lane 4), and a nonimmune control (lane 3). (Lanes 5–7) PDZ protein, mLin7, preferentially binds to β-catenin–α-catenin dimer: metabolically labeled Rat1+Wnt lysates were affinity precipitated with (lane 5) anti–β-catenin pAb, (lane 6) control GST, and (lane 7) mLin7-GST.
Figure Legend Snippet: The α-catenin–free, monomeric form of β-catenin exhibits preferential binding to TCF compared with cadherin in Wnt cells. (A) Rat1 cells were labeled to steady-state with [ 35 S]methionine/cysteine, and a cytosolic fraction was prepared from each condition (−Wnt, +Wnt, 10 mM LiCl, 12 h) and immunoprecipitated with the designated antibodies or affinity precipitated with GST proteins. Note that immunoprecipitation of endogenous E-cadherin (from the 100,000 g membrane pellet, lanes 5, 10, and 16) and TCF (lane 11) are also shown. Non-specific bands were not seen with a GST control (not depicted). Overnight incubation with LiCl (10 mM) allows the α-catenin–free pool of β-catenin to bind cad-GST, TCF-GST, and the endogenous E-cadherin (lanes 14–16), equivalently. (B). COOH-terminal epitopes of β-catenin are masked in the α-catenin–free fraction of β-catenin. Equivalent amounts of an S100 fraction from [ 35 S]methionine/cysteine steady-state–labeled Rat1+Wnt cells were immunoprecipitated with the following antibodies: anti–β-catenin NH 2 -terminal mAb (1.1.1; lane 1), anti–β-catenin COOH-terminal mAb (M5.2; lane 2), anti–α-catenin mAb (lane 4), and a nonimmune control (lane 3). (Lanes 5–7) PDZ protein, mLin7, preferentially binds to β-catenin–α-catenin dimer: metabolically labeled Rat1+Wnt lysates were affinity precipitated with (lane 5) anti–β-catenin pAb, (lane 6) control GST, and (lane 7) mLin7-GST.

Techniques Used: Binding Assay, Labeling, Immunoprecipitation, Incubation, Metabolic Labelling

Differential binding activity of recombinant β-catenin as revealed by deletion analysis. (A) Schematic representation of β-catenin constructs. WT-myc- Xenopus β-catenin and GSK3β mutant (S/T > A residues 33, 37, 41, and 45) β-catenin were described previously by Guger and Gumbiner (2000) . WT-human β-catenin-flag, ΔC695-flag and ΔN89-flag constructs were described in Kolligs et al. (1999) . The myc-tagged, Xenopus β-catenin construct encoding only the arm repeat region of β-catenin was described previously ( Funayama et al., 1995 ). (B) Recombinant β-catenin binding to cad-GST versus TCF-GST proteins. HEK293T cells were transfected with decreasing amounts of β-catenin plasmid and incubated in the presence (+) of Wnt3a conditioned media (CM). Cytosolic fractions were affinity precipitated and immunoblotted with anti-myc, -flag, or β-catenin antibodies. Input amounts of wild-type β-catenin, −ΔC695, and arm 12 constructs were the same in accordance with similar expression levels (not depicted).
Figure Legend Snippet: Differential binding activity of recombinant β-catenin as revealed by deletion analysis. (A) Schematic representation of β-catenin constructs. WT-myc- Xenopus β-catenin and GSK3β mutant (S/T > A residues 33, 37, 41, and 45) β-catenin were described previously by Guger and Gumbiner (2000) . WT-human β-catenin-flag, ΔC695-flag and ΔN89-flag constructs were described in Kolligs et al. (1999) . The myc-tagged, Xenopus β-catenin construct encoding only the arm repeat region of β-catenin was described previously ( Funayama et al., 1995 ). (B) Recombinant β-catenin binding to cad-GST versus TCF-GST proteins. HEK293T cells were transfected with decreasing amounts of β-catenin plasmid and incubated in the presence (+) of Wnt3a conditioned media (CM). Cytosolic fractions were affinity precipitated and immunoblotted with anti-myc, -flag, or β-catenin antibodies. Input amounts of wild-type β-catenin, −ΔC695, and arm 12 constructs were the same in accordance with similar expression levels (not depicted).

Techniques Used: Binding Assay, Activity Assay, Recombinant, Construct, Mutagenesis, Transfection, Plasmid Preparation, Incubation, Expressing

Wnt signaling generates a form of β-catenin that binds preferentially to TCF-GST compared with cadherin-GST. (A) Detergent-free supernatants were prepared from C57MG and Rat1 cells stably expressing Wnt-1, and HEK293T cells incubated overnight ± Wnt3a-conditioned media (CM). Samples were affinity precipitated using equimolar amounts of cad-GST or TCF-GST fusion proteins. GST gives no binding and is not depicted. A fivefold excess of parental cell lysates was required to detect a signal in lanes 5 and 6. Cytosolic β-catenin from C57MG parentals binds cad-GST and TCF-GST proteins equivalently, like the Rat1 and HEK293 controls (not depicted). The blot was probed with a pAb to β-catenin. (B) Preferential binding of β-catenin to TCF-GST over cadherin-GST is not observed with purified, recombinant β-catenin. Recombinant, purified Xenopus β-catenin ( Suh and Gumbiner, 2003 ) and β-catenin from a C57MG/Wnt cytosolic fraction were affinity precipitated with cad-GST and TCF-GST proteins, and blotted with an antibody to β-catenin.
Figure Legend Snippet: Wnt signaling generates a form of β-catenin that binds preferentially to TCF-GST compared with cadherin-GST. (A) Detergent-free supernatants were prepared from C57MG and Rat1 cells stably expressing Wnt-1, and HEK293T cells incubated overnight ± Wnt3a-conditioned media (CM). Samples were affinity precipitated using equimolar amounts of cad-GST or TCF-GST fusion proteins. GST gives no binding and is not depicted. A fivefold excess of parental cell lysates was required to detect a signal in lanes 5 and 6. Cytosolic β-catenin from C57MG parentals binds cad-GST and TCF-GST proteins equivalently, like the Rat1 and HEK293 controls (not depicted). The blot was probed with a pAb to β-catenin. (B) Preferential binding of β-catenin to TCF-GST over cadherin-GST is not observed with purified, recombinant β-catenin. Recombinant, purified Xenopus β-catenin ( Suh and Gumbiner, 2003 ) and β-catenin from a C57MG/Wnt cytosolic fraction were affinity precipitated with cad-GST and TCF-GST proteins, and blotted with an antibody to β-catenin.

Techniques Used: Stable Transfection, Expressing, Incubation, Binding Assay, Purification, Recombinant

7) Product Images from "Inhibition of CED-3 zymogen activation and apoptosis in Caenorhabditis elegans by caspase homolog CSP-3"

Article Title: Inhibition of CED-3 zymogen activation and apoptosis in Caenorhabditis elegans by caspase homolog CSP-3

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.1488

CSP-3 associates with CED-3 in vitro and in C. elegans ( a ) CSP-3 binds to the CED-3 zymogen. GST–CSP-3, GST–CSP-3(F57D) or GST was coexpressed in bacteria with the CED-3 zymogen tagged with a Flag epitode (CED-3–Flag). One portion of the soluble fraction was analyzed by western blot (IB) to examine the expression levels of GST fusion proteins and CED-3–Flag. The remaining portion of the soluble fraction was used for GST protein pull-down experiment, and the amount of CED-3–Flag pulled down was analyzed by western blot analysis. ( b ) CSP-3 associates specifically with the large subunit of CED-3 in vitro . GST–CSP-3, GST–CSP-3(F57D) or GST was coexpressed in bacteria with the CED-3 large subunit (p17) or the small subunit (p13), both tagged with a Flag epitode (gray box). Analysis of expression levels as well as the amounts of two CED-3 subunits coprecipitated with GST fusion proteins was conducted as described in a . The diagram above depicts the domain structure of the CED-3 zymogen, with arrows indicating the three proteolytic cleavage sites that lead to the activation of the CED-3 zymogen. The three CED-3 cleavage products are shown below as boxes. ( c ) CSP-3 associates with CED-3 in C. elegans . Lysates from C. elegans animals expressing CED-3::GFP or CEH-30::GFP were prepared as described in Methods . One portion of the worm lysate was used in the western blot analysis to examine the expression levels of CSP-3 and GFP fusion proteins. The remaining portion of the lysate was incubated with a mouse anti-GFP monoclonal antibody and precipitated using Protein G Sepharose beads. The amount of the CSP-3 protein pulled down with the GFP fusion proteins was analyzed by western blot using purified anti–CSP-3 antibody. A small amount of full-length CED-3::GFP fusion was detected in the lysate (data not shown). The predominant species detected was CED-3::GFP fusion without its prodomain but containing both large and small subunits.
Figure Legend Snippet: CSP-3 associates with CED-3 in vitro and in C. elegans ( a ) CSP-3 binds to the CED-3 zymogen. GST–CSP-3, GST–CSP-3(F57D) or GST was coexpressed in bacteria with the CED-3 zymogen tagged with a Flag epitode (CED-3–Flag). One portion of the soluble fraction was analyzed by western blot (IB) to examine the expression levels of GST fusion proteins and CED-3–Flag. The remaining portion of the soluble fraction was used for GST protein pull-down experiment, and the amount of CED-3–Flag pulled down was analyzed by western blot analysis. ( b ) CSP-3 associates specifically with the large subunit of CED-3 in vitro . GST–CSP-3, GST–CSP-3(F57D) or GST was coexpressed in bacteria with the CED-3 large subunit (p17) or the small subunit (p13), both tagged with a Flag epitode (gray box). Analysis of expression levels as well as the amounts of two CED-3 subunits coprecipitated with GST fusion proteins was conducted as described in a . The diagram above depicts the domain structure of the CED-3 zymogen, with arrows indicating the three proteolytic cleavage sites that lead to the activation of the CED-3 zymogen. The three CED-3 cleavage products are shown below as boxes. ( c ) CSP-3 associates with CED-3 in C. elegans . Lysates from C. elegans animals expressing CED-3::GFP or CEH-30::GFP were prepared as described in Methods . One portion of the worm lysate was used in the western blot analysis to examine the expression levels of CSP-3 and GFP fusion proteins. The remaining portion of the lysate was incubated with a mouse anti-GFP monoclonal antibody and precipitated using Protein G Sepharose beads. The amount of the CSP-3 protein pulled down with the GFP fusion proteins was analyzed by western blot using purified anti–CSP-3 antibody. A small amount of full-length CED-3::GFP fusion was detected in the lysate (data not shown). The predominant species detected was CED-3::GFP fusion without its prodomain but containing both large and small subunits.

Techniques Used: In Vitro, Western Blot, Expressing, Activation Assay, Incubation, Purification

8) Product Images from "Analysis of the Secretomes of Paracoccidioides Mycelia and Yeast Cells"

Article Title: Analysis of the Secretomes of Paracoccidioides Mycelia and Yeast Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052470

Enzymatic activity analysis validates the secretome data for Paracoccidioides mycelia and yeast cells. Activity assay results of ( A ) formamidase (FMD), ( B ) superoxide dismutase (SOD) and ( C ) glutathione S-transferase (GST) assessed for mycelia and yeast protein extracts. FMD activity was assessed by measuring the levels of ammonia released using a standard curve. The SOD and GST Assay Kit were used to determine SOD and GST enzymatic activity, respectively. The student's t test was used for statistical comparisons, and the observed differences were statistically significants ( p ≤0.05). The erros bars represent the standard deviation of three biological replicates.
Figure Legend Snippet: Enzymatic activity analysis validates the secretome data for Paracoccidioides mycelia and yeast cells. Activity assay results of ( A ) formamidase (FMD), ( B ) superoxide dismutase (SOD) and ( C ) glutathione S-transferase (GST) assessed for mycelia and yeast protein extracts. FMD activity was assessed by measuring the levels of ammonia released using a standard curve. The SOD and GST Assay Kit were used to determine SOD and GST enzymatic activity, respectively. The student's t test was used for statistical comparisons, and the observed differences were statistically significants ( p ≤0.05). The erros bars represent the standard deviation of three biological replicates.

Techniques Used: Activity Assay, Glutathione S-Transferase Assay, Standard Deviation

Blocking the conventional protein secretion pathway leads to a decrease in Paracoccidioides yeast cell phagocytosis. A - The protein profile of the cell-free supernatant samples reveals the effect of blocking the protein secretion pathway on yeast cells. Paracoccidioides yeast cells were cultivated in Fava Netto's liquid medium in either the absence (control) (lanes 1, 3 and 5) or presence of Brefeldin A (BFA) at 6 μg/mL (lanes 2, 4 and 6) for 6, 12 and 24 hours, respectively. The cell-free supernatant samples were prepared (as described in the Materials and Methods section), reduced to equal final volumes (1 mL), and processed for one-dimensional electrophoresis (SDS-PAGE). Thirty microliters of each sample was separated via SDS-PAGE and visualized using Coomassie brilliant blue staining. The numbers on the left side correspond to the molecular mass standard. B - The average number of internalized/adhered Paracoccidioides cells by macrophages was determined. Macrophages were infected with Paracoccidioides yeast cells, which were pre-cultivated previously without BFA (control), in the presence of BFA or the presence of concentrated culture supernatant containing extracellular proteins (EP). The adhered/internalized cells were analyzed as described in the materials and methods section. C - The number of viable yeast cells after phagocytosis by macrophages was evaluated by counting the number of colony forming units (CFUs). The results are representative of triplicate biological samples. Statistical significance (* p ≤0.05) was determined by comparing the results with the control group.
Figure Legend Snippet: Blocking the conventional protein secretion pathway leads to a decrease in Paracoccidioides yeast cell phagocytosis. A - The protein profile of the cell-free supernatant samples reveals the effect of blocking the protein secretion pathway on yeast cells. Paracoccidioides yeast cells were cultivated in Fava Netto's liquid medium in either the absence (control) (lanes 1, 3 and 5) or presence of Brefeldin A (BFA) at 6 μg/mL (lanes 2, 4 and 6) for 6, 12 and 24 hours, respectively. The cell-free supernatant samples were prepared (as described in the Materials and Methods section), reduced to equal final volumes (1 mL), and processed for one-dimensional electrophoresis (SDS-PAGE). Thirty microliters of each sample was separated via SDS-PAGE and visualized using Coomassie brilliant blue staining. The numbers on the left side correspond to the molecular mass standard. B - The average number of internalized/adhered Paracoccidioides cells by macrophages was determined. Macrophages were infected with Paracoccidioides yeast cells, which were pre-cultivated previously without BFA (control), in the presence of BFA or the presence of concentrated culture supernatant containing extracellular proteins (EP). The adhered/internalized cells were analyzed as described in the materials and methods section. C - The number of viable yeast cells after phagocytosis by macrophages was evaluated by counting the number of colony forming units (CFUs). The results are representative of triplicate biological samples. Statistical significance (* p ≤0.05) was determined by comparing the results with the control group.

Techniques Used: Blocking Assay, Electrophoresis, SDS Page, Staining, Infection

Validation of the extracellular protein extraction method. A- The viability of Paracoccidioides yeast cells incubated in Fava Netto's liquid medium (dark gray square) and the incubation of yeast cells in Fava Netto's liquid medium containing 6 µg/mL Brefeldin A (light gray square). Viability was assessed using trypan blue staining. The error bars represent the standard deviation of three biological replicates. B- The growth of Paracoccidioides yeast cells in liquid medium in either the absence (dark line) or presence of 6 µg/mL Brefeldin A (light gray line). Culture growth was evaluated by quantifying the number of yeast cells per mL. The error bars represent the standard deviation of three biological replicates. C- PCR sensitivity for the formamidase gene was assessed using Paracoccidiodes Pb01 genomic DNA (at five dilutions) as a template (50 ng to 1 pg). Lanes: 1 −50 ng; 2 −5 ng; 3 −50 pg; 4 −5 pg; 5 −1 pg; 6 - negative control (without genomic DNA). The formamidase PCR amplicons were assessed via 1% agarose gel electrophoresis and stained with ethidium bromide. D- The yeast and mycelia cell-free supernatant samples (2 µL) were assessed for the presence of Paracoccidiodes DNA via PCR using oligonucleotides specific for the formamidase gene.
Figure Legend Snippet: Validation of the extracellular protein extraction method. A- The viability of Paracoccidioides yeast cells incubated in Fava Netto's liquid medium (dark gray square) and the incubation of yeast cells in Fava Netto's liquid medium containing 6 µg/mL Brefeldin A (light gray square). Viability was assessed using trypan blue staining. The error bars represent the standard deviation of three biological replicates. B- The growth of Paracoccidioides yeast cells in liquid medium in either the absence (dark line) or presence of 6 µg/mL Brefeldin A (light gray line). Culture growth was evaluated by quantifying the number of yeast cells per mL. The error bars represent the standard deviation of three biological replicates. C- PCR sensitivity for the formamidase gene was assessed using Paracoccidiodes Pb01 genomic DNA (at five dilutions) as a template (50 ng to 1 pg). Lanes: 1 −50 ng; 2 −5 ng; 3 −50 pg; 4 −5 pg; 5 −1 pg; 6 - negative control (without genomic DNA). The formamidase PCR amplicons were assessed via 1% agarose gel electrophoresis and stained with ethidium bromide. D- The yeast and mycelia cell-free supernatant samples (2 µL) were assessed for the presence of Paracoccidiodes DNA via PCR using oligonucleotides specific for the formamidase gene.

Techniques Used: Protein Extraction, Incubation, Staining, Standard Deviation, Polymerase Chain Reaction, Negative Control, Agarose Gel Electrophoresis

9) Product Images from "Up-regulation of Thrombospondin-2 in Akt1-null Mice Contributes to Compromised Tissue Repair Due to Abnormalities in Fibroblast Function *"

Article Title: Up-regulation of Thrombospondin-2 in Akt1-null Mice Contributes to Compromised Tissue Repair Due to Abnormalities in Fibroblast Function *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.618421

Increased TSP2 regulates Rac1 activity in Akt1 KO fibroblasts. A , pull-down assay of active Rac1 in fibroblasts shows reduced levels in Akt1 KO fibroblasts and DKO fibroblasts treated with exogenous TSP2. Active Rac1 is shown as Rac1-GTP. B , Rac1 activity
Figure Legend Snippet: Increased TSP2 regulates Rac1 activity in Akt1 KO fibroblasts. A , pull-down assay of active Rac1 in fibroblasts shows reduced levels in Akt1 KO fibroblasts and DKO fibroblasts treated with exogenous TSP2. Active Rac1 is shown as Rac1-GTP. B , Rac1 activity

Techniques Used: Activity Assay, Pull Down Assay

10) Product Images from "Scaffold-mediated gating of Cdc42 signalling flux"

Article Title: Scaffold-mediated gating of Cdc42 signalling flux

Journal: eLife

doi: 10.7554/eLife.25257

Expression of Cdc24-mEOS in vivo and the activity of cdc24 phospho-mutants in vitro. ( A ) A Western blot showing the levels of expression of the Cdc24-mEOS constructs. The constructs contain a His tag that was used for detection. ( B ) Representative in vitro GEF reactions of indicated cdc24 phospho-mutant proteins in the presence and absence of Bem1. ( C and D ) Quantification of pull-down experiments to assess the interaction between GST-Bem1 and the Cdc24 phospho-mutant proteins. DOI: http://dx.doi.org/10.7554/eLife.25257.011 10.7554/eLife.25257.012 Excel file showing the band intensity of the data presented in Figure 3—figure supplement 1D . DOI: http://dx.doi.org/10.7554/eLife.25257.012
Figure Legend Snippet: Expression of Cdc24-mEOS in vivo and the activity of cdc24 phospho-mutants in vitro. ( A ) A Western blot showing the levels of expression of the Cdc24-mEOS constructs. The constructs contain a His tag that was used for detection. ( B ) Representative in vitro GEF reactions of indicated cdc24 phospho-mutant proteins in the presence and absence of Bem1. ( C and D ) Quantification of pull-down experiments to assess the interaction between GST-Bem1 and the Cdc24 phospho-mutant proteins. DOI: http://dx.doi.org/10.7554/eLife.25257.011 10.7554/eLife.25257.012 Excel file showing the band intensity of the data presented in Figure 3—figure supplement 1D . DOI: http://dx.doi.org/10.7554/eLife.25257.012

Techniques Used: Expressing, In Vivo, Activity Assay, In Vitro, Western Blot, Construct, Mutagenesis

11) Product Images from "Klf4 methylated by Prmt1 is required for lineage segregation of epiblast and primitive endoderm"

Article Title: Klf4 methylated by Prmt1 is required for lineage segregation of epiblast and primitive endoderm

Journal: bioRxiv

doi: 10.1101/2020.04.24.059055

Klf4 is methylated by Prmt1 at arginine 396, and methylated Klf4 is required for Prmt1-mediated expression of PrE genes. (A) Co-IP of Prmt1 and Klf4. Whole-cell extracts (WCEs) of HEK293T cells transfected with (+) or without (-) Myc-Klf4 and/or FLAG-Prmt1 were subjected to IP with an anti-Myc antibody and blotted with antibodies specific for FLAG for Prmt1 and Myc for Klf4. (B) GST pulldown assays to detect the interaction of Prmt1 with Klf4. GST or GST-Prmt1 was incubated with WCEs of HEK293T cells expressing FLAG-Klf4 and then blotted with antibodies specific for FLAG or GST. (C) Purified GST-Prmt1 was incubated with WCEs of HEK293T cells expressing FLAG-Klf4 and its truncations. The GST pulldown products were immunoblotted with an anti-FLAG antibody, and WCEs that were not subjected to IP were used as the input. (D) Purified GST-Klf4 and its derivatives were incubated with WCEs of HEK293T cells expressing FLAG-Prmt1 or vector. The GST pulldown products were then immunoblotted with an anti-FLAG antibody for Prmt1 or an anti-GST antibody. (E) Autoradiography of in vitro methylation assays using purified GST-Klf4 and its derivatives with Prmt1 or Prmt1m (an inactive enzymatic mutant). Total amounts of GST-Klf4 and Klf4 truncations were visualized by Coomassie brilliant blue (C.B.B.) staining. (F) MS analysis of a Klf4 peptide mixture to identify methylated sites in vitro. Dimethylated arginine (Rme2) is displayed in green. (G) Autoradiography of in vitro methylation assays using purified GST-Klf4 and an R396K mutant of Klf4 with Prmt1 or Prmt1m. (H) RT-qPCR analysis showed the effects of Klf4-WT, Klf4-R396A, Klf4-R396K, Klf4-396F, and vector on the expression levels of Gata4 and Gata6 in E14 cells. (I) ChIP assays showed the recruitment of mSin3a, HDAC1, and HDAC2 to the promoters of Gata4 and Gata6 in E14 cells transfected with Klf4 (WT) or Klf4 point mutants at R396 (R/K or R/A). (J) Co-IP assays of Klf4 with the mSin3a complex. WCEs of E14 cells transfected with FLAG-Klf4 (WT) or its R396 mutants (Klf4-R/K or Klf4 R/A) were subjected to IP with an anti-FLAG antibody and then blotted with antibodies specific for HDAC1, HDAC2, mSin3a and FLAG for Klf4. WCEs that were not subjected to IP were used as the input. IgG was used as a negative control. (K) ChIP/re-ChIP assays showed that Klf4-mediated mSin3a/HDAC recruitment to the promoter of Gata6 is arginine methylation dependent. E14 cells were transfected with FLAG-Klf4 (WT) or its mutant at R396 (R/K). An anti-FLAG antibody was used for the initial ChIP (1st) to obtain the Klf4-associated chromatin fragments. Then, these fragments were subjected to re-ChIP (2nd) using mSin3a, HDAC1, and HDAC2 antibodies. IgG was used as a ChIP control. (L) FACS showed the impact of mutant R396 of Klf4 in E14 cells transfected with Klf4 and its mutants R396A and R396F. The percentage of Gata6+/Nanog- cells is shown.
Figure Legend Snippet: Klf4 is methylated by Prmt1 at arginine 396, and methylated Klf4 is required for Prmt1-mediated expression of PrE genes. (A) Co-IP of Prmt1 and Klf4. Whole-cell extracts (WCEs) of HEK293T cells transfected with (+) or without (-) Myc-Klf4 and/or FLAG-Prmt1 were subjected to IP with an anti-Myc antibody and blotted with antibodies specific for FLAG for Prmt1 and Myc for Klf4. (B) GST pulldown assays to detect the interaction of Prmt1 with Klf4. GST or GST-Prmt1 was incubated with WCEs of HEK293T cells expressing FLAG-Klf4 and then blotted with antibodies specific for FLAG or GST. (C) Purified GST-Prmt1 was incubated with WCEs of HEK293T cells expressing FLAG-Klf4 and its truncations. The GST pulldown products were immunoblotted with an anti-FLAG antibody, and WCEs that were not subjected to IP were used as the input. (D) Purified GST-Klf4 and its derivatives were incubated with WCEs of HEK293T cells expressing FLAG-Prmt1 or vector. The GST pulldown products were then immunoblotted with an anti-FLAG antibody for Prmt1 or an anti-GST antibody. (E) Autoradiography of in vitro methylation assays using purified GST-Klf4 and its derivatives with Prmt1 or Prmt1m (an inactive enzymatic mutant). Total amounts of GST-Klf4 and Klf4 truncations were visualized by Coomassie brilliant blue (C.B.B.) staining. (F) MS analysis of a Klf4 peptide mixture to identify methylated sites in vitro. Dimethylated arginine (Rme2) is displayed in green. (G) Autoradiography of in vitro methylation assays using purified GST-Klf4 and an R396K mutant of Klf4 with Prmt1 or Prmt1m. (H) RT-qPCR analysis showed the effects of Klf4-WT, Klf4-R396A, Klf4-R396K, Klf4-396F, and vector on the expression levels of Gata4 and Gata6 in E14 cells. (I) ChIP assays showed the recruitment of mSin3a, HDAC1, and HDAC2 to the promoters of Gata4 and Gata6 in E14 cells transfected with Klf4 (WT) or Klf4 point mutants at R396 (R/K or R/A). (J) Co-IP assays of Klf4 with the mSin3a complex. WCEs of E14 cells transfected with FLAG-Klf4 (WT) or its R396 mutants (Klf4-R/K or Klf4 R/A) were subjected to IP with an anti-FLAG antibody and then blotted with antibodies specific for HDAC1, HDAC2, mSin3a and FLAG for Klf4. WCEs that were not subjected to IP were used as the input. IgG was used as a negative control. (K) ChIP/re-ChIP assays showed that Klf4-mediated mSin3a/HDAC recruitment to the promoter of Gata6 is arginine methylation dependent. E14 cells were transfected with FLAG-Klf4 (WT) or its mutant at R396 (R/K). An anti-FLAG antibody was used for the initial ChIP (1st) to obtain the Klf4-associated chromatin fragments. Then, these fragments were subjected to re-ChIP (2nd) using mSin3a, HDAC1, and HDAC2 antibodies. IgG was used as a ChIP control. (L) FACS showed the impact of mutant R396 of Klf4 in E14 cells transfected with Klf4 and its mutants R396A and R396F. The percentage of Gata6+/Nanog- cells is shown.

Techniques Used: Methylation, Expressing, Co-Immunoprecipitation Assay, Transfection, Incubation, Purification, Plasmid Preparation, Autoradiography, In Vitro, Mutagenesis, Staining, Quantitative RT-PCR, Chromatin Immunoprecipitation, Negative Control, FACS

12) Product Images from "Termination of non-coding transcription in yeast relies on both a CTD-interaction domain and a CTD-mimic in Sen1"

Article Title: Termination of non-coding transcription in yeast relies on both a CTD-interaction domain and a CTD-mimic in Sen1

Journal: bioRxiv

doi: 10.1101/433045

The N-terminal domain of Sen1 can recognize the S5-phosphorylated form of RNAPII CTD and Sen1 C-terminal domain. A) Deletion of Sen1 N-terminal domain does not prevent the interaction of Sen1 with RNAPII. CoIP experiments using Rbp3-FLAG as the bait. Assays were performed in a Sen1-AID strain harboring a plasmid expressing either SEN1 or sen1ΔNter upon depletion of Sen1-AID in the presence of IAA for 2h. An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1ΔNter. Nrd1 is detected as a positive control. Representative gel of one out of two independent experiments. B ) Deletion of the Sen1 N-terminal domain reduces the interaction of Sen1 with the S5P-CTD. CoIP experiments using TAP-Sen1 as the bait. Sen1 proteins were expressed from pGAL in the presence of galactose. Nab3 is detected as a positive control. Representative gel of one out of three independent experiments. In both A ) and B ) protein extracts were treated with RNaseA before immunoprecipitation. C ) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability. Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1 -plasmid carrying the SEN1 versions indicated in the scheme on the left. The growth of the strain expressing the Nrd1 CID-sen1 Δ Nter chimera in 5-FOA implies that this gene can support viability. D ) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1ΔNter mutant. Northern blot assays performed in a Sen1-AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B . Experiments performed in a Δrrp6 background. Representative gel of one out of two independent experiments.The U4 RNA is used as a loading control. Probes used for RNA detection are described in table S14. E ) Sen1 Nter interacts with the C-terminal domain (Cter) of Sen1 both in the presence and in the absence of the NIM in vitro . Pull-down experiments using either a wt or a Δ NIM version of recombinant Sen1 Cter immobilized on glutathione-sepharose beads and a TAP-tagged version of Sen1 Nter expressed in yeast. Representative gel of one out of three independent experiments. Antibodies used for protein detection are listed in table S11.
Figure Legend Snippet: The N-terminal domain of Sen1 can recognize the S5-phosphorylated form of RNAPII CTD and Sen1 C-terminal domain. A) Deletion of Sen1 N-terminal domain does not prevent the interaction of Sen1 with RNAPII. CoIP experiments using Rbp3-FLAG as the bait. Assays were performed in a Sen1-AID strain harboring a plasmid expressing either SEN1 or sen1ΔNter upon depletion of Sen1-AID in the presence of IAA for 2h. An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1ΔNter. Nrd1 is detected as a positive control. Representative gel of one out of two independent experiments. B ) Deletion of the Sen1 N-terminal domain reduces the interaction of Sen1 with the S5P-CTD. CoIP experiments using TAP-Sen1 as the bait. Sen1 proteins were expressed from pGAL in the presence of galactose. Nab3 is detected as a positive control. Representative gel of one out of three independent experiments. In both A ) and B ) protein extracts were treated with RNaseA before immunoprecipitation. C ) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability. Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1 -plasmid carrying the SEN1 versions indicated in the scheme on the left. The growth of the strain expressing the Nrd1 CID-sen1 Δ Nter chimera in 5-FOA implies that this gene can support viability. D ) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1ΔNter mutant. Northern blot assays performed in a Sen1-AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B . Experiments performed in a Δrrp6 background. Representative gel of one out of two independent experiments.The U4 RNA is used as a loading control. Probes used for RNA detection are described in table S14. E ) Sen1 Nter interacts with the C-terminal domain (Cter) of Sen1 both in the presence and in the absence of the NIM in vitro . Pull-down experiments using either a wt or a Δ NIM version of recombinant Sen1 Cter immobilized on glutathione-sepharose beads and a TAP-tagged version of Sen1 Nter expressed in yeast. Representative gel of one out of three independent experiments. Antibodies used for protein detection are listed in table S11.

Techniques Used: Co-Immunoprecipitation Assay, Plasmid Preparation, Expressing, Positive Control, Immunoprecipitation, Mutagenesis, Northern Blot, RNA Detection, In Vitro, Recombinant

13) Product Images from "Krüppel-like factor 4 interacts with p300 to activate mitofusin 2 gene expression induced by all-trans retinoic acid in VSMCs"

Article Title: Krüppel-like factor 4 interacts with p300 to activate mitofusin 2 gene expression induced by all-trans retinoic acid in VSMCs

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2010.96

Acetylation of KLF4 by p300 enhances its binding to the mfn-2 promoter. (A) Identification of anti-Ac-lys antibody. VSMCs were treated with 10 μmol/L ATRA for 2 h, cell lysates were immunoprecipitated using an irrelevant antibody (nonimmune IgG) or the antibodies against KLF4 and acetylated lysine, the precipitates were detected via Western blotting using anti-KLF4 or anti-acetylated lysine antibodies. (B) ATRA induced KLF4 acetylation. VSMCs were treated with 10 μmol/L ATRA for the indicated times. Cell lysates were immunoprecipitated with anti-KLF4 antibody, and acetylated KLF4 was detected via Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (C) ATRA increased the interaction of KLF4 with p300. VSMCs were treated with ATRA for 1 h. Cell lysates were immunoprecipitated with anti-KLF4 or anti-p300 antibodies as indicated (IP). The precipitates were analyzed by Western blotting (IB) with anti-p300 and anti-KLF4 antibodies, respectively. (D) KLF4 was acetylated by p300 in vitro . KLF4 or its acetylation-deficient mutants (300 ng) and p300 (100 ng) were incubated with acetyl-CoA for 30 min at 30 °C, and reaction products were separated by SDS-PAGE, and then acetylated KLF4 was determined by Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (E) The interaction of p300 with KLF4 and its acetylation-deficient mutants. The lysates of VSMCs treated with or without ATRA were incubated with GST, GST-KLF4, GST-KLF4 (K225R), GST-KLF4 (K229R), or GST-KLF4 (K225/229R). After pull down with GST beads, p300 was detected via Western blotting using anti-p300 antibody, and KLF4 was detected with anti-GST antibody. (F) Acetylation of KLF4 enhanced the mfn-2 promoter activity. Luciferase assay was performed in A293 cells transfected with the mfn-2 promoter-reporter plasmid (containing nucleotides −441 to +15 of the mfn-2 promoter), along with different combinations of expression plasmids for KLF4 or its acetylation-deficient mutant (K225/229R), p300, and deacetylase HDAC2. The bars represent the means±SE from three independent experiments. b P
Figure Legend Snippet: Acetylation of KLF4 by p300 enhances its binding to the mfn-2 promoter. (A) Identification of anti-Ac-lys antibody. VSMCs were treated with 10 μmol/L ATRA for 2 h, cell lysates were immunoprecipitated using an irrelevant antibody (nonimmune IgG) or the antibodies against KLF4 and acetylated lysine, the precipitates were detected via Western blotting using anti-KLF4 or anti-acetylated lysine antibodies. (B) ATRA induced KLF4 acetylation. VSMCs were treated with 10 μmol/L ATRA for the indicated times. Cell lysates were immunoprecipitated with anti-KLF4 antibody, and acetylated KLF4 was detected via Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (C) ATRA increased the interaction of KLF4 with p300. VSMCs were treated with ATRA for 1 h. Cell lysates were immunoprecipitated with anti-KLF4 or anti-p300 antibodies as indicated (IP). The precipitates were analyzed by Western blotting (IB) with anti-p300 and anti-KLF4 antibodies, respectively. (D) KLF4 was acetylated by p300 in vitro . KLF4 or its acetylation-deficient mutants (300 ng) and p300 (100 ng) were incubated with acetyl-CoA for 30 min at 30 °C, and reaction products were separated by SDS-PAGE, and then acetylated KLF4 was determined by Western blotting using anti-Ac-lys antibody. Blots for total KLF4 are also shown. (E) The interaction of p300 with KLF4 and its acetylation-deficient mutants. The lysates of VSMCs treated with or without ATRA were incubated with GST, GST-KLF4, GST-KLF4 (K225R), GST-KLF4 (K229R), or GST-KLF4 (K225/229R). After pull down with GST beads, p300 was detected via Western blotting using anti-p300 antibody, and KLF4 was detected with anti-GST antibody. (F) Acetylation of KLF4 enhanced the mfn-2 promoter activity. Luciferase assay was performed in A293 cells transfected with the mfn-2 promoter-reporter plasmid (containing nucleotides −441 to +15 of the mfn-2 promoter), along with different combinations of expression plasmids for KLF4 or its acetylation-deficient mutant (K225/229R), p300, and deacetylase HDAC2. The bars represent the means±SE from three independent experiments. b P

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, In Vitro, Incubation, SDS Page, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Histone Deacetylase Assay

14) Product Images from "Conserved Residues of the Human Mitochondrial Holocytochrome c Synthase Mediate Interactions with Heme"

Article Title: Conserved Residues of the Human Mitochondrial Holocytochrome c Synthase Mediate Interactions with Heme

Journal: Biochemistry

doi: 10.1021/bi500704p

Mutation of HCCS Domain I residues alter heme interactions. Recombinant GST-HCCS protein (alone) and GST-HCCS: cytochrome c cocomplexes were purified from Δccm E. coli and prepared for UV/vis absorption spectroscopy and SDS-PAGE. Shown are spectra for (A) WT HCCS/cyt c, (B) W118A HCCS/cyt c, and (C) N128A/M130A HCCS/cyt c following purification (black line), chemical reduction with sodium dithionite (red), and extraction with pyridine (inset). (D) Soret peak spectra were obtained from cocomplexes representing WT HCCS/cyt c (left) and W118A HCCS/cyt c (right) following purification (black) and treatment with 100 mM imidazole (purple). UV/vis spectra between 500–580 nm (alpha/beta region) of cocomplexes treated with 100 mM imidazole following chemical reduction with sodium dithionite are shown for (E) WT HCCS/cyt c, (F) W118A HCCS/cyt c, (G) N128A/M130A HCCS/cyt c, (H) N128A HCCS/cyt c, and (I) M130A HCCS/cyt c. (J) Heme stain of the indicated purified cocomplexes following SDS-PAGE and transfer to nitrocellulose. (K) Heme stain (top) and GST-HCCS immunoblot (bottom) of the indicated purified cocomplexes following SDS-PAGE and transfer to nitrocellulose. (L) UV–vis spectra of sodium dithionite reduced purified cocomplexes from WT HCCS (black), Y120A HCCS (red), and P121A HCCS (blue). (M) UV–vis spectra of HCCS proteins (alone) purified from WT HCCS (black) and Y120A/P121A HCCS (green). (N) UV–vis spectra of purified cocomplexes from WT HCCS/cyt c (black) and Y120A/P121A HCCS/cyt c (orange). (O) Heme stain (top) and GST-HCCS immunoblot (bottom) of the indicated purified cocomplexes following SDS-PAGE and transfer to nitrocellulose. (P) UV–vis spectra of HCCS proteins (alone) purified from WT HCCS (black), Y120A HCCS (red), and P121A HCCS (blue). Arrows indicate wavelength (nm) of peak absorption maxima. All spectra were performed with equal amounts (50–100 μg) of total purified protein. All SDS-PAGE samples were equally loaded (2–5 μg of total purified protein each). For all proteins, Bradford quantitation was confirmed by Coomassie staining, which also indicated that GST-HCCS proteins were obtained at > 90% purity.
Figure Legend Snippet: Mutation of HCCS Domain I residues alter heme interactions. Recombinant GST-HCCS protein (alone) and GST-HCCS: cytochrome c cocomplexes were purified from Δccm E. coli and prepared for UV/vis absorption spectroscopy and SDS-PAGE. Shown are spectra for (A) WT HCCS/cyt c, (B) W118A HCCS/cyt c, and (C) N128A/M130A HCCS/cyt c following purification (black line), chemical reduction with sodium dithionite (red), and extraction with pyridine (inset). (D) Soret peak spectra were obtained from cocomplexes representing WT HCCS/cyt c (left) and W118A HCCS/cyt c (right) following purification (black) and treatment with 100 mM imidazole (purple). UV/vis spectra between 500–580 nm (alpha/beta region) of cocomplexes treated with 100 mM imidazole following chemical reduction with sodium dithionite are shown for (E) WT HCCS/cyt c, (F) W118A HCCS/cyt c, (G) N128A/M130A HCCS/cyt c, (H) N128A HCCS/cyt c, and (I) M130A HCCS/cyt c. (J) Heme stain of the indicated purified cocomplexes following SDS-PAGE and transfer to nitrocellulose. (K) Heme stain (top) and GST-HCCS immunoblot (bottom) of the indicated purified cocomplexes following SDS-PAGE and transfer to nitrocellulose. (L) UV–vis spectra of sodium dithionite reduced purified cocomplexes from WT HCCS (black), Y120A HCCS (red), and P121A HCCS (blue). (M) UV–vis spectra of HCCS proteins (alone) purified from WT HCCS (black) and Y120A/P121A HCCS (green). (N) UV–vis spectra of purified cocomplexes from WT HCCS/cyt c (black) and Y120A/P121A HCCS/cyt c (orange). (O) Heme stain (top) and GST-HCCS immunoblot (bottom) of the indicated purified cocomplexes following SDS-PAGE and transfer to nitrocellulose. (P) UV–vis spectra of HCCS proteins (alone) purified from WT HCCS (black), Y120A HCCS (red), and P121A HCCS (blue). Arrows indicate wavelength (nm) of peak absorption maxima. All spectra were performed with equal amounts (50–100 μg) of total purified protein. All SDS-PAGE samples were equally loaded (2–5 μg of total purified protein each). For all proteins, Bradford quantitation was confirmed by Coomassie staining, which also indicated that GST-HCCS proteins were obtained at > 90% purity.

Techniques Used: Mutagenesis, Recombinant, Purification, Spectroscopy, SDS Page, Staining, Quantitation Assay

Mutation of HCCS Domain II residues alter heme interactions. Recombinant GST-HCCS protein (alone) and GST-HCCS: cytochrome c cocomplexes were purified from E. coli and prepared for UV/vis absorption spectroscopy and SDS-PAGE. (A) UV–vis spectra of purified (black), sodium dithionite reduced (red), and pyridine extracted (inset) N155A HCCS/cyt cocomplexes. (B) Heme stain (top) and GST-HCCS immunoblot (bottom) of the indicated purified cocomplexes following SDS-PAGE and transfer to nitrocellulose. (C) UV–vis spectra of purified WT HCCS protein (black) and N155A HCCS protein (orange). (D) UV–vis spectra of purified cocomplexes from WT HCCS/cyt c (black), E159D HCCS/cyt c (orange), E159K HCCS/cyt c (red), and E159A (green). (E) Heme stain (top) of the indicated purified cocomplexes following SDS-PAGE and transfer to nitrocellulose, and corresponding Coomassie stain (bottom). (F) UV–vis spectra of purified WT HCCS protein (black), E159D HCCS protein (orange), E159K HCCS protein (red), and E159A HCCS protein (green). Arrows indicate wavelength (nm) of absorption maxima. All spectra were performed with equal amounts (50–100 μg) of total purified protein. All SDS-PAGE samples were equally loaded (2–5 μgof total purified protein each). For all proteins, Bradford quantitation was confirmed by Coomassie staining, which also indicated that GST-HCCS proteins were obtained at > 90% purity.
Figure Legend Snippet: Mutation of HCCS Domain II residues alter heme interactions. Recombinant GST-HCCS protein (alone) and GST-HCCS: cytochrome c cocomplexes were purified from E. coli and prepared for UV/vis absorption spectroscopy and SDS-PAGE. (A) UV–vis spectra of purified (black), sodium dithionite reduced (red), and pyridine extracted (inset) N155A HCCS/cyt cocomplexes. (B) Heme stain (top) and GST-HCCS immunoblot (bottom) of the indicated purified cocomplexes following SDS-PAGE and transfer to nitrocellulose. (C) UV–vis spectra of purified WT HCCS protein (black) and N155A HCCS protein (orange). (D) UV–vis spectra of purified cocomplexes from WT HCCS/cyt c (black), E159D HCCS/cyt c (orange), E159K HCCS/cyt c (red), and E159A (green). (E) Heme stain (top) of the indicated purified cocomplexes following SDS-PAGE and transfer to nitrocellulose, and corresponding Coomassie stain (bottom). (F) UV–vis spectra of purified WT HCCS protein (black), E159D HCCS protein (orange), E159K HCCS protein (red), and E159A HCCS protein (green). Arrows indicate wavelength (nm) of absorption maxima. All spectra were performed with equal amounts (50–100 μg) of total purified protein. All SDS-PAGE samples were equally loaded (2–5 μgof total purified protein each). For all proteins, Bradford quantitation was confirmed by Coomassie staining, which also indicated that GST-HCCS proteins were obtained at > 90% purity.

Techniques Used: Mutagenesis, Recombinant, Purification, Spectroscopy, SDS Page, Staining, Quantitation Assay

15) Product Images from "Mitofilin and CHCHD6 physically interact with Sam50 to sustain cristae structure"

Article Title: Mitofilin and CHCHD6 physically interact with Sam50 to sustain cristae structure

Journal: Scientific Reports

doi: 10.1038/srep16064

Altered cristae morphology caused by Mitofilin knockdown or CHCHD6 knockout. ( A , B ) Electron microscopy of mitochondria in control and Mitofilin-knockdown ( A ) or CHCHD6-knockout cells ( B ). The black arrows indicate disrupted mitochondria. ( C ) Electron tomography of mitochondrial morphological changes in Mitofilin-knockdown cells. The OMM is depicted in light blue, the IBM is shown in pink, and cristae are shown in green. These images are rotations of surface-rendered views of tomographic reconstructions of mitochondria.
Figure Legend Snippet: Altered cristae morphology caused by Mitofilin knockdown or CHCHD6 knockout. ( A , B ) Electron microscopy of mitochondria in control and Mitofilin-knockdown ( A ) or CHCHD6-knockout cells ( B ). The black arrows indicate disrupted mitochondria. ( C ) Electron tomography of mitochondrial morphological changes in Mitofilin-knockdown cells. The OMM is depicted in light blue, the IBM is shown in pink, and cristae are shown in green. These images are rotations of surface-rendered views of tomographic reconstructions of mitochondria.

Techniques Used: Knock-Out, Electron Microscopy

Generation of Mitofilin-knockdown and CHCHD6-knockout HeLa cell clones with TALENs ( A ) The boxes indicate the TALEN binding sites for Mitofilin, targeting exon 3 ( a ), and for CHCHD6, targeting exon 2 ( b ).Deletions in alleles of each clone are indicated. ( B ) Immunoblot analysis using whole-cell lysates from wild-type (WT) cells and the two Mitofilin-knockdown (a) or two CHCHD6-knockout ( b ) HeLa cell lines. Full-length blots/gels are presented in Supplementary Figure 4 .
Figure Legend Snippet: Generation of Mitofilin-knockdown and CHCHD6-knockout HeLa cell clones with TALENs ( A ) The boxes indicate the TALEN binding sites for Mitofilin, targeting exon 3 ( a ), and for CHCHD6, targeting exon 2 ( b ).Deletions in alleles of each clone are indicated. ( B ) Immunoblot analysis using whole-cell lysates from wild-type (WT) cells and the two Mitofilin-knockdown (a) or two CHCHD6-knockout ( b ) HeLa cell lines. Full-length blots/gels are presented in Supplementary Figure 4 .

Techniques Used: Knock-Out, Clone Assay, TALENs, Binding Assay

CHCHD6 interacts with OPA1 and directly interacts with Sam50. ( A ) The IP samples of Mitofilin and CHCHD6 were analyzed via SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. ( B ) Schematic illustration of the full-length and deletion variants of the CHCHD6 protein. ( C ) Coomassie Blue staining of purified GST, GST-Sam50, and His-tagged full-length and deletion variants of CHCHD6. The protein products are indicated by asterisks . ( D ) Mapping of the CHCHD6 and Sam50 interaction region on CHCHD6. Left , purified His-tagged full-length CHCHD6 and deletion variants were incubated with GST or GST-tagged Sam50. Then, GST pull-downs were conducted. Right , western blot analysis of 1% input of purified GST, GST-Sam50, and His-tagged full-length and deletion variants of CHCHD6. E, Sam50 directly interacts with Mitofilin. Left , the GST pull-down was performed by incubating His-tagged Mitofilin 123–758 with GST-tagged Sam50 or GST. The pull-down protein products were analyzed by Western blot using anti-GST and anti-His antibodies. Right , western blot analysis of 1% input of His-Mitofilin 123–758 . Full-length blots/gels are presented in Supplementary Figure 6 .
Figure Legend Snippet: CHCHD6 interacts with OPA1 and directly interacts with Sam50. ( A ) The IP samples of Mitofilin and CHCHD6 were analyzed via SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. ( B ) Schematic illustration of the full-length and deletion variants of the CHCHD6 protein. ( C ) Coomassie Blue staining of purified GST, GST-Sam50, and His-tagged full-length and deletion variants of CHCHD6. The protein products are indicated by asterisks . ( D ) Mapping of the CHCHD6 and Sam50 interaction region on CHCHD6. Left , purified His-tagged full-length CHCHD6 and deletion variants were incubated with GST or GST-tagged Sam50. Then, GST pull-downs were conducted. Right , western blot analysis of 1% input of purified GST, GST-Sam50, and His-tagged full-length and deletion variants of CHCHD6. E, Sam50 directly interacts with Mitofilin. Left , the GST pull-down was performed by incubating His-tagged Mitofilin 123–758 with GST-tagged Sam50 or GST. The pull-down protein products were analyzed by Western blot using anti-GST and anti-His antibodies. Right , western blot analysis of 1% input of His-Mitofilin 123–758 . Full-length blots/gels are presented in Supplementary Figure 6 .

Techniques Used: SDS Page, Staining, Purification, Incubation, Western Blot

Steady-state levels several MICOS components in Mitofilin-knockdown or CHCHD6-knockout cells. Equal amounts of protein samples in control cells and Mitofilin knockdown cells ( A ) or CHCHD6 knockout cells ( B ) were analyzed via SDS-PAGE followed by immunoblot with indicated antibodies. The values represent the average protein expression ± SD from three independent experiments. GAPDH was used as a loading control. Full-length blots/gels are presented in Supplementary Figure 5 .
Figure Legend Snippet: Steady-state levels several MICOS components in Mitofilin-knockdown or CHCHD6-knockout cells. Equal amounts of protein samples in control cells and Mitofilin knockdown cells ( A ) or CHCHD6 knockout cells ( B ) were analyzed via SDS-PAGE followed by immunoblot with indicated antibodies. The values represent the average protein expression ± SD from three independent experiments. GAPDH was used as a loading control. Full-length blots/gels are presented in Supplementary Figure 5 .

Techniques Used: Knock-Out, SDS Page, Expressing

Effects of stable Mitofilin depletion and CHCHD6 knockout on mitochondrial function. ( A ) Effects of Mitofilin knockdown and CHCHD6 knockout on ΔΨm. ΔΨm was determined by the fluorescence ratio of red to green using the JC-1 assay. ( B ) Effects of Mitofilin knockdown and CHCHD6 knockout on intracellular ATP levels. Intracellular ATP production was measured using a luciferase-based assay; ATP levels were normalized to protein levels. Statistical analysis was performed using Student’s t-test (*P
Figure Legend Snippet: Effects of stable Mitofilin depletion and CHCHD6 knockout on mitochondrial function. ( A ) Effects of Mitofilin knockdown and CHCHD6 knockout on ΔΨm. ΔΨm was determined by the fluorescence ratio of red to green using the JC-1 assay. ( B ) Effects of Mitofilin knockdown and CHCHD6 knockout on intracellular ATP levels. Intracellular ATP production was measured using a luciferase-based assay; ATP levels were normalized to protein levels. Statistical analysis was performed using Student’s t-test (*P

Techniques Used: Knock-Out, Fluorescence, Luciferase

Mitofilin, Sam50, and CHCHD 3 and 6 are in the same complex, ( A ) a and b show MS data for the indicated proteins from the Mitofilin and CHCHD6 IPs. The number of total peptides (Total) and total unique peptides (Unique) identified by MS are shown. ( B ) The IP sample of endogenous Mitofilin ( a ) and CHCHD6 ( b ) were analyzed via SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. Asterisks indicated that the blot of CHCHD3 was cropped from a different gel, as CHCHD3 and CHCHD6 have similar sizes. Full-length blots/gels are presented in Supplementary Figure 3 .
Figure Legend Snippet: Mitofilin, Sam50, and CHCHD 3 and 6 are in the same complex, ( A ) a and b show MS data for the indicated proteins from the Mitofilin and CHCHD6 IPs. The number of total peptides (Total) and total unique peptides (Unique) identified by MS are shown. ( B ) The IP sample of endogenous Mitofilin ( a ) and CHCHD6 ( b ) were analyzed via SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. Asterisks indicated that the blot of CHCHD3 was cropped from a different gel, as CHCHD3 and CHCHD6 have similar sizes. Full-length blots/gels are presented in Supplementary Figure 3 .

Techniques Used: Mass Spectrometry, SDS Page

Proposed model of how Mitofilin and CHCHD6 function in cristae formation and preservation. Mitofilin and CHCHD6 forms a complex with OPA1 at CJs thereby influencing CJs formation and stability. Mitofilin and CHCHD6 directly connects MICOS with Sam50 would sustain cristae architecture. The direct interaction between Mitofilin and CHCHD6 was reported by Jie An, et al. 12 .
Figure Legend Snippet: Proposed model of how Mitofilin and CHCHD6 function in cristae formation and preservation. Mitofilin and CHCHD6 forms a complex with OPA1 at CJs thereby influencing CJs formation and stability. Mitofilin and CHCHD6 directly connects MICOS with Sam50 would sustain cristae architecture. The direct interaction between Mitofilin and CHCHD6 was reported by Jie An, et al. 12 .

Techniques Used: Preserving

16) Product Images from "APOBEC4 Enhances the Replication of HIV-1"

Article Title: APOBEC4 Enhances the Replication of HIV-1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0155422

A4 interacts weakly with single-stranded DNA. EMSA with purified, GST-A3C (a), GST-A4 and GST-A4ΔKK (b) performed with 30 nt single stranded target DNA labeled with 3’-labeled with biotin. Indicated amounts of protein (at the bottom of blot) were titrated with 10 nM of DNA. (+) indicates presence of competitor DNA, which is unlabeled 80 nt DNA (200-fold molar excess), as used for deamination assay to demonstrate specific binding of protein to DNA being causative for the shift. For GST-A3C (a) a separate panel was added for reactions containing 0.05% NP-40 detergent. (c) A4-HA crosslinking by DSS. DSS was added to the cleared cell lysates to reach the indicated DSS concentrations. The blot was probed with anti HA antibody to detect monomeric and dimeric forms of A4-HA.
Figure Legend Snippet: A4 interacts weakly with single-stranded DNA. EMSA with purified, GST-A3C (a), GST-A4 and GST-A4ΔKK (b) performed with 30 nt single stranded target DNA labeled with 3’-labeled with biotin. Indicated amounts of protein (at the bottom of blot) were titrated with 10 nM of DNA. (+) indicates presence of competitor DNA, which is unlabeled 80 nt DNA (200-fold molar excess), as used for deamination assay to demonstrate specific binding of protein to DNA being causative for the shift. For GST-A3C (a) a separate panel was added for reactions containing 0.05% NP-40 detergent. (c) A4-HA crosslinking by DSS. DSS was added to the cleared cell lysates to reach the indicated DSS concentrations. The blot was probed with anti HA antibody to detect monomeric and dimeric forms of A4-HA.

Techniques Used: Purification, Labeling, Binding Assay

17) Product Images from "Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A"

Article Title: Interferon-inducible protein SCOTIN interferes with HCV replication through the autolysosomal degradation of NS5A

Journal: Nature Communications

doi: 10.1038/ncomms10631

SCOTIN promotes NS5A trafficking to autophagosomes. ( a , b ) Huh-7 cells were transfected with the indicated plasmids or siRNAs for 48 h, and total cell lysates were subjected to immunoblotting using the indicated antibodies. A GST-expressing pEBG ( a ) or LacZ-V5 ( b ) plasmid was included to monitor transfection efficiency. ( c – f ) Huh-7 cells were transfected with the indicated plasmids followed by treatment with MG132 (1 μM; c ), 3-MA (10 mM; d ), BFA (100 μM; e ) or CQ (50 μM; f ) for 12 h. Total cell lysates were subjected to immunoblotting using the indicated antibodies. Distilled water (DW) was used as a control for 3-MA and CQ, and dimethylsulphoxide (DMSO) was used as a control for BFA and MG132. c-MYC was used as a positive control for MG132. The relative ratios (FLAG-NS5A/LacZ-V5) are shown based on the intensity values quantified using the Multigauge programme (Fuji Film). ( g ) Huh-7 cells transfected with the indicated plasmids along with control or ATG7 siRNA. Extracted lysates were subjected to immunoblotting using the indicated antibodies. ( c – g ) To determine the transfection efficiency, LacZ-V5 was co-transfected with the indicated plasmids. ( h – j ) Huh-7 cells were transfected with FLAG-NS5A along with control or SCOTIN siRNA, and then were treated with rapamycin (2 μM) for 6 h. ( h ) Cellular localization of endogenous LC3 (green), FLAG-NS5A (red) and the nucleus (Hoechst) was detected using confocal fluorescence microscopy. A colocalization image was obtained using the Co-localization Image J Plugin. Scale bars, 10 μm. ( i ) Total cell lysates from the same cell populations were subjected to immunoblotting using the indicated antibodies. ( j ) The extent of colocalization of FLAG-NS5A and LC3 in each cell was measured using Pearson's correlation coefficient with the JAcoP Image J Plugin. The coefficient values were plotted using a whiskers box plot. The box extends from the 25th to 75th percentiles, and the error bars depict the minimum and maximum values. Cells transfected with siCON ( N =46) or with siSCOTIN ( N =50) were analysed. The asterisk denotes the P value calculated using the t -test (*** P value
Figure Legend Snippet: SCOTIN promotes NS5A trafficking to autophagosomes. ( a , b ) Huh-7 cells were transfected with the indicated plasmids or siRNAs for 48 h, and total cell lysates were subjected to immunoblotting using the indicated antibodies. A GST-expressing pEBG ( a ) or LacZ-V5 ( b ) plasmid was included to monitor transfection efficiency. ( c – f ) Huh-7 cells were transfected with the indicated plasmids followed by treatment with MG132 (1 μM; c ), 3-MA (10 mM; d ), BFA (100 μM; e ) or CQ (50 μM; f ) for 12 h. Total cell lysates were subjected to immunoblotting using the indicated antibodies. Distilled water (DW) was used as a control for 3-MA and CQ, and dimethylsulphoxide (DMSO) was used as a control for BFA and MG132. c-MYC was used as a positive control for MG132. The relative ratios (FLAG-NS5A/LacZ-V5) are shown based on the intensity values quantified using the Multigauge programme (Fuji Film). ( g ) Huh-7 cells transfected with the indicated plasmids along with control or ATG7 siRNA. Extracted lysates were subjected to immunoblotting using the indicated antibodies. ( c – g ) To determine the transfection efficiency, LacZ-V5 was co-transfected with the indicated plasmids. ( h – j ) Huh-7 cells were transfected with FLAG-NS5A along with control or SCOTIN siRNA, and then were treated with rapamycin (2 μM) for 6 h. ( h ) Cellular localization of endogenous LC3 (green), FLAG-NS5A (red) and the nucleus (Hoechst) was detected using confocal fluorescence microscopy. A colocalization image was obtained using the Co-localization Image J Plugin. Scale bars, 10 μm. ( i ) Total cell lysates from the same cell populations were subjected to immunoblotting using the indicated antibodies. ( j ) The extent of colocalization of FLAG-NS5A and LC3 in each cell was measured using Pearson's correlation coefficient with the JAcoP Image J Plugin. The coefficient values were plotted using a whiskers box plot. The box extends from the 25th to 75th percentiles, and the error bars depict the minimum and maximum values. Cells transfected with siCON ( N =46) or with siSCOTIN ( N =50) were analysed. The asterisk denotes the P value calculated using the t -test (*** P value

Techniques Used: Transfection, Expressing, Plasmid Preparation, Positive Control, Fluorescence, Microscopy

Physical interaction between NS5A and SCOTIN is required for control of degradation. ( a ) HEK293 cells were transfected with GST-NS5A along with an empty (pcDNA3.1-MYC) or SCOTIN-MYC plasmid for 48 h. GST-NS5A was pulled down from total cell lysates using glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( b ) Schematic representation of GST-tagged NS5A deletion constructs. ( c ) HEK293 cells were transfected with the indicated plasmids and subjected to a GST pulldown assay. ( d ) Huh-7 cells were transfected with the indicated plasmids for 48 h, followed by immunoblotting analysis using the indicated antibodies. EGFP was used to monitor transfection efficiency. ( e ) An illustration of the truncated SCOTIN constructs is shown. ( f ) Huh-7 cells were transfected with the indicated SCOTIN mutant constructs, and immunofluorescence analysis was performed using LC3 and MYC antibodies, followed by Hoechst staining. Cellular localization of LC3 (green), MYC (red) and the nucleus (blue) was determined using fluorescence microscopy. Representative images are shown. Higher-magnification images are shown in the right corner. Scale bar, 10 μm. ( g ) Western blot analysis of Huh-7 cells transfected with the indicated plasmids. EGFP-N1 was co-transfected to monitor transfection efficiency. ( h ) HEK293 cells were transfected with GST-NS5A along with the indicated plasmids for 48 h. Total cell lysates were incubated with glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( i , j ) Huh-7 cells were transfected with the indicated plasmids followed by HCVcc infection (10 MOI) for 3 days before harvesting. The intracellular HCV RNA levels were determined using RT–qPCR ( i ), and total cell lysates were subjected to immunoblotting using the indicated antibodies ( j ). The bars indicate the mean value±s.d. obtained from triplicate experiments. The asterisks indicate the P values calculated using the t -test. ** P value
Figure Legend Snippet: Physical interaction between NS5A and SCOTIN is required for control of degradation. ( a ) HEK293 cells were transfected with GST-NS5A along with an empty (pcDNA3.1-MYC) or SCOTIN-MYC plasmid for 48 h. GST-NS5A was pulled down from total cell lysates using glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( b ) Schematic representation of GST-tagged NS5A deletion constructs. ( c ) HEK293 cells were transfected with the indicated plasmids and subjected to a GST pulldown assay. ( d ) Huh-7 cells were transfected with the indicated plasmids for 48 h, followed by immunoblotting analysis using the indicated antibodies. EGFP was used to monitor transfection efficiency. ( e ) An illustration of the truncated SCOTIN constructs is shown. ( f ) Huh-7 cells were transfected with the indicated SCOTIN mutant constructs, and immunofluorescence analysis was performed using LC3 and MYC antibodies, followed by Hoechst staining. Cellular localization of LC3 (green), MYC (red) and the nucleus (blue) was determined using fluorescence microscopy. Representative images are shown. Higher-magnification images are shown in the right corner. Scale bar, 10 μm. ( g ) Western blot analysis of Huh-7 cells transfected with the indicated plasmids. EGFP-N1 was co-transfected to monitor transfection efficiency. ( h ) HEK293 cells were transfected with GST-NS5A along with the indicated plasmids for 48 h. Total cell lysates were incubated with glutathione-Sepharose beads, and the interacting proteins were analysed by immunoblotting. ( i , j ) Huh-7 cells were transfected with the indicated plasmids followed by HCVcc infection (10 MOI) for 3 days before harvesting. The intracellular HCV RNA levels were determined using RT–qPCR ( i ), and total cell lysates were subjected to immunoblotting using the indicated antibodies ( j ). The bars indicate the mean value±s.d. obtained from triplicate experiments. The asterisks indicate the P values calculated using the t -test. ** P value

Techniques Used: Transfection, Plasmid Preparation, Construct, GST Pulldown Assay, Mutagenesis, Immunofluorescence, Staining, Fluorescence, Microscopy, Western Blot, Incubation, Infection, Quantitative RT-PCR

18) Product Images from "Digitor/dASCIZ Has Multiple Roles in Drosophila Development"

Article Title: Digitor/dASCIZ Has Multiple Roles in Drosophila Development

Journal: PLoS ONE

doi: 10.1371/journal.pone.0166829

Digitor/dASCIZ interact with Skeletor and Cut up in pull down assays. (A) Diagrams of full-length Skeletor and Digitor/dASCIZ and the fragments used for yeast two-hybrid assays. The region of Skeletor corresponding to the yeast two-hybrid (Y2H) bait and Skeletor-His is indicated in green. Full-length Digitor/dASCIZ has four zinc-finger domains (ZN) and a SCD with six TQT-motifs and the two GST- and His-tagged COOH-terminal fragments, respectively, are indicated below. (B) A Digitor-GST construct pulls down Skeletor-His as detected by His antibody (lane 4). Beads-only and GST-only pulldown controls were negative (lane 2 and 3). Lane 1 shows the position of the Skeletor-His fusion protein. (C) A Skeletor-His construct pulls down Digitor-GST as detected by GST antibody (lane 4). Beads only and JIL-1-His pulldown controls were negative (lane 2 and 3). Lane 1 shows the position of the Digitor-GST fusion protein. (D) A Cut up-MBP construct pulls down Digitor-His as detected by His antibody (lane 4). Beads only and MBP only pulldown controls were negative (lane 2 and 3). Lane 1 shows the position of the Digitor-His fusion protein. (E) A Digitor-His construct pulls down Cut up-MBP as detected by MBP antibody (lane 4). Beads-only and Skeletor-His pulldown controls were negative (lane 2 and 3). Lane 1 shows the position of the Cut up-MBP fusion protein.
Figure Legend Snippet: Digitor/dASCIZ interact with Skeletor and Cut up in pull down assays. (A) Diagrams of full-length Skeletor and Digitor/dASCIZ and the fragments used for yeast two-hybrid assays. The region of Skeletor corresponding to the yeast two-hybrid (Y2H) bait and Skeletor-His is indicated in green. Full-length Digitor/dASCIZ has four zinc-finger domains (ZN) and a SCD with six TQT-motifs and the two GST- and His-tagged COOH-terminal fragments, respectively, are indicated below. (B) A Digitor-GST construct pulls down Skeletor-His as detected by His antibody (lane 4). Beads-only and GST-only pulldown controls were negative (lane 2 and 3). Lane 1 shows the position of the Skeletor-His fusion protein. (C) A Skeletor-His construct pulls down Digitor-GST as detected by GST antibody (lane 4). Beads only and JIL-1-His pulldown controls were negative (lane 2 and 3). Lane 1 shows the position of the Digitor-GST fusion protein. (D) A Cut up-MBP construct pulls down Digitor-His as detected by His antibody (lane 4). Beads only and MBP only pulldown controls were negative (lane 2 and 3). Lane 1 shows the position of the Digitor-His fusion protein. (E) A Digitor-His construct pulls down Cut up-MBP as detected by MBP antibody (lane 4). Beads-only and Skeletor-His pulldown controls were negative (lane 2 and 3). Lane 1 shows the position of the Cut up-MBP fusion protein.

Techniques Used: Construct

19) Product Images from "Integrin ?1?1 Promotes Caveolin-1 Dephosphorylation by Activating T Cell Protein-tyrosine Phosphatase *"

Article Title: Integrin ?1?1 Promotes Caveolin-1 Dephosphorylation by Activating T Cell Protein-tyrosine Phosphatase *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.156729

Constitutively active TCPTP dephosphorylates caveolin-1. A , Coomassie staining of purified GST-pCav-1 and GST-Cav-1 (2 μg/lane). 20 ng of purified GST-pCav-1 and GST-Cav-1 were separated in 12% SDS-PAGE, transferred to nitrocellulose, and blotted with anti-Tyr(P) 14 -pCav-1 or anti-Cav-1 antibodies. The arrow indicates full-length constructs, whereas the asterisk indicates a cleaved product still retaining the Cav-1 phosphorylation site (Tyr 14 ). B , GST-pCav-1 (0.4 μg/ml) was incubated with constitutively active TCPTP (TC-37, 0.4 μg/ml) or PTP1B (0.4 μg/ml) with or without tyrosine phosphatase inhibitors. 30 min later, the samples were analyzed by Western blot for levels of phosphorylated and total Cav-1. C , GST-pCav-1, coated at the concentrations indicated, was incubated with either phosphatase buffer or TCPTP-37 followed by incubation with anti-phosphorylated or anti-Cav-1 antibodies. One representative experiment performed in triplicate is shown. Two experiments were performed with similar results. *, indicates significant differences ( p
Figure Legend Snippet: Constitutively active TCPTP dephosphorylates caveolin-1. A , Coomassie staining of purified GST-pCav-1 and GST-Cav-1 (2 μg/lane). 20 ng of purified GST-pCav-1 and GST-Cav-1 were separated in 12% SDS-PAGE, transferred to nitrocellulose, and blotted with anti-Tyr(P) 14 -pCav-1 or anti-Cav-1 antibodies. The arrow indicates full-length constructs, whereas the asterisk indicates a cleaved product still retaining the Cav-1 phosphorylation site (Tyr 14 ). B , GST-pCav-1 (0.4 μg/ml) was incubated with constitutively active TCPTP (TC-37, 0.4 μg/ml) or PTP1B (0.4 μg/ml) with or without tyrosine phosphatase inhibitors. 30 min later, the samples were analyzed by Western blot for levels of phosphorylated and total Cav-1. C , GST-pCav-1, coated at the concentrations indicated, was incubated with either phosphatase buffer or TCPTP-37 followed by incubation with anti-phosphorylated or anti-Cav-1 antibodies. One representative experiment performed in triplicate is shown. Two experiments were performed with similar results. *, indicates significant differences ( p

Techniques Used: Staining, Purification, SDS Page, Construct, Incubation, Western Blot

20) Product Images from "Sld2 binds to origin single-stranded DNA and stimulates DNA annealing"

Article Title: Sld2 binds to origin single-stranded DNA and stimulates DNA annealing

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq1222

Sld2T84D binds to ss ARS1 . ( A ) Schematic of ARS1. The complete nucleotide sequences are found in Supplementary Table S1 . ( B ) Purified GST–Sld2T84D, GST–Sld2 or GST were studied for interaction with radiolabeled DNA as indicated to the left of the gel. GST–Sld2T84D, GST–Sld2 or GST (13 pmol) and varying amounts of DNA, as indicated in the figure, were mixed with 1.3 mg glutathione agarose beads and incubated for 5 min at 30°C. The beads were washed and analyzed as described in ‘Materials and Methods’ section. The results from experiments similar to (B) were quantified and plotted as pmol of DNA bound versus pmol of input DNA ( C, D and E ). Sld2T84D binds tighter to ss ARS1 -1 than to either ss ARS1 -2 or ds ARS1 . Sld2T84D binds tighter than Sld2 to ss ARS1 -1.
Figure Legend Snippet: Sld2T84D binds to ss ARS1 . ( A ) Schematic of ARS1. The complete nucleotide sequences are found in Supplementary Table S1 . ( B ) Purified GST–Sld2T84D, GST–Sld2 or GST were studied for interaction with radiolabeled DNA as indicated to the left of the gel. GST–Sld2T84D, GST–Sld2 or GST (13 pmol) and varying amounts of DNA, as indicated in the figure, were mixed with 1.3 mg glutathione agarose beads and incubated for 5 min at 30°C. The beads were washed and analyzed as described in ‘Materials and Methods’ section. The results from experiments similar to (B) were quantified and plotted as pmol of DNA bound versus pmol of input DNA ( C, D and E ). Sld2T84D binds tighter to ss ARS1 -1 than to either ss ARS1 -2 or ds ARS1 . Sld2T84D binds tighter than Sld2 to ss ARS1 -1.

Techniques Used: Purification, Incubation

Sld2T84D binds to ss ARS305 bottom strand (ss ARS305 -1). ( A ) Purified GST–Sld2T84D, GST–Sld2 or GST were studied for interaction with radiolabeled ss ARS305 -1 (top panel), ss ARS305 -2 (middle panel) or ds ARS305 (bottom panel). GST–Sld2T84D, GST–Sld2 or GST (13 pmol) and varying amounts of DNA, as indicated in the figure, were mixed with 1.3 mg glutathione agarose beads and incubated for 5 min at 30°C. The beads were washed and analyzed as described in ‘Materials and Methods’ section. The results from experiments similar to (A) were quantified and plotted as pmol of DNA bound versus pmol of input DNA ( B and C ). Sld2T84D binds tighter to ss ARS305 -1 than to either ss ARS305 -2 or ds ARS305 . Sld2T84D binds tighter than Sld2 to ss ARS305 -1.
Figure Legend Snippet: Sld2T84D binds to ss ARS305 bottom strand (ss ARS305 -1). ( A ) Purified GST–Sld2T84D, GST–Sld2 or GST were studied for interaction with radiolabeled ss ARS305 -1 (top panel), ss ARS305 -2 (middle panel) or ds ARS305 (bottom panel). GST–Sld2T84D, GST–Sld2 or GST (13 pmol) and varying amounts of DNA, as indicated in the figure, were mixed with 1.3 mg glutathione agarose beads and incubated for 5 min at 30°C. The beads were washed and analyzed as described in ‘Materials and Methods’ section. The results from experiments similar to (A) were quantified and plotted as pmol of DNA bound versus pmol of input DNA ( B and C ). Sld2T84D binds tighter to ss ARS305 -1 than to either ss ARS305 -2 or ds ARS305 . Sld2T84D binds tighter than Sld2 to ss ARS305 -1.

Techniques Used: Purification, Incubation

21) Product Images from "The role of IAP antagonist proteins in the core apoptosis pathway of the mosquito disease vector Aedes aegypti"

Article Title: The role of IAP antagonist proteins in the core apoptosis pathway of the mosquito disease vector Aedes aegypti

Journal: Apoptosis : an international journal on programmed cell death

doi: 10.1007/s10495-011-0575-3

AeIAP1 and DIAP1 inhibit the activity of AeDronc and effector caspases CASPS7 and CASPS8. a C6/36 cells were transfected with CASPS7 or CASPS8 constructs, with or without a plasmid expressing AeIAP1 or DIAP1 or a control plasmid. Cell lysates were prepared
Figure Legend Snippet: AeIAP1 and DIAP1 inhibit the activity of AeDronc and effector caspases CASPS7 and CASPS8. a C6/36 cells were transfected with CASPS7 or CASPS8 constructs, with or without a plasmid expressing AeIAP1 or DIAP1 or a control plasmid. Cell lysates were prepared

Techniques Used: Activity Assay, Transfection, Construct, Plasmid Preparation, Expressing

Silencing expression of mx or imp protects Aag2 cells from apoptotic stimuli. Aag2 cells were treated with the indicated dsRNAs for 24 h, and then 50 ng ml −1 of Act D ( a ) or 5 μg ml −1 of Aeiap1 dsRNA ( b ) were added. Twelve hours
Figure Legend Snippet: Silencing expression of mx or imp protects Aag2 cells from apoptotic stimuli. Aag2 cells were treated with the indicated dsRNAs for 24 h, and then 50 ng ml −1 of Act D ( a ) or 5 μg ml −1 of Aeiap1 dsRNA ( b ) were added. Twelve hours

Techniques Used: Expressing, Activated Clotting Time Assay

A. aegypti IAP antagonists directly bind to AeIAP1, and binding is dependent on the IBM. a The indicated His-tagged recombinant proteins were incubated with 35 S-labeled AeIAP1, after which protein complexes were purified using Talon resin and examined
Figure Legend Snippet: A. aegypti IAP antagonists directly bind to AeIAP1, and binding is dependent on the IBM. a The indicated His-tagged recombinant proteins were incubated with 35 S-labeled AeIAP1, after which protein complexes were purified using Talon resin and examined

Techniques Used: Binding Assay, Recombinant, Incubation, Labeling, Purification

AeIAP1 and DIAP1 inhibit IAP antagonist-induced caspase activation. C6/36 cells ( a ) or SF-21 cells ( b ) were co-transfected with IAP antagonist constructs and plasmids expressing AeIAP1 or DIAP1, or a control irrelevant plasmid. Cell lysates were prepared
Figure Legend Snippet: AeIAP1 and DIAP1 inhibit IAP antagonist-induced caspase activation. C6/36 cells ( a ) or SF-21 cells ( b ) were co-transfected with IAP antagonist constructs and plasmids expressing AeIAP1 or DIAP1, or a control irrelevant plasmid. Cell lysates were prepared

Techniques Used: Activation Assay, Transfection, Construct, Expressing, Plasmid Preparation

IAP antagonists release AeDronc from inhibition by AeIAP1. a Recombinant GST-AeIAP1 (10 μM) was incubated with 0.5 μM of active AeDronc, followed by addition of recombinant IAP antagonists (Mx, IMP or Rpr) (10 μM) or control GB
Figure Legend Snippet: IAP antagonists release AeDronc from inhibition by AeIAP1. a Recombinant GST-AeIAP1 (10 μM) was incubated with 0.5 μM of active AeDronc, followed by addition of recombinant IAP antagonists (Mx, IMP or Rpr) (10 μM) or control GB

Techniques Used: Inhibition, Recombinant, Incubation

Interactions between AeIAP1 and initiator or effector caspases. a In vitro translated caspases were incubated with recombinant proteins (GST-BIR1, GST-BIR2, GST-BIR1+2, GST-AeIAP1, or GST) or buffer alone, and protein complexes were purified using glutathione-agarose
Figure Legend Snippet: Interactions between AeIAP1 and initiator or effector caspases. a In vitro translated caspases were incubated with recombinant proteins (GST-BIR1, GST-BIR2, GST-BIR1+2, GST-AeIAP1, or GST) or buffer alone, and protein complexes were purified using glutathione-agarose

Techniques Used: In Vitro, Incubation, Recombinant, Purification

22) Product Images from "Identification of a Novel Microtubule-destabilizing Motif in CPAP That Binds to Tubulin Heterodimers and Inhibits Microtubule Assembly D⃞"

Article Title: Identification of a Novel Microtubule-destabilizing Motif in CPAP That Binds to Tubulin Heterodimers and Inhibits Microtubule Assembly D⃞

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-02-0121

(A) Testing the effects of PN2-3 polypeptide on microtubule polymerization by fluorescence microscopy. Tubulin (3 mg/ml) was incubated with different GST-CPAP–truncated proteins (0.45 μM) or nocodazole (15 μM). After incubation, the polymerized MTs were fixed and sedimented onto acid-treated coverslips as described in MATERIALS AND METHODS. MTs were analyzed by immunofluorescence assay using FITC-conjugated anti-α-tubulin antibodies (DM1A-FITC). Bar, 10 μm. Microtubules sedimentation assay (B and C). (B) Purified tubulins (15 μM) were incubated without (control) or with various amounts of GST-PN2-3 (top) or GST-PN2-2 (bottom) recombinant proteins. After centrifugation, the supernatants (S) and the pellets (P) were analyzed by SDS-PAGE, and the separated proteins were stained by Coomassie Blue. (C) Taxol-stabilized MTs (15 μM) were incubated with or without (control) indicated GST recombinant proteins and centrifuged through a glycerol cushion. After centrifugation, the supernatants (S) and the pellets (P) were analyzed by SDS-PAGE.
Figure Legend Snippet: (A) Testing the effects of PN2-3 polypeptide on microtubule polymerization by fluorescence microscopy. Tubulin (3 mg/ml) was incubated with different GST-CPAP–truncated proteins (0.45 μM) or nocodazole (15 μM). After incubation, the polymerized MTs were fixed and sedimented onto acid-treated coverslips as described in MATERIALS AND METHODS. MTs were analyzed by immunofluorescence assay using FITC-conjugated anti-α-tubulin antibodies (DM1A-FITC). Bar, 10 μm. Microtubules sedimentation assay (B and C). (B) Purified tubulins (15 μM) were incubated without (control) or with various amounts of GST-PN2-3 (top) or GST-PN2-2 (bottom) recombinant proteins. After centrifugation, the supernatants (S) and the pellets (P) were analyzed by SDS-PAGE, and the separated proteins were stained by Coomassie Blue. (C) Taxol-stabilized MTs (15 μM) were incubated with or without (control) indicated GST recombinant proteins and centrifuged through a glycerol cushion. After centrifugation, the supernatants (S) and the pellets (P) were analyzed by SDS-PAGE.

Techniques Used: Fluorescence, Microscopy, Incubation, Immunofluorescence, Sedimentation, Purification, Recombinant, Centrifugation, SDS Page, Staining

23) Product Images from "GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP"

Article Title: GIPC, a PDZ domain containing protein, interacts specifically with the C terminus of RGS-GAIP

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

doi:

GIPC interacts specifically with GAIP in vitro . GST-fusion proteins bound to glutathione-agarose beads were incubated with in vitro- translated, radiolabeled GIPC as described. GIPC bound specifically to GAIP (lane 3) but not to GST alone (lane 2). GIPC did not bind to RGS4 (lane 4), to the RGS domain of GAIP (GAIP 80–206 , lane 5), or the N-terminal domain of GAIP (GAIP 1–79 , lane 6). GIPC appears as a 40-kDa doublet (lane 1) in 35 S-labeled in vitro- translated GIPC product.
Figure Legend Snippet: GIPC interacts specifically with GAIP in vitro . GST-fusion proteins bound to glutathione-agarose beads were incubated with in vitro- translated, radiolabeled GIPC as described. GIPC bound specifically to GAIP (lane 3) but not to GST alone (lane 2). GIPC did not bind to RGS4 (lane 4), to the RGS domain of GAIP (GAIP 80–206 , lane 5), or the N-terminal domain of GAIP (GAIP 1–79 , lane 6). GIPC appears as a 40-kDa doublet (lane 1) in 35 S-labeled in vitro- translated GIPC product.

Techniques Used: In Vitro, Incubation, Labeling

GIPC interacts with the C terminus of GAIP. GST–GAIP fusion protein bound to glutathione-agarose beads was incubated with in vitro- translated GIPC (lane 3) as described. Addition of 30 μg (lane 4) or 300 μg (lane 5) of anti-GAIP C-terminal IgG reduced the binding of GIPC to GAIP, whereas 300 μg anti-CALNUC antibody (lane 6) had minimal effect. When 10 μM (lane 7) or 1 mM (lane 8) of GAIP C-terminal peptide were added the binding of GIPC to GAIP was also reduced 16% and 43%, respectively. A control peptide (1 mM, lane 9) had little effect on binding. Binding to GST alone (lane 2) was taken as background, and the signal obtained after binding of GIPC to GST–GAIP (lane 3) (with background subtracted) was defined as 100% in arbitrary units. Lane 1, 35 S-labeled in vitro- translated GIPC product.
Figure Legend Snippet: GIPC interacts with the C terminus of GAIP. GST–GAIP fusion protein bound to glutathione-agarose beads was incubated with in vitro- translated GIPC (lane 3) as described. Addition of 30 μg (lane 4) or 300 μg (lane 5) of anti-GAIP C-terminal IgG reduced the binding of GIPC to GAIP, whereas 300 μg anti-CALNUC antibody (lane 6) had minimal effect. When 10 μM (lane 7) or 1 mM (lane 8) of GAIP C-terminal peptide were added the binding of GIPC to GAIP was also reduced 16% and 43%, respectively. A control peptide (1 mM, lane 9) had little effect on binding. Binding to GST alone (lane 2) was taken as background, and the signal obtained after binding of GIPC to GST–GAIP (lane 3) (with background subtracted) was defined as 100% in arbitrary units. Lane 1, 35 S-labeled in vitro- translated GIPC product.

Techniques Used: Incubation, In Vitro, Binding Assay, Labeling

24) Product Images from "Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans"

Article Title: Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans

Journal: The EMBO Journal

doi: 10.1093/emboj/19.14.3618

Fig. 3. CBP1 binds to calcineurin in vitro and in vivo . ( A ) Purified GST–CBP fusion protein bound to glutathione agarose was incu bated with bovine calcineurin in the presence (+) or absence (–) of calmodulin, FKBP12, FK506 or EGTA for 2 h at 4°C. Reactions were separated on 10% SDS–PAGE and transferred to PVDF membranes. Membranes were incubated with anti-calcineurin (bovine) antibody to detect binding of CBP to calcineurin or anti-GST antibody to detect the GST–CBP fusion protein. The arrow indicates the position of calcineurin. The panel on the right indicates the position of the GST–CBP fusion protein. ( B ) Wild-type strain H99 ( CNA1 ) and the isogenic Δcna1 mutant strain expressing wild-type CBP1 (H99, AO4) or a CBP1–GFP fusion protein (JMC4, JMC6) were grown over night in rich medium, cells were mechanically disrupted, and immuno precipitation experiments were conducted with total cell extracts for 1 h at 4°C in the presence (+) and absence (–) of anti-GFP antisera (α-GFP Ab). Proteins bound to the antibody were subsequently precipitated with protein A–Sepharose (PAS) and separated on 12% SDS–PAGE. The calcineurin A protein (CNA1) was detected by incubating the western blots with [ 125 I]calmodulin. The first two lanes of the right and left panels are total extract controls that were not incubated with the anti-GFP antisera.
Figure Legend Snippet: Fig. 3. CBP1 binds to calcineurin in vitro and in vivo . ( A ) Purified GST–CBP fusion protein bound to glutathione agarose was incu bated with bovine calcineurin in the presence (+) or absence (–) of calmodulin, FKBP12, FK506 or EGTA for 2 h at 4°C. Reactions were separated on 10% SDS–PAGE and transferred to PVDF membranes. Membranes were incubated with anti-calcineurin (bovine) antibody to detect binding of CBP to calcineurin or anti-GST antibody to detect the GST–CBP fusion protein. The arrow indicates the position of calcineurin. The panel on the right indicates the position of the GST–CBP fusion protein. ( B ) Wild-type strain H99 ( CNA1 ) and the isogenic Δcna1 mutant strain expressing wild-type CBP1 (H99, AO4) or a CBP1–GFP fusion protein (JMC4, JMC6) were grown over night in rich medium, cells were mechanically disrupted, and immuno precipitation experiments were conducted with total cell extracts for 1 h at 4°C in the presence (+) and absence (–) of anti-GFP antisera (α-GFP Ab). Proteins bound to the antibody were subsequently precipitated with protein A–Sepharose (PAS) and separated on 12% SDS–PAGE. The calcineurin A protein (CNA1) was detected by incubating the western blots with [ 125 I]calmodulin. The first two lanes of the right and left panels are total extract controls that were not incubated with the anti-GFP antisera.

Techniques Used: In Vitro, In Vivo, Purification, SDS Page, Incubation, Binding Assay, Mutagenesis, Expressing, Immunoprecipitation, Western Blot

Fig. 1. Calcineurin and CBP1 interact in the two-hybrid assay. ( A ) The C.neoformans calcineurin A protein (cCNA1) specifically interacts with CBP1 and this interaction is inhibited by FK506. β-galactosidase assays were conducted in the presence and absence of 1 µg/ml FK506 as indicated. ( B ) FK506 inhibition of calcineurin–CBP1 binding requires FKBP12. An S.cerevisiae strain lacking FKBP12 ( fpr1 strain SMY87-4) was transformed with plasmids expressing the GAL4 activation domain (AD) or DNA binding domain (BD) fused to the indicated proteins. To detect expression of the GAL-ADE2 reporter gene, cells were grown on synthetic medium minus leucine, tryptophan and adenine (SD-leu-trp-ade) for 7 days at 30°C. ‘y’ denotes S.cerevisiae proteins and ‘c’ denotes C.neoformans proteins. FK506 stimulated FKBP12 binding to calcineurin A as expected. FK506 did not inhibit CBP1 binding to calcineurin in these cells lacking endogenous FKBP12. Calcineurin A (cCNA1) binding to calcineurin B (yCNB) was not affected by FK506. ( C ) Saccharomyces cerevisiae calcineurin B interacts with CBP1 and is required for CBP1 binding to calcineurin A. Isogenic S.cerevisiae strains expressing (PJ69-4A) or lacking calcineurin B (ΔCnB, SMY3) were transformed with plasmids expressing the GAL4 AD or BD fused to the indicated proteins. Cells were grown on medium lacking adenine (SD-leu-trp-ade) to detect expression of the GAL-ADE2 reporter gene, and on medium lacking histidine (SD-leu-trp-his + 5 mM 3-AT) to detect expression of the GAL-HIS3 reporter gene. ( D ) Two-hybrid analysis of the binding of truncated forms of CBP1 to calcineurin A. The black box represents the highly conserved region of CBP1. Fragment length is indicated in amino acid residues. * indicates the GST–CBP1 fusion protein.
Figure Legend Snippet: Fig. 1. Calcineurin and CBP1 interact in the two-hybrid assay. ( A ) The C.neoformans calcineurin A protein (cCNA1) specifically interacts with CBP1 and this interaction is inhibited by FK506. β-galactosidase assays were conducted in the presence and absence of 1 µg/ml FK506 as indicated. ( B ) FK506 inhibition of calcineurin–CBP1 binding requires FKBP12. An S.cerevisiae strain lacking FKBP12 ( fpr1 strain SMY87-4) was transformed with plasmids expressing the GAL4 activation domain (AD) or DNA binding domain (BD) fused to the indicated proteins. To detect expression of the GAL-ADE2 reporter gene, cells were grown on synthetic medium minus leucine, tryptophan and adenine (SD-leu-trp-ade) for 7 days at 30°C. ‘y’ denotes S.cerevisiae proteins and ‘c’ denotes C.neoformans proteins. FK506 stimulated FKBP12 binding to calcineurin A as expected. FK506 did not inhibit CBP1 binding to calcineurin in these cells lacking endogenous FKBP12. Calcineurin A (cCNA1) binding to calcineurin B (yCNB) was not affected by FK506. ( C ) Saccharomyces cerevisiae calcineurin B interacts with CBP1 and is required for CBP1 binding to calcineurin A. Isogenic S.cerevisiae strains expressing (PJ69-4A) or lacking calcineurin B (ΔCnB, SMY3) were transformed with plasmids expressing the GAL4 AD or BD fused to the indicated proteins. Cells were grown on medium lacking adenine (SD-leu-trp-ade) to detect expression of the GAL-ADE2 reporter gene, and on medium lacking histidine (SD-leu-trp-his + 5 mM 3-AT) to detect expression of the GAL-HIS3 reporter gene. ( D ) Two-hybrid analysis of the binding of truncated forms of CBP1 to calcineurin A. The black box represents the highly conserved region of CBP1. Fragment length is indicated in amino acid residues. * indicates the GST–CBP1 fusion protein.

Techniques Used: Two Hybrid Assay, Inhibition, Binding Assay, Transformation Assay, Expressing, Activation Assay

25) Product Images from "Role of SH3 Domain-Containing Proteins in Clathrin-Mediated Vesicle Trafficking in Arabidopsis"

Article Title: Role of SH3 Domain-Containing Proteins in Clathrin-Mediated Vesicle Trafficking in Arabidopsis

Journal: The Plant Cell

doi: 10.1105/tpc.010279

The AtSH3P1-Associating Auxilin-Like Protein Binds and Uncoats Clathrin. (A) Clathrin uncoating. Arabidopsis microsomal membrane was incubated with AtSH3P1, auxilin-like protein, animal Hsc70, or combinations of these proteins. The supernatant (S) or pellet (P) of a 100,000 g centrifugation of the treated membrane was collected and subjected to protein gel blotting for the presence of clathrin (as shown at top) and each GST fusion protein (data not shown). (B) GST pulldown binding assay. Glutathione resins with no recombinant protein (lane 1), with GST-auxilin (lanes 2 and 3), GST-AtSH3P1 (lanes 4 and 5), and GST (lanes 6 and 7) were incubated with binding buffer (even-numbered lanes) or Arabidopsis cytosol (odd-numbered lanes) as described in Methods. The presence of “pulled down” clathrin was determined by protein gel blotting using the anti-plant clathrin antibodies. Ten micrograms of Arabidopsis cytosol was loaded in lane 8 as a positive control for the immunoblotting.
Figure Legend Snippet: The AtSH3P1-Associating Auxilin-Like Protein Binds and Uncoats Clathrin. (A) Clathrin uncoating. Arabidopsis microsomal membrane was incubated with AtSH3P1, auxilin-like protein, animal Hsc70, or combinations of these proteins. The supernatant (S) or pellet (P) of a 100,000 g centrifugation of the treated membrane was collected and subjected to protein gel blotting for the presence of clathrin (as shown at top) and each GST fusion protein (data not shown). (B) GST pulldown binding assay. Glutathione resins with no recombinant protein (lane 1), with GST-auxilin (lanes 2 and 3), GST-AtSH3P1 (lanes 4 and 5), and GST (lanes 6 and 7) were incubated with binding buffer (even-numbered lanes) or Arabidopsis cytosol (odd-numbered lanes) as described in Methods. The presence of “pulled down” clathrin was determined by protein gel blotting using the anti-plant clathrin antibodies. Ten micrograms of Arabidopsis cytosol was loaded in lane 8 as a positive control for the immunoblotting.

Techniques Used: Incubation, Centrifugation, Binding Assay, Recombinant, Positive Control

AtSH3P1 Binds to Specific Lipid Types. Five micrograms of lipids was spotted onto nitrocellulose membranes and subjected to in vitro lipid binding assay as described in Methods using full-length AtSH3P1 (A) , AtSH3P1 without the SH3 domain (B) , and GST-SH3 domain as probes (C) . The lipids used were phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), phosphatidylinositol-4,5-bisphosphate (PIP 2 ), cholesterol (CS), sphingomyelin (SP), and fatty acid CoA (FA). Chloroform (CHCl 3 ), the solvent for the lipids, also was spotted as a negative control. The relative position of each sample on the blots is indicated in (D) .
Figure Legend Snippet: AtSH3P1 Binds to Specific Lipid Types. Five micrograms of lipids was spotted onto nitrocellulose membranes and subjected to in vitro lipid binding assay as described in Methods using full-length AtSH3P1 (A) , AtSH3P1 without the SH3 domain (B) , and GST-SH3 domain as probes (C) . The lipids used were phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), phosphatidylinositol-4,5-bisphosphate (PIP 2 ), cholesterol (CS), sphingomyelin (SP), and fatty acid CoA (FA). Chloroform (CHCl 3 ), the solvent for the lipids, also was spotted as a negative control. The relative position of each sample on the blots is indicated in (D) .

Techniques Used: In Vitro, Binding Assay, Negative Control

AtSH3P1 Binds Actin at a Region Distinct from the SH3 Domain. Recombinant proteins of AtSH3P1, AtSH3P1 without the SH3 domain (AtSH3P1-ΔSH3), and the GST-SH3 domain fusion were incubated with (right) or without (left) F-actin and centrifuged at 280,000 g . The presence of AtSH3P1 or actin in either the supernatant (S) or the pellet (P) was determined by anti-AtSH3P1 or anti-actin protein gel blotting.
Figure Legend Snippet: AtSH3P1 Binds Actin at a Region Distinct from the SH3 Domain. Recombinant proteins of AtSH3P1, AtSH3P1 without the SH3 domain (AtSH3P1-ΔSH3), and the GST-SH3 domain fusion were incubated with (right) or without (left) F-actin and centrifuged at 280,000 g . The presence of AtSH3P1 or actin in either the supernatant (S) or the pellet (P) was determined by anti-AtSH3P1 or anti-actin protein gel blotting.

Techniques Used: Recombinant, Incubation

26) Product Images from "Trichoplax adhaerens reveals a network of nuclear receptors sensitive to 9-cis-retinoic acid at the base of metazoan evolution"

Article Title: Trichoplax adhaerens reveals a network of nuclear receptors sensitive to 9-cis-retinoic acid at the base of metazoan evolution

Journal: PeerJ

doi: 10.7717/peerj.3789

Binding of retinoic acids to TaRXR. (A) Single point analysis of binding preference of T. adhaerens RXR (thrombin cleaved) to 3 H-labelled 9- cis -RA over all- trans -RA. Radioactive 9- cis -RA (9- cis -RA ∗ ) binds at a concentration of 4 nM to 200 nanograms of T. adhaerens RXR. 200-fold excess of unlabeled 9-cis -RA displaces more than 80% of labeled 9-cis -RA from binding to T. adhaerens RXR (9- cis -RA ∗ + 9- cis -RA) while the same molar excess of all- trans -RA (9- cis -RA ∗ + AT-RA) which is likely to contain approximately 1% spontaneously isomerized 9- cis - RA, competes away less than 50 % of bound 3 H-labeled 9- cis -RA. Radioactive 3 H-labeld all- trans -RA (AT-RA ∗ ) at identical conditions binds only slightly more than the observed non-specific binding. This interaction is not displaced by the excess of non-labeled 9- cis -RA (AT-RA ∗ + 9- cis -RA) nor non-labeled all- trans -RA (AT-RA ∗ + AT-RA). Results are expressed as a ratio of the radioactivity bound to TaRXR/total radioactivity used for the binding at the given condition. (B) Analysis of binding properties of T. adhaerens RXR (in the form of GST-TaRXR) to 3 H-labelled 9- cis -RA and 3 H-labelled all- trans -RA. The experiment differs from the experiment shown in A in 5-fold greater amount of radioactive all- trans -RA (and therefore only 40-fold excess of non-radioactive competitors). The experiment shows identical binding properties of GST-TaRXR as those observed with thrombin cleaved TaRXR. (C) Kinetic analysis of binding of 3 H-labeled 9- cis -RA to T. adhaerens RXR prepared as GST-fusion protein (GST-TaRXR). The plateau is reached at around 3 to 5 × 10 −9 M.
Figure Legend Snippet: Binding of retinoic acids to TaRXR. (A) Single point analysis of binding preference of T. adhaerens RXR (thrombin cleaved) to 3 H-labelled 9- cis -RA over all- trans -RA. Radioactive 9- cis -RA (9- cis -RA ∗ ) binds at a concentration of 4 nM to 200 nanograms of T. adhaerens RXR. 200-fold excess of unlabeled 9-cis -RA displaces more than 80% of labeled 9-cis -RA from binding to T. adhaerens RXR (9- cis -RA ∗ + 9- cis -RA) while the same molar excess of all- trans -RA (9- cis -RA ∗ + AT-RA) which is likely to contain approximately 1% spontaneously isomerized 9- cis - RA, competes away less than 50 % of bound 3 H-labeled 9- cis -RA. Radioactive 3 H-labeld all- trans -RA (AT-RA ∗ ) at identical conditions binds only slightly more than the observed non-specific binding. This interaction is not displaced by the excess of non-labeled 9- cis -RA (AT-RA ∗ + 9- cis -RA) nor non-labeled all- trans -RA (AT-RA ∗ + AT-RA). Results are expressed as a ratio of the radioactivity bound to TaRXR/total radioactivity used for the binding at the given condition. (B) Analysis of binding properties of T. adhaerens RXR (in the form of GST-TaRXR) to 3 H-labelled 9- cis -RA and 3 H-labelled all- trans -RA. The experiment differs from the experiment shown in A in 5-fold greater amount of radioactive all- trans -RA (and therefore only 40-fold excess of non-radioactive competitors). The experiment shows identical binding properties of GST-TaRXR as those observed with thrombin cleaved TaRXR. (C) Kinetic analysis of binding of 3 H-labeled 9- cis -RA to T. adhaerens RXR prepared as GST-fusion protein (GST-TaRXR). The plateau is reached at around 3 to 5 × 10 −9 M.

Techniques Used: Binding Assay, Concentration Assay, Labeling, Radioactivity

27) Product Images from "Scaffold-mediated gating of Cdc42 signalling flux"

Article Title: Scaffold-mediated gating of Cdc42 signalling flux

Journal: eLife

doi: 10.7554/eLife.25257

Expression of Cdc24-mEOS in vivo and the activity of cdc24 phospho-mutants in vitro. ( A ) A Western blot showing the levels of expression of the Cdc24-mEOS constructs. The constructs contain a His tag that was used for detection. ( B ) Representative in vitro GEF reactions of indicated cdc24 phospho-mutant proteins in the presence and absence of Bem1. ( C and D ) Quantification of pull-down experiments to assess the interaction between GST-Bem1 and the Cdc24 phospho-mutant proteins. DOI: http://dx.doi.org/10.7554/eLife.25257.011 10.7554/eLife.25257.012 Excel file showing the band intensity of the data presented in Figure 3—figure supplement 1D . DOI: http://dx.doi.org/10.7554/eLife.25257.012
Figure Legend Snippet: Expression of Cdc24-mEOS in vivo and the activity of cdc24 phospho-mutants in vitro. ( A ) A Western blot showing the levels of expression of the Cdc24-mEOS constructs. The constructs contain a His tag that was used for detection. ( B ) Representative in vitro GEF reactions of indicated cdc24 phospho-mutant proteins in the presence and absence of Bem1. ( C and D ) Quantification of pull-down experiments to assess the interaction between GST-Bem1 and the Cdc24 phospho-mutant proteins. DOI: http://dx.doi.org/10.7554/eLife.25257.011 10.7554/eLife.25257.012 Excel file showing the band intensity of the data presented in Figure 3—figure supplement 1D . DOI: http://dx.doi.org/10.7554/eLife.25257.012

Techniques Used: Expressing, In Vivo, Activity Assay, In Vitro, Western Blot, Construct, Mutagenesis

28) Product Images from "Adapter Protein SH2B1? Cross-Links Actin Filaments and Regulates Actin Cytoskeleton"

Article Title: Adapter Protein SH2B1? Cross-Links Actin Filaments and Regulates Actin Cytoskeleton

Journal:

doi: 10.1210/me.2008-0428

Both actin-binding sites of SH2B1β are required for maximal GH-induced membrane ruffling. A–F, 3T3 F44A cells expressing GFP or the indicated forms of myc-tagged forms of SH2B1β were serum deprived (A, C, and E) and treated with
Figure Legend Snippet: Both actin-binding sites of SH2B1β are required for maximal GH-induced membrane ruffling. A–F, 3T3 F44A cells expressing GFP or the indicated forms of myc-tagged forms of SH2B1β were serum deprived (A, C, and E) and treated with

Techniques Used: Binding Assay, Expressing

Maximal phagokinesis of 3T3 F442A cells requires SH2B1β. A–F, 3T3 F442A cells expressing GFP or the indicated forms of myc-tagged SH2B1β were plated on colloid gold-covered coverslips, and the areas that became free of colloid
Figure Legend Snippet: Maximal phagokinesis of 3T3 F442A cells requires SH2B1β. A–F, 3T3 F442A cells expressing GFP or the indicated forms of myc-tagged SH2B1β were plated on colloid gold-covered coverslips, and the areas that became free of colloid

Techniques Used: Expressing

Actin-binding sites of SH2B1β are involved in PRL-induced membrane ruffling. A–F, T47D cells expressing GFP or the indicated forms of myc-tagged forms of SH2B1β were serum deprived (A, C, and E) and treated with 150 ng/ml PRL for
Figure Legend Snippet: Actin-binding sites of SH2B1β are involved in PRL-induced membrane ruffling. A–F, T47D cells expressing GFP or the indicated forms of myc-tagged forms of SH2B1β were serum deprived (A, C, and E) and treated with 150 ng/ml PRL for

Techniques Used: Binding Assay, Expressing

SH2B1β binds F-actin. Before each experiment, WT SH2B1β and mutants were centrifuged in the absence of F-actin to remove any insoluble protein aggregates. A defined concentration of F-actin (4 μ m ) was mixed with increasing concentrations
Figure Legend Snippet: SH2B1β binds F-actin. Before each experiment, WT SH2B1β and mutants were centrifuged in the absence of F-actin to remove any insoluble protein aggregates. A defined concentration of F-actin (4 μ m ) was mixed with increasing concentrations

Techniques Used: Concentration Assay

SH2B1β cross-links actin filaments. For the low-speed centrifugation assay (A and B), 0.1 μ m GST-SH2B1β WT or GST-SH2B1β mutants were incubated with 8 μ m F-actin for 30 min in actin polymerizing buffer (F-buffer)
Figure Legend Snippet: SH2B1β cross-links actin filaments. For the low-speed centrifugation assay (A and B), 0.1 μ m GST-SH2B1β WT or GST-SH2B1β mutants were incubated with 8 μ m F-actin for 30 min in actin polymerizing buffer (F-buffer)

Techniques Used: Centrifugation, Incubation

Intracellular localization of SH2B1β depends on both the first actin-binding domain of SH2B1β and VASP protein. A–F, 3T3 F442A cells overexpressing the indicated forms of myc-SH2B1β were stained with α-myc ( green
Figure Legend Snippet: Intracellular localization of SH2B1β depends on both the first actin-binding domain of SH2B1β and VASP protein. A–F, 3T3 F442A cells overexpressing the indicated forms of myc-SH2B1β were stained with α-myc ( green

Techniques Used: Binding Assay, Staining

SH2B1β enhances tyrosyl phosphorylation of JAK2 in response to prolactin. A, SH2B1β was either overexpressed (lanes 3 and 4) or was not (lanes 1 and 2) in T47D cells. The cells were deprived of serum and treated without (lanes 1 and 3)
Figure Legend Snippet: SH2B1β enhances tyrosyl phosphorylation of JAK2 in response to prolactin. A, SH2B1β was either overexpressed (lanes 3 and 4) or was not (lanes 1 and 2) in T47D cells. The cells were deprived of serum and treated without (lanes 1 and 3)

Techniques Used:

Schematic representation of WT and mutant forms of rat SH2B1β used in the study. Actin-binding domains (amino acids 150–200 and 615–670) are shown in gray. PH is the PH domain (amino acids 274–376), and SH2 is the SH2 domain
Figure Legend Snippet: Schematic representation of WT and mutant forms of rat SH2B1β used in the study. Actin-binding domains (amino acids 150–200 and 615–670) are shown in gray. PH is the PH domain (amino acids 274–376), and SH2 is the SH2 domain

Techniques Used: Mutagenesis, Binding Assay

29) Product Images from "?-Arrestin2 Plays Permissive Roles in the Inhibitory Activities of RGS9-2 on G Protein-Coupled Receptors by Maintaining RGS9-2 in the Open Conformation ▿"

Article Title: ?-Arrestin2 Plays Permissive Roles in the Inhibitory Activities of RGS9-2 on G Protein-Coupled Receptors by Maintaining RGS9-2 in the Open Conformation ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.05690-11

Role of affinity for β-arrestin2 in the regulation of D 3 R signaling through RGS9-2. (A) Interaction between β-arrestin2 and D 3 R was determined by GST pulldown assay. Bacterial lysates containing the GST fusion proteins of the third intracellular
Figure Legend Snippet: Role of affinity for β-arrestin2 in the regulation of D 3 R signaling through RGS9-2. (A) Interaction between β-arrestin2 and D 3 R was determined by GST pulldown assay. Bacterial lysates containing the GST fusion proteins of the third intracellular

Techniques Used: GST Pulldown Assay

Roles of β-arrestins in the regulatory effects of RGS9-2 on the signaling of D 3 R. (A) Interactions between β-arrestin2 and RGS9-2 proteins were assessed by immunoprecipitation in cells expressing FLAG-β-arrestin2 along with FL-RGS9-2-EGFP,
Figure Legend Snippet: Roles of β-arrestins in the regulatory effects of RGS9-2 on the signaling of D 3 R. (A) Interactions between β-arrestin2 and RGS9-2 proteins were assessed by immunoprecipitation in cells expressing FLAG-β-arrestin2 along with FL-RGS9-2-EGFP,

Techniques Used: Immunoprecipitation, Expressing

Dopamine D 2 R and D 3 R differently interact with RGS9-2. (A) Interaction between RGS9-2 and D 2 R or D 3 R was determined by immunoprecipitation. HEK293 cells were transfected with RGS9-2-EGFP together with FLAG-tagged D 2 R or D 3 R in pCMV5. Cell lysates were
Figure Legend Snippet: Dopamine D 2 R and D 3 R differently interact with RGS9-2. (A) Interaction between RGS9-2 and D 2 R or D 3 R was determined by immunoprecipitation. HEK293 cells were transfected with RGS9-2-EGFP together with FLAG-tagged D 2 R or D 3 R in pCMV5. Cell lysates were

Techniques Used: Immunoprecipitation, Transfection

Roles of β-arrestin2 in the subcellular localization of RGS9-2. (A) Effects of KD of endogenous β-arrestins on the subcellular localization of RGS9-2. Con-KD and β-arr2-KD cells were transfected with RGS9-2-EGFP. (B) Effects of
Figure Legend Snippet: Roles of β-arrestin2 in the subcellular localization of RGS9-2. (A) Effects of KD of endogenous β-arrestins on the subcellular localization of RGS9-2. Con-KD and β-arr2-KD cells were transfected with RGS9-2-EGFP. (B) Effects of

Techniques Used: Transfection

Proposed working model of the D 3 R regulatory complex. When Gβ5 and the DEP domain of the RGS9 protein are associated, RGS9-2 is in the inactive (closed) conformation and cannot regulate G protein cycling. If the interaction between Gβ5
Figure Legend Snippet: Proposed working model of the D 3 R regulatory complex. When Gβ5 and the DEP domain of the RGS9 protein are associated, RGS9-2 is in the inactive (closed) conformation and cannot regulate G protein cycling. If the interaction between Gβ5

Techniques Used:

Effects of RGS9-2 on the signaling of D2-like receptors. (A) Effects of RGS9-2 on the signaling of D 2 R were determined in cells expressing D 2 R together with EGFP-Gβ5 (0.6 μg) and/or RGS9-2-EGFP (3 μg). Cellular cAMP was measured
Figure Legend Snippet: Effects of RGS9-2 on the signaling of D2-like receptors. (A) Effects of RGS9-2 on the signaling of D 2 R were determined in cells expressing D 2 R together with EGFP-Gβ5 (0.6 μg) and/or RGS9-2-EGFP (3 μg). Cellular cAMP was measured

Techniques Used: Expressing

Roles of specific subdomains of RGS9-2 in the regulation of D 3 ). The numbers at the top of the diagram indicate the positions of the domains in the original wild-type protein starting from
Figure Legend Snippet: Roles of specific subdomains of RGS9-2 in the regulation of D 3 ). The numbers at the top of the diagram indicate the positions of the domains in the original wild-type protein starting from

Techniques Used:

Determination of receptor regions responsible for RGS9-2-mediated inhibition of D 3 R signaling. (A) Schematic representation of chimeric receptors consisting of D 2 R and D 3 R, whose second and third intracellular loops were switched. Signaling of the chimeric
Figure Legend Snippet: Determination of receptor regions responsible for RGS9-2-mediated inhibition of D 3 R signaling. (A) Schematic representation of chimeric receptors consisting of D 2 R and D 3 R, whose second and third intracellular loops were switched. Signaling of the chimeric

Techniques Used: Inhibition

Role of β-arrestin2 in the interaction of Gβ5 with adjacent proteins. (A) Cooperative activities of β-arrestin2 and RGS9-2 in the inhibition of D 3 R signaling. Cells were transfected with low doses of β-arrestin2 (1 μg)
Figure Legend Snippet: Role of β-arrestin2 in the interaction of Gβ5 with adjacent proteins. (A) Cooperative activities of β-arrestin2 and RGS9-2 in the inhibition of D 3 R signaling. Cells were transfected with low doses of β-arrestin2 (1 μg)

Techniques Used: Inhibition, Transfection

30) Product Images from "Association of Class II Histone Deacetylases with Heterochromatin Protein 1: Potential Role for Histone Methylation in Control of Muscle Differentiation"

Article Title: Association of Class II Histone Deacetylases with Heterochromatin Protein 1: Potential Role for Histone Methylation in Control of Muscle Differentiation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.20.7302-7312.2002

Effects of promyogenic signals on histone H3 methylation and MITR or HDAC-HP1 complexes. (A) Methylation and acetylation states of histone H3 lysine 9 on a MEF2-binding element during myogenesis. Soluble chromatin from C2 myoblasts in growth medium (GM) or from confluent myotubes in differentiation medium (DM) was subjected to immunoprecipitation with antibodies specific for dimethylated lysine 9 of histone H3 [α-MeH3(K9)] or acetylated lysine 9 and 14 of histone H3 [α-AcH3(K9/K14)]. Precipitated DNA was used as a template for PCR with primers spanning either the MEF2-binding site on the myogenin gene promoter or the transcription start site of the GAPDH gene promoter. The positions of the primer-binding sites (numbers) relative to the transcription start sites (arrows) are indicated. The position of the MEF2-binding element within the myogenin promoter is shown. PCR was also performed using genomic DNA prior to immunoprecipitation to control for equal input of chromatin (bottom panel). (B) CaMK-mediated dissociation of HP1α from MITR and HDAC5. COS cells were cotransfected with expression vectors encoding FLAG-tagged HP1α and Myc-tagged HDAC5 or Myc-tagged versions of wild-type MITR (MITR) or a mutant of MITR containing alanines in place of serines 218 and 448 (MITR S218/448A) in the absence or presence of an expression vector encoding activated CaMKI. Myc-tagged proteins were immunoprecipitated from cell lysates with a polyclonal anti-Myc antibody, and coimmunoprecipitating FLAG-tagged HP1α was detected by immunoblotting with a monoclonal anti-FLAG antibody (top panel). The membrane was reprobed with anti-Myc antibody to reveal immunoprecipitated HDAC5 and MITR (bottom panel).
Figure Legend Snippet: Effects of promyogenic signals on histone H3 methylation and MITR or HDAC-HP1 complexes. (A) Methylation and acetylation states of histone H3 lysine 9 on a MEF2-binding element during myogenesis. Soluble chromatin from C2 myoblasts in growth medium (GM) or from confluent myotubes in differentiation medium (DM) was subjected to immunoprecipitation with antibodies specific for dimethylated lysine 9 of histone H3 [α-MeH3(K9)] or acetylated lysine 9 and 14 of histone H3 [α-AcH3(K9/K14)]. Precipitated DNA was used as a template for PCR with primers spanning either the MEF2-binding site on the myogenin gene promoter or the transcription start site of the GAPDH gene promoter. The positions of the primer-binding sites (numbers) relative to the transcription start sites (arrows) are indicated. The position of the MEF2-binding element within the myogenin promoter is shown. PCR was also performed using genomic DNA prior to immunoprecipitation to control for equal input of chromatin (bottom panel). (B) CaMK-mediated dissociation of HP1α from MITR and HDAC5. COS cells were cotransfected with expression vectors encoding FLAG-tagged HP1α and Myc-tagged HDAC5 or Myc-tagged versions of wild-type MITR (MITR) or a mutant of MITR containing alanines in place of serines 218 and 448 (MITR S218/448A) in the absence or presence of an expression vector encoding activated CaMKI. Myc-tagged proteins were immunoprecipitated from cell lysates with a polyclonal anti-Myc antibody, and coimmunoprecipitating FLAG-tagged HP1α was detected by immunoblotting with a monoclonal anti-FLAG antibody (top panel). The membrane was reprobed with anti-Myc antibody to reveal immunoprecipitated HDAC5 and MITR (bottom panel).

Techniques Used: Methylation, Binding Assay, Immunoprecipitation, Polymerase Chain Reaction, Expressing, Mutagenesis, Plasmid Preparation

Association of MITR and class II HDACs with SUV39H1 and HP1α-mediated repression of MEF2C. (A) Coimmunoprecipitation of MITR and HDAC4 and -5 with SUV39H1. COS cells were cotransfected with expression vectors encoding HA-tagged SUV39H1 and the indicated FLAG-tagged proteins. FLAG-tagged proteins were immunoprecipitated from cell lysates with a monoclonal anti-FLAG antibody, and coimmunoprecipitating SUV39H1 was detected by immunoblotting with an anti-HA antibody (top panel). The membrane was reprobed with anti-FLAG antibody to reveal immunoprecipitated FLAG-tagged proteins (bottom panel). Arrows indicate the positions of full-length CtBP, HP1α, HDAC4, HDAC5, and MITR. (B) Association of HP1α and SUV39H1 with MITR by GST pull-down assays. Residues 389 to 506 of MITR were fused to GST and expressed in bacteria. The GST-MITR fusion protein was then conjugated to glutathione agarose beads and used in pull-down assays with 35 S-labeled HP1α and SUV39H1. The associated proteins were resolved by SDS-PAGE and visualized using a phosphorimager. GST alone was used as negative control. Ten percent of the 35 S-labeled protein was also directly applied to the gel to control for protein input. The positions of labeled HP1α and SUV39H1, which appears as a doublet, are indicated with arrows. (C) Inhibition of MEF2 transcriptional activity by HP1α. COS cells were transiently transfected with expression vectors for HP1α (0.1 to 0.8 μg), MEF2C (0.2 μg), MITR (5 ng), a MEF2-dependent reporter plasmid (3XMEF2-luciferase; 0.1 μg), and a CMV- lacZ reporter (0.1 μg) to control for differences in transfection efficiency. Luciferase activity was determined as described in Materials and Methods. Values represent means ± standard deviations.
Figure Legend Snippet: Association of MITR and class II HDACs with SUV39H1 and HP1α-mediated repression of MEF2C. (A) Coimmunoprecipitation of MITR and HDAC4 and -5 with SUV39H1. COS cells were cotransfected with expression vectors encoding HA-tagged SUV39H1 and the indicated FLAG-tagged proteins. FLAG-tagged proteins were immunoprecipitated from cell lysates with a monoclonal anti-FLAG antibody, and coimmunoprecipitating SUV39H1 was detected by immunoblotting with an anti-HA antibody (top panel). The membrane was reprobed with anti-FLAG antibody to reveal immunoprecipitated FLAG-tagged proteins (bottom panel). Arrows indicate the positions of full-length CtBP, HP1α, HDAC4, HDAC5, and MITR. (B) Association of HP1α and SUV39H1 with MITR by GST pull-down assays. Residues 389 to 506 of MITR were fused to GST and expressed in bacteria. The GST-MITR fusion protein was then conjugated to glutathione agarose beads and used in pull-down assays with 35 S-labeled HP1α and SUV39H1. The associated proteins were resolved by SDS-PAGE and visualized using a phosphorimager. GST alone was used as negative control. Ten percent of the 35 S-labeled protein was also directly applied to the gel to control for protein input. The positions of labeled HP1α and SUV39H1, which appears as a doublet, are indicated with arrows. (C) Inhibition of MEF2 transcriptional activity by HP1α. COS cells were transiently transfected with expression vectors for HP1α (0.1 to 0.8 μg), MEF2C (0.2 μg), MITR (5 ng), a MEF2-dependent reporter plasmid (3XMEF2-luciferase; 0.1 μg), and a CMV- lacZ reporter (0.1 μg) to control for differences in transfection efficiency. Luciferase activity was determined as described in Materials and Methods. Values represent means ± standard deviations.

Techniques Used: Expressing, Immunoprecipitation, Labeling, SDS Page, Negative Control, Inhibition, Activity Assay, Transfection, Plasmid Preparation, Luciferase

Association of HP1α with MITR and class II HDACs. (A) Interaction of HP1α and MITR in yeast. A mutant of MITR containing alanines in place of serines 218 and 448 (MITR S218/448A) was fused to the GAL4 DNA-binding domain and used as bait in a yeast two-hybrid screen (see Materials and Methods). A positive clone encoding full-length HP1α was rescued as a prey. The relative association of HP1α with the indicated MITR baits was determined by measuring the activity of a β-galactosidase reporter in yeast filter lift or liquid culture assays. Values from the liquid culture assay are expressed as activity relative to that seen with MITR S218/448A and HP1α. (B) Schematic diagrams of MITR and HDACs. The MEF2-binding region and nuclear localization signals (NLS) of MITR, HDAC4, and HDAC5 are indicated by the blue and green boxes, respectively. The red boxes indicate the HDAC catalytic domain. The number of amino acids in each protein is shown at the right. (C) GST pull-down assays. GST-HP1α (top panel) or GST alone (middle panel) was expressed in E. coli, conjugated to glutathione-agarose beads, and incubated with [ 35 S]methionine-labeled MITR and HDACs, as described in Materials and Methods. Associated proteins were resolved by SDS-PAGE and analyzed by autoradiography (top and middle panels). In the bottom panel, 10% of the [ 35 S]methionine-labeled protein was applied directly to the gel to control for input. (D) COS cells were cotransfected with expression vectors encoding Myc-tagged HP1α and the indicated FLAG-tagged proteins (1 μg each). FLAG-tagged proteins were immunoprecipitated from cell lysates with a monoclonal anti-FLAG antibody, and coimmunoprecipitating Myc-tagged proteins were detected by immunoblotting with a polyclonal anti-Myc antibody (upper panel). The positions of Myc-HP1α and the light chain of immunoglobulin (IgL) are indicated. The membrane was reprobed with anti-FLAG antibody to reveal total immunoprecipitated FLAG-tagged protein (bottom panel). (E) 293T cells were transiently transfected with expression vectors encoding Myc-tagged HP1α or GFP. At 48 h posttransfection, cell extracts were immunoprecipitated with anti-Myc antibody and immune complexes were assayed for HDAC activity as described in Materials and Methods. Values represent means ± standard deviations.
Figure Legend Snippet: Association of HP1α with MITR and class II HDACs. (A) Interaction of HP1α and MITR in yeast. A mutant of MITR containing alanines in place of serines 218 and 448 (MITR S218/448A) was fused to the GAL4 DNA-binding domain and used as bait in a yeast two-hybrid screen (see Materials and Methods). A positive clone encoding full-length HP1α was rescued as a prey. The relative association of HP1α with the indicated MITR baits was determined by measuring the activity of a β-galactosidase reporter in yeast filter lift or liquid culture assays. Values from the liquid culture assay are expressed as activity relative to that seen with MITR S218/448A and HP1α. (B) Schematic diagrams of MITR and HDACs. The MEF2-binding region and nuclear localization signals (NLS) of MITR, HDAC4, and HDAC5 are indicated by the blue and green boxes, respectively. The red boxes indicate the HDAC catalytic domain. The number of amino acids in each protein is shown at the right. (C) GST pull-down assays. GST-HP1α (top panel) or GST alone (middle panel) was expressed in E. coli, conjugated to glutathione-agarose beads, and incubated with [ 35 S]methionine-labeled MITR and HDACs, as described in Materials and Methods. Associated proteins were resolved by SDS-PAGE and analyzed by autoradiography (top and middle panels). In the bottom panel, 10% of the [ 35 S]methionine-labeled protein was applied directly to the gel to control for input. (D) COS cells were cotransfected with expression vectors encoding Myc-tagged HP1α and the indicated FLAG-tagged proteins (1 μg each). FLAG-tagged proteins were immunoprecipitated from cell lysates with a monoclonal anti-FLAG antibody, and coimmunoprecipitating Myc-tagged proteins were detected by immunoblotting with a polyclonal anti-Myc antibody (upper panel). The positions of Myc-HP1α and the light chain of immunoglobulin (IgL) are indicated. The membrane was reprobed with anti-FLAG antibody to reveal total immunoprecipitated FLAG-tagged protein (bottom panel). (E) 293T cells were transiently transfected with expression vectors encoding Myc-tagged HP1α or GFP. At 48 h posttransfection, cell extracts were immunoprecipitated with anti-Myc antibody and immune complexes were assayed for HDAC activity as described in Materials and Methods. Values represent means ± standard deviations.

Techniques Used: Mutagenesis, Binding Assay, Two Hybrid Screening, Activity Assay, Incubation, Labeling, SDS Page, Autoradiography, Expressing, Immunoprecipitation, Transfection

Mapping of the HP1α-binding region of MITR. (A) Association of GST-HP1α with amino- and carboxy-terminal MITR deletions. GST-HP1α (top panel) or GST alone (middle panel) was expressed in E. coli , conjugated to glutathione-agarose beads, and incubated with the indicated [ 35 S]methionine-labeled MITR deletion mutants. Associated proteins were resolved by SDS-PAGE and analyzed by autoradiography (top and middle panels). The only MITR mutant that failed to interact with HP1α in this assay (mutant 1-300) is indicated with an asterisk. In the bottom panel, 20% of the [ 35 S]methionine-labeled protein was applied directly to the gel to control for input. (B) Association of GST-HP1α with internal deletion mutants of MITR. The ability of GST-HP1α (top panel) to associate with the indicated MITR proteins was determined as described for panel A. In the bottom panel, 10% of the 35 S-labeled MITR was applied directly to the gel to control for input. None of the MITR proteins exhibited significant binding to GST alone (data not shown). (C) COS cells were cotransfected with expression vectors encoding FLAG-tagged HP1α and the indicated Myc-tagged MITR proteins or CtBP (1 μg each). Myc-tagged proteins were immunoprecipitated from cell lysates with a polyclonal anti-Myc antibody, and coimmunoprecipitating FLAG-tagged proteins were detected by immunoblotting with a monoclonal anti-FLAG antibody (top panel). The positions of FLAG-HP1α and the light chain of immunoglobulin (IgL) are indicated. The membrane was reprobed with anti-Myc antibody to reveal the total amount of immunoprecipitated Myc-tagged protein (bottom panel). (D) Schematic representations of MITR proteins and their interactions with HP1α. There are two adjacent domains that are predicted to form α-helices (I and II); each is sufficient to mediate HP1α-binding. NLS, nuclear localization signal.
Figure Legend Snippet: Mapping of the HP1α-binding region of MITR. (A) Association of GST-HP1α with amino- and carboxy-terminal MITR deletions. GST-HP1α (top panel) or GST alone (middle panel) was expressed in E. coli , conjugated to glutathione-agarose beads, and incubated with the indicated [ 35 S]methionine-labeled MITR deletion mutants. Associated proteins were resolved by SDS-PAGE and analyzed by autoradiography (top and middle panels). The only MITR mutant that failed to interact with HP1α in this assay (mutant 1-300) is indicated with an asterisk. In the bottom panel, 20% of the [ 35 S]methionine-labeled protein was applied directly to the gel to control for input. (B) Association of GST-HP1α with internal deletion mutants of MITR. The ability of GST-HP1α (top panel) to associate with the indicated MITR proteins was determined as described for panel A. In the bottom panel, 10% of the 35 S-labeled MITR was applied directly to the gel to control for input. None of the MITR proteins exhibited significant binding to GST alone (data not shown). (C) COS cells were cotransfected with expression vectors encoding FLAG-tagged HP1α and the indicated Myc-tagged MITR proteins or CtBP (1 μg each). Myc-tagged proteins were immunoprecipitated from cell lysates with a polyclonal anti-Myc antibody, and coimmunoprecipitating FLAG-tagged proteins were detected by immunoblotting with a monoclonal anti-FLAG antibody (top panel). The positions of FLAG-HP1α and the light chain of immunoglobulin (IgL) are indicated. The membrane was reprobed with anti-Myc antibody to reveal the total amount of immunoprecipitated Myc-tagged protein (bottom panel). (D) Schematic representations of MITR proteins and their interactions with HP1α. There are two adjacent domains that are predicted to form α-helices (I and II); each is sufficient to mediate HP1α-binding. NLS, nuclear localization signal.

Techniques Used: Binding Assay, Incubation, Labeling, SDS Page, Autoradiography, Mutagenesis, Expressing, Immunoprecipitation

31) Product Images from "Human AP-endonuclease 1 and hnRNP-L interact with a nCaRE-like repressor element in the AP-endonuclease 1 promoter"

Article Title: Human AP-endonuclease 1 and hnRNP-L interact with a nCaRE-like repressor element in the AP-endonuclease 1 promoter

Journal: Nucleic Acids Research

doi:

Reconstitution of the nCaRE-B2-binding complex and peptide interference assay. ( A ) Domain organization of the GST–hnRNP-L fusion protein. ( B ) Purification of recombinant hnRNP-L protein from Sf9 cells. (Left) Coomassie brilliant blue staining: lanes 1 and 4, protein standards; lane 2, uninfected Sf9 extract; lane 3, Sf9 extract expressing GST–hnRNP-L; lanes 5 and 6, purified GST–hnRNP-L before (lane 5) and after (lane 6) cleavage with thrombin. (Right) Western analysis of the proteins in lanes 5 and 6 with anti-hnRNP-L antibody. ( C ) In vitro reconstitution of nCaRE-B2-binding activity by EMSA. Lane 1, no protein; lane 2, HeLa nuclear extract; lane 3, APE1 and hnRNP-L; lane 4, APE1 alone; lane 5, hnRNP-L alone.
Figure Legend Snippet: Reconstitution of the nCaRE-B2-binding complex and peptide interference assay. ( A ) Domain organization of the GST–hnRNP-L fusion protein. ( B ) Purification of recombinant hnRNP-L protein from Sf9 cells. (Left) Coomassie brilliant blue staining: lanes 1 and 4, protein standards; lane 2, uninfected Sf9 extract; lane 3, Sf9 extract expressing GST–hnRNP-L; lanes 5 and 6, purified GST–hnRNP-L before (lane 5) and after (lane 6) cleavage with thrombin. (Right) Western analysis of the proteins in lanes 5 and 6 with anti-hnRNP-L antibody. ( C ) In vitro reconstitution of nCaRE-B2-binding activity by EMSA. Lane 1, no protein; lane 2, HeLa nuclear extract; lane 3, APE1 and hnRNP-L; lane 4, APE1 alone; lane 5, hnRNP-L alone.

Techniques Used: Binding Assay, Purification, Recombinant, Staining, Expressing, Western Blot, In Vitro, Activity Assay

32) Product Images from "The Detergent-Soluble Maltose Transporter Is Activated by Maltose Binding Protein and Verapamil"

Article Title: The Detergent-Soluble Maltose Transporter Is Activated by Maltose Binding Protein and Verapamil

Journal: Journal of Bacteriology

doi:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection of the purified FGK2 complex. The FGK2 complex was purified as described in Materials and Methods. The purified complex was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane for immunoblotting with specific antibodies (Ab). (A) Coomassie staining of the gel. Lanes: 1, proteins eluted by glutathione; 2, proteins eluted by thrombin cleavage. (B) Immunoblotting of the purified complex with specific antibodies to GST-MalG, MalF, and MalK individually. Lanes: 1, proteins bound to the glutathione agarose resin; 2, proteins eluted by thrombin cleavage; 3, proteins retained on the column following thrombin cleavage. The values on the left are molecular weights (M.W.) in thousands.
Figure Legend Snippet: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection of the purified FGK2 complex. The FGK2 complex was purified as described in Materials and Methods. The purified complex was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane for immunoblotting with specific antibodies (Ab). (A) Coomassie staining of the gel. Lanes: 1, proteins eluted by glutathione; 2, proteins eluted by thrombin cleavage. (B) Immunoblotting of the purified complex with specific antibodies to GST-MalG, MalF, and MalK individually. Lanes: 1, proteins bound to the glutathione agarose resin; 2, proteins eluted by thrombin cleavage; 3, proteins retained on the column following thrombin cleavage. The values on the left are molecular weights (M.W.) in thousands.

Techniques Used: Polyacrylamide Gel Electrophoresis, Immunodetection, Purification, Staining

33) Product Images from "BID Binds to Replication Protein A and Stimulates ATR Function following Replicative Stress ▿"

Article Title: BID Binds to Replication Protein A and Stimulates ATR Function following Replicative Stress ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.05737-11

BID facilitates the RPA-ATRIP interaction in vitro in a dose-dependent manner. (A) His-GST fused wild-type RPA70N or RPA70N harboring R41E/R43E mutations (250 pmol) was bound to glutathione-agarose beads. Then, the nuclear fraction was purified from U2OS
Figure Legend Snippet: BID facilitates the RPA-ATRIP interaction in vitro in a dose-dependent manner. (A) His-GST fused wild-type RPA70N or RPA70N harboring R41E/R43E mutations (250 pmol) was bound to glutathione-agarose beads. Then, the nuclear fraction was purified from U2OS

Techniques Used: Recombinase Polymerase Amplification, In Vitro, Purification

34) Product Images from "Developmentally Essential Protein Flightless I Is a Nuclear Receptor Coactivator with Actin Binding Activity"

Article Title: Developmentally Essential Protein Flightless I Is a Nuclear Receptor Coactivator with Actin Binding Activity

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.5.2103-2117.2004

Binding of NRs to Fli-I in vitro and in vivo. (A) In vitro binding with ER. LRR and gelsolin-like fragments of Fli-I were translated in vitro and were tested for binding to bead-bound GST or GST-ER in the absence or presence of E2. (B) Coimmunoprecipitation with ER. Flag-tagged LRR or gelsolin-like fragments of Fli-I (2.5 μg of plasmid) were coexpressed with ER (2.5 μg of plasmid) in Cos-7 cells in the presence or absence of E2. Immunoprecipitation (IP) by anti-Flag antibody was followed by immunoblotting (W) with anti-ER antibody (upper panel). Expression of ER in the transfected-cell extracts before immunoprecipitation (input) was examined by immunoblotting with anti-ER antibodies (lower panel). (C) Coimmunoprecipitation with TR and CARM1. Coimmunoprecipitation of the Fli-I fragments with TR (2.5 μg of plasmid) or HA-tagged CARM1 (2.5 μg of plasmid) was tested as done for panel B in the presence and absence of T3, except that anti-TR (lanes 1 to 6) or anti-HA (lanes 7 and 8) was used for immunoprecipitation, and Fli-I fragments were detected on the subsequent immunoblot (W) with anti-Flag antibodies. Expression of LRR and gelsolin-like fragments of Fli-I in the transfected-cell extracts was examined before immunoprecipitation (input) by immunoblot with anti-Flag antibodies (lower panel).
Figure Legend Snippet: Binding of NRs to Fli-I in vitro and in vivo. (A) In vitro binding with ER. LRR and gelsolin-like fragments of Fli-I were translated in vitro and were tested for binding to bead-bound GST or GST-ER in the absence or presence of E2. (B) Coimmunoprecipitation with ER. Flag-tagged LRR or gelsolin-like fragments of Fli-I (2.5 μg of plasmid) were coexpressed with ER (2.5 μg of plasmid) in Cos-7 cells in the presence or absence of E2. Immunoprecipitation (IP) by anti-Flag antibody was followed by immunoblotting (W) with anti-ER antibody (upper panel). Expression of ER in the transfected-cell extracts before immunoprecipitation (input) was examined by immunoblotting with anti-ER antibodies (lower panel). (C) Coimmunoprecipitation with TR and CARM1. Coimmunoprecipitation of the Fli-I fragments with TR (2.5 μg of plasmid) or HA-tagged CARM1 (2.5 μg of plasmid) was tested as done for panel B in the presence and absence of T3, except that anti-TR (lanes 1 to 6) or anti-HA (lanes 7 and 8) was used for immunoprecipitation, and Fli-I fragments were detected on the subsequent immunoblot (W) with anti-Flag antibodies. Expression of LRR and gelsolin-like fragments of Fli-I in the transfected-cell extracts was examined before immunoprecipitation (input) by immunoblot with anti-Flag antibodies (lower panel).

Techniques Used: Binding Assay, In Vitro, In Vivo, Plasmid Preparation, Immunoprecipitation, Expressing, Transfection

Binding of CARM1 to Fli-I in vitro and in vivo. (A) The LRR region (vertical stripes) and two gelsolin-like motifs (black boxes) of Fli-I protein are indicated. (B) In vitro binding assay. [ 35 S]Fli-I protein synthesized in vitro was incubated with GST or GST-CARM1 immobilized on glutathione-agarose beads; bound proteins were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography. A portion of the in vitro translated Fli-I protein before incubation with the beads is shown at left (Input, 10%). The position of the 145-kDa full-length Fli-I protein is indicated. (C) Coimmunoprecipitation assay. pSG5.Flag-FliI(LRR) or pSG5.Flag-FliI(gelsolin) (2.5 μg) was transiently transfected along with pSG5.HA-CARM1 or pSG5.HA-PRMT1 (2.5 μg) into Cos-7 cells. Immunoprecipitation (IP) was performed on transfected-cell extracts with antibodies against the Flag epitope, and the precipitated proteins were analyzed by immunoblotting (W) with anti-HA antibodies (upper panel). A sample of the transfected-cell extract before immunoprecipitation (2% of the volume used for the immunoprecipitation shown in lane 3) is shown in lane 1. The relative expression of HA-CARM1 and HA-PRMT1 before immunoprecipitation is shown in the lower panel. Results are representative of two independent experiments. LRR, LRR fragment of Fli-I; GEL, gelsolin-like fragment of Fli-I; and IgG, position of the immunoglobulin heavy chain on the immunoblot.
Figure Legend Snippet: Binding of CARM1 to Fli-I in vitro and in vivo. (A) The LRR region (vertical stripes) and two gelsolin-like motifs (black boxes) of Fli-I protein are indicated. (B) In vitro binding assay. [ 35 S]Fli-I protein synthesized in vitro was incubated with GST or GST-CARM1 immobilized on glutathione-agarose beads; bound proteins were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography. A portion of the in vitro translated Fli-I protein before incubation with the beads is shown at left (Input, 10%). The position of the 145-kDa full-length Fli-I protein is indicated. (C) Coimmunoprecipitation assay. pSG5.Flag-FliI(LRR) or pSG5.Flag-FliI(gelsolin) (2.5 μg) was transiently transfected along with pSG5.HA-CARM1 or pSG5.HA-PRMT1 (2.5 μg) into Cos-7 cells. Immunoprecipitation (IP) was performed on transfected-cell extracts with antibodies against the Flag epitope, and the precipitated proteins were analyzed by immunoblotting (W) with anti-HA antibodies (upper panel). A sample of the transfected-cell extract before immunoprecipitation (2% of the volume used for the immunoprecipitation shown in lane 3) is shown in lane 1. The relative expression of HA-CARM1 and HA-PRMT1 before immunoprecipitation is shown in the lower panel. Results are representative of two independent experiments. LRR, LRR fragment of Fli-I; GEL, gelsolin-like fragment of Fli-I; and IgG, position of the immunoglobulin heavy chain on the immunoblot.

Techniques Used: Binding Assay, In Vitro, In Vivo, Synthesized, Incubation, Polyacrylamide Gel Electrophoresis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, FLAG-tag, Expressing

35) Product Images from "Developmentally Essential Protein Flightless I Is a Nuclear Receptor Coactivator with Actin Binding Activity"

Article Title: Developmentally Essential Protein Flightless I Is a Nuclear Receptor Coactivator with Actin Binding Activity

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.5.2103-2117.2004

Binding of NRs to Fli-I in vitro and in vivo. (A) In vitro binding with ER. LRR and gelsolin-like fragments of Fli-I were translated in vitro and were tested for binding to bead-bound GST or GST-ER in the absence or presence of E2. (B) Coimmunoprecipitation with ER. Flag-tagged LRR or gelsolin-like fragments of Fli-I (2.5 μg of plasmid) were coexpressed with ER (2.5 μg of plasmid) in Cos-7 cells in the presence or absence of E2. Immunoprecipitation (IP) by anti-Flag antibody was followed by immunoblotting (W) with anti-ER antibody (upper panel). Expression of ER in the transfected-cell extracts before immunoprecipitation (input) was examined by immunoblotting with anti-ER antibodies (lower panel). (C) Coimmunoprecipitation with TR and CARM1. Coimmunoprecipitation of the Fli-I fragments with TR (2.5 μg of plasmid) or HA-tagged CARM1 (2.5 μg of plasmid) was tested as done for panel B in the presence and absence of T3, except that anti-TR (lanes 1 to 6) or anti-HA (lanes 7 and 8) was used for immunoprecipitation, and Fli-I fragments were detected on the subsequent immunoblot (W) with anti-Flag antibodies. Expression of LRR and gelsolin-like fragments of Fli-I in the transfected-cell extracts was examined before immunoprecipitation (input) by immunoblot with anti-Flag antibodies (lower panel).
Figure Legend Snippet: Binding of NRs to Fli-I in vitro and in vivo. (A) In vitro binding with ER. LRR and gelsolin-like fragments of Fli-I were translated in vitro and were tested for binding to bead-bound GST or GST-ER in the absence or presence of E2. (B) Coimmunoprecipitation with ER. Flag-tagged LRR or gelsolin-like fragments of Fli-I (2.5 μg of plasmid) were coexpressed with ER (2.5 μg of plasmid) in Cos-7 cells in the presence or absence of E2. Immunoprecipitation (IP) by anti-Flag antibody was followed by immunoblotting (W) with anti-ER antibody (upper panel). Expression of ER in the transfected-cell extracts before immunoprecipitation (input) was examined by immunoblotting with anti-ER antibodies (lower panel). (C) Coimmunoprecipitation with TR and CARM1. Coimmunoprecipitation of the Fli-I fragments with TR (2.5 μg of plasmid) or HA-tagged CARM1 (2.5 μg of plasmid) was tested as done for panel B in the presence and absence of T3, except that anti-TR (lanes 1 to 6) or anti-HA (lanes 7 and 8) was used for immunoprecipitation, and Fli-I fragments were detected on the subsequent immunoblot (W) with anti-Flag antibodies. Expression of LRR and gelsolin-like fragments of Fli-I in the transfected-cell extracts was examined before immunoprecipitation (input) by immunoblot with anti-Flag antibodies (lower panel).

Techniques Used: Binding Assay, In Vitro, In Vivo, Plasmid Preparation, Immunoprecipitation, Expressing, Transfection

Binding of CARM1 to Fli-I in vitro and in vivo. (A) The LRR region (vertical stripes) and two gelsolin-like motifs (black boxes) of Fli-I protein are indicated. (B) In vitro binding assay. [ 35 S]Fli-I protein synthesized in vitro was incubated with GST or GST-CARM1 immobilized on glutathione-agarose beads; bound proteins were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography. A portion of the in vitro translated Fli-I protein before incubation with the beads is shown at left (Input, 10%). The position of the 145-kDa full-length Fli-I protein is indicated. (C) Coimmunoprecipitation assay. pSG5.Flag-FliI(LRR) or pSG5.Flag-FliI(gelsolin) (2.5 μg) was transiently transfected along with pSG5.HA-CARM1 or pSG5.HA-PRMT1 (2.5 μg) into Cos-7 cells. Immunoprecipitation (IP) was performed on transfected-cell extracts with antibodies against the Flag epitope, and the precipitated proteins were analyzed by immunoblotting (W) with anti-HA antibodies (upper panel). A sample of the transfected-cell extract before immunoprecipitation (2% of the volume used for the immunoprecipitation shown in lane 3) is shown in lane 1. The relative expression of HA-CARM1 and HA-PRMT1 before immunoprecipitation is shown in the lower panel. Results are representative of two independent experiments. LRR, LRR fragment of Fli-I; GEL, gelsolin-like fragment of Fli-I; and IgG, position of the immunoglobulin heavy chain on the immunoblot.
Figure Legend Snippet: Binding of CARM1 to Fli-I in vitro and in vivo. (A) The LRR region (vertical stripes) and two gelsolin-like motifs (black boxes) of Fli-I protein are indicated. (B) In vitro binding assay. [ 35 S]Fli-I protein synthesized in vitro was incubated with GST or GST-CARM1 immobilized on glutathione-agarose beads; bound proteins were eluted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography. A portion of the in vitro translated Fli-I protein before incubation with the beads is shown at left (Input, 10%). The position of the 145-kDa full-length Fli-I protein is indicated. (C) Coimmunoprecipitation assay. pSG5.Flag-FliI(LRR) or pSG5.Flag-FliI(gelsolin) (2.5 μg) was transiently transfected along with pSG5.HA-CARM1 or pSG5.HA-PRMT1 (2.5 μg) into Cos-7 cells. Immunoprecipitation (IP) was performed on transfected-cell extracts with antibodies against the Flag epitope, and the precipitated proteins were analyzed by immunoblotting (W) with anti-HA antibodies (upper panel). A sample of the transfected-cell extract before immunoprecipitation (2% of the volume used for the immunoprecipitation shown in lane 3) is shown in lane 1. The relative expression of HA-CARM1 and HA-PRMT1 before immunoprecipitation is shown in the lower panel. Results are representative of two independent experiments. LRR, LRR fragment of Fli-I; GEL, gelsolin-like fragment of Fli-I; and IgG, position of the immunoglobulin heavy chain on the immunoblot.

Techniques Used: Binding Assay, In Vitro, In Vivo, Synthesized, Incubation, Polyacrylamide Gel Electrophoresis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, FLAG-tag, Expressing

36) Product Images from "Loss of Oncogenic H-ras-Induced Cell Cycle Arrest and p38 Mitogen-Activated Protein Kinase Activation by Disruption of Gadd45a"

Article Title: Loss of Oncogenic H-ras-Induced Cell Cycle Arrest and p38 Mitogen-Activated Protein Kinase Activation by Disruption of Gadd45a

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.11.3859-3871.2003

The central region of Gadd45a is required for p38 activation by H-ras. Gadd45a −/− MEF were cotransfected with p38α-Flag, different forms of Myc-Gadd45a, and plasmids with either puromycin (puro) or H-ras (1:3:9 ratio). Two days later, p38 activity was analyzed after immunoprecipitation with anti-Flag Ab in an in vitro kinase reaction with GST-ATF2 as the substrate.
Figure Legend Snippet: The central region of Gadd45a is required for p38 activation by H-ras. Gadd45a −/− MEF were cotransfected with p38α-Flag, different forms of Myc-Gadd45a, and plasmids with either puromycin (puro) or H-ras (1:3:9 ratio). Two days later, p38 activity was analyzed after immunoprecipitation with anti-Flag Ab in an in vitro kinase reaction with GST-ATF2 as the substrate.

Techniques Used: Activation Assay, Activity Assay, Immunoprecipitation, In Vitro

The central region of Gadd45a protein is involved in the interaction with p38. RKO cells were transiently transfected with Flag-p38α and Myc-tagged Gadd45a deletion proteins (1-71)Gadd45a, (1-96)Gadd45a, (1-124)Gadd45a, (48-132)Gadd45a, and (48-165)Gadd45a as well as full-size Myc-tagged Gadd45a. The total lysates of transfected cells were immunoprecipitated (IP) with either anti-Myc (A and C, top panels) or anti-Flag (B, top panel) immunomatrix, and the presence of deletion mutant proteins in precipitate was analyzed by immunoblotting (IB) with anti-Flag or anti-Myc Ab, respectively. The abundant expression of Flag-tagged p38α (A and C, middle panels) and Myc-tagged Gadd45a proteins (B, middle panel, and C, bottom panel) in total lysates was confirmed by immunoblotting with anti-Flag and anti-Myc Abs, respectively. The blots presented at the top of panels A and B were also reprobed with anti-Myc (bands that correspond to various forms of Gadd45a are marked with asterisks in lanes where there is more than one band) and anti-Flag Abs, respectively (A and B, bottom panels) to confirm comparable levels of protein in the primary immunoprecipitates. Cotransfection with Myc vector and Flag-p38α or Flag vector and full-size Myc-Gadd45a was used as for controls.
Figure Legend Snippet: The central region of Gadd45a protein is involved in the interaction with p38. RKO cells were transiently transfected with Flag-p38α and Myc-tagged Gadd45a deletion proteins (1-71)Gadd45a, (1-96)Gadd45a, (1-124)Gadd45a, (48-132)Gadd45a, and (48-165)Gadd45a as well as full-size Myc-tagged Gadd45a. The total lysates of transfected cells were immunoprecipitated (IP) with either anti-Myc (A and C, top panels) or anti-Flag (B, top panel) immunomatrix, and the presence of deletion mutant proteins in precipitate was analyzed by immunoblotting (IB) with anti-Flag or anti-Myc Ab, respectively. The abundant expression of Flag-tagged p38α (A and C, middle panels) and Myc-tagged Gadd45a proteins (B, middle panel, and C, bottom panel) in total lysates was confirmed by immunoblotting with anti-Flag and anti-Myc Abs, respectively. The blots presented at the top of panels A and B were also reprobed with anti-Myc (bands that correspond to various forms of Gadd45a are marked with asterisks in lanes where there is more than one band) and anti-Flag Abs, respectively (A and B, bottom panels) to confirm comparable levels of protein in the primary immunoprecipitates. Cotransfection with Myc vector and Flag-p38α or Flag vector and full-size Myc-Gadd45a was used as for controls.

Techniques Used: Transfection, Immunoprecipitation, Mutagenesis, Expressing, Cotransfection, Plasmid Preparation

Gadd45a and p38 are required for p53 activation after H-ras overexpression. (A) wt and Gadd45a −/− MEF were incubated in the presence of a MEK1 (50 μM PD98059) or p38 (10 μM SB202190) inhibitor, and protein extracts were obtained on day 5 after selection (see Materials and Methods). The levels of p16/Ink4a, p21/Waf1, and p53 proteins were analyzed. DMSO, dimethyl sulfoxide; puro, puromycin. (B) wt and Gadd45a −/− MEF were cotransfected with p53RE-CAT reporter plasmid and expression vectors containing either puromycin or H-ras. Some cells were additionally transfected with a dominant-negative p38α vector (p38DN). Four days later, cells were treated with either a MEK1 (PD90859) or p38 (SB202190) inhibitor, and CAT assays were carried out 12 h later. (C) wt and Gadd45a −/− MEF were cotransfected with p53RE-CAT reporter plasmid and expression vectors containing either puromycin or MKK6(E). Four days later, CAT activity was analyzed, and representative results are shown. Relative induction, as measured by increased CAT activity, was consistently twofold or greater in wt MEF compared to that in Gadd45a −/− MEF.
Figure Legend Snippet: Gadd45a and p38 are required for p53 activation after H-ras overexpression. (A) wt and Gadd45a −/− MEF were incubated in the presence of a MEK1 (50 μM PD98059) or p38 (10 μM SB202190) inhibitor, and protein extracts were obtained on day 5 after selection (see Materials and Methods). The levels of p16/Ink4a, p21/Waf1, and p53 proteins were analyzed. DMSO, dimethyl sulfoxide; puro, puromycin. (B) wt and Gadd45a −/− MEF were cotransfected with p53RE-CAT reporter plasmid and expression vectors containing either puromycin or H-ras. Some cells were additionally transfected with a dominant-negative p38α vector (p38DN). Four days later, cells were treated with either a MEK1 (PD90859) or p38 (SB202190) inhibitor, and CAT assays were carried out 12 h later. (C) wt and Gadd45a −/− MEF were cotransfected with p53RE-CAT reporter plasmid and expression vectors containing either puromycin or MKK6(E). Four days later, CAT activity was analyzed, and representative results are shown. Relative induction, as measured by increased CAT activity, was consistently twofold or greater in wt MEF compared to that in Gadd45a −/− MEF.

Techniques Used: Activation Assay, Over Expression, Incubation, Selection, Plasmid Preparation, Expressing, Transfection, Dominant Negative Mutation, Activity Assay

37) Product Images from "Phosphorylation of PEA-15 switches its binding specificity from ERK/MAPK to FADD"

Article Title: Phosphorylation of PEA-15 switches its binding specificity from ERK/MAPK to FADD

Journal: Biochemical Journal

doi: 10.1042/BJ20050378

Phosphorylation of PEA-15 at Ser-104 impairs its association with ERK Purified agarose-bound fusion proteins GST or GST–PEA-15 were in vitro phosphorylated using purified PKC ( A ) or CamKII ( B ) for 15 min, washed and incubated with Cos-7 cell lysates for 2 h. Amount of ERK1/2 associated with the fusion proteins was detected by Western blotting. An aliquot of GST or GST–PEA-15 (saved before pull down) was immunoblotted with phospho-Ser-104 or phospho-Ser-116 antibodies respectively. The section shown was blotted for phospho-Ser-104 and Ser-116 in the region corresponding to the 45 kDa band of GST–PEA-15 and the 27 kDa band of GST respectively. Blots were also probed with PEA-15 antibody to verify equal input fusion proteins. Bottom panel shows a Coomassie Blue-stained gel of the GST control and GST–PEA-15 fusion proteins. The amount of total ERK associated with PEA-15 was determined by densitometry and the net intensity in the presence or absence of PKC or CamKII treatment is plotted as a graph. Blots are representative of three independent experiments.
Figure Legend Snippet: Phosphorylation of PEA-15 at Ser-104 impairs its association with ERK Purified agarose-bound fusion proteins GST or GST–PEA-15 were in vitro phosphorylated using purified PKC ( A ) or CamKII ( B ) for 15 min, washed and incubated with Cos-7 cell lysates for 2 h. Amount of ERK1/2 associated with the fusion proteins was detected by Western blotting. An aliquot of GST or GST–PEA-15 (saved before pull down) was immunoblotted with phospho-Ser-104 or phospho-Ser-116 antibodies respectively. The section shown was blotted for phospho-Ser-104 and Ser-116 in the region corresponding to the 45 kDa band of GST–PEA-15 and the 27 kDa band of GST respectively. Blots were also probed with PEA-15 antibody to verify equal input fusion proteins. Bottom panel shows a Coomassie Blue-stained gel of the GST control and GST–PEA-15 fusion proteins. The amount of total ERK associated with PEA-15 was determined by densitometry and the net intensity in the presence or absence of PKC or CamKII treatment is plotted as a graph. Blots are representative of three independent experiments.

Techniques Used: Purification, In Vitro, Incubation, Western Blot, Staining

Characterization of phospho-PEA-15 antibodies ( A ) A schematic representation of PEA-15. The protein is 130 amino acids in length and consists of a DED that constitutes amino acids 1–80. The DED is a homotypic binding domain found primarily in proteins that control extrinsic apoptosis. PEA-15 is phosphorylated at two serine residues. It is phosphorylated at Ser-104 by PKC and at Ser-116 by either CamKII or AKT. These phosphorylations are depicted by arrows. PEA-15 binding partners ERK and FADD are shown by connecting lines. ( B ) Cos-7 cells were transfected with the wild-type HA–PEA-15 or the mutant His–S104A and His–S116A constructs; 30 h post-transfection, cells were serum-starved for 18–20 h, and they were either left unstimulated (−) or stimulated (+) with 50 ng/ml PMA for 15 min and lysates were prepared. Lysates were subjected to SDS/PAGE and Western blotting. Phospho-epitope-specific antibodies were used to detect phosphorylation of PEA-15 at Ser-104 and Ser-116. PEA-15 expression was verified by immunoblotting. ( C ) Time course of phosphorylation of PEA-15 at Ser-104. Cos-7 cells were transfected with HA–PEA-15 construct, and 30 h post-transfection they were serum-starved overnight. Cells were then either left untreated (−) or treated with 50 ng/ml PMA for 15 min, washed twice with serum-free DMEM and left in serum-free DMEM for various time periods, namely 30 min and 1, 2, 5, 16 and 25 h. Cells were lysed and phospho-PEA-15 was detected by Western blotting with phospho-Ser-104 and phospho-Ser-116 antibodies. Total PEA-15 was also detected with PEA-15 antibody.
Figure Legend Snippet: Characterization of phospho-PEA-15 antibodies ( A ) A schematic representation of PEA-15. The protein is 130 amino acids in length and consists of a DED that constitutes amino acids 1–80. The DED is a homotypic binding domain found primarily in proteins that control extrinsic apoptosis. PEA-15 is phosphorylated at two serine residues. It is phosphorylated at Ser-104 by PKC and at Ser-116 by either CamKII or AKT. These phosphorylations are depicted by arrows. PEA-15 binding partners ERK and FADD are shown by connecting lines. ( B ) Cos-7 cells were transfected with the wild-type HA–PEA-15 or the mutant His–S104A and His–S116A constructs; 30 h post-transfection, cells were serum-starved for 18–20 h, and they were either left unstimulated (−) or stimulated (+) with 50 ng/ml PMA for 15 min and lysates were prepared. Lysates were subjected to SDS/PAGE and Western blotting. Phospho-epitope-specific antibodies were used to detect phosphorylation of PEA-15 at Ser-104 and Ser-116. PEA-15 expression was verified by immunoblotting. ( C ) Time course of phosphorylation of PEA-15 at Ser-104. Cos-7 cells were transfected with HA–PEA-15 construct, and 30 h post-transfection they were serum-starved overnight. Cells were then either left untreated (−) or treated with 50 ng/ml PMA for 15 min, washed twice with serum-free DMEM and left in serum-free DMEM for various time periods, namely 30 min and 1, 2, 5, 16 and 25 h. Cells were lysed and phospho-PEA-15 was detected by Western blotting with phospho-Ser-104 and phospho-Ser-116 antibodies. Total PEA-15 was also detected with PEA-15 antibody.

Techniques Used: Binding Assay, Transfection, Mutagenesis, Construct, SDS Page, Western Blot, Expressing

38) Product Images from "Tax Recruitment of CBP/p300, via the KIX Domain, Reveals a Potent Requirement for Acetyltransferase Activity That Is Chromatin Dependent and Histone Tail Independent"

Article Title: Tax Recruitment of CBP/p300, via the KIX Domain, Reveals a Potent Requirement for Acetyltransferase Activity That Is Chromatin Dependent and Histone Tail Independent

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.10.3392-3404.2003

Exogenous p300 relieves KIX and SREBP-1a-mediated inhibition of Tax-activated transcription. Transcription reaction mixtures contained the chromatin-assembled p4TxRE-G-less template, CEM nuclear extract, and Tax/CREB, as indicated. GST-KIX and GST-SREBP-1a were added to the indicated transcription reaction mixtures at a fivefold molar excess (++) relative to the Tax/CREB complex. Recombinant p300 was added at 0.05 and 0.1 pmol. Size markers and the position of the full-length G-less transcript are indicated.
Figure Legend Snippet: Exogenous p300 relieves KIX and SREBP-1a-mediated inhibition of Tax-activated transcription. Transcription reaction mixtures contained the chromatin-assembled p4TxRE-G-less template, CEM nuclear extract, and Tax/CREB, as indicated. GST-KIX and GST-SREBP-1a were added to the indicated transcription reaction mixtures at a fivefold molar excess (++) relative to the Tax/CREB complex. Recombinant p300 was added at 0.05 and 0.1 pmol. Size markers and the position of the full-length G-less transcript are indicated.

Techniques Used: Inhibition, Recombinant

KIX, SREBP-1a, and Lys-CoA inhibit Tax-activated transcription on chromatin templates lacking histone amino-terminal tails. Transcription reaction mixtures contained the p4TxRE-G-less template assembled into chromatin by using tailless histones, CEM nuclear extract, and Tax/CREB, as indicated. The recovery standard and full-length G-less transcripts are indicated. (A) The KIX polypeptide specifically inhibits Tax/CREB transcription on tailless chromatin. GST-KIX or GST-KIXmut was added at an equimolar concentration (+) or fivefold molar excess (++) relative to the Tax/CREB complex, as indicated. (B) SREBP-1a specifically inhibits Tax/CREB transcription on tailless chromatin. GST-SREBP-1a and GST were added at equimolar concentration (+) or fivefold molar excess (++) relative to the Tax/CREB complex, as indicated. (C) Exogenous p300 relieves KIX and SREBP-1a-mediated inhibition of Tax/CREB transcription on tailless chromatin. GST-KIX and GST-SREBP-1a were added to the indicated transcription reaction mixtures at fivefold molar excess (++) relative to the Tax/CREB complex. Recombinant p300 was added at 0.05 and 0.1 pmol. (D) Lys-CoA inhibits Tax/CREB transcription on tailless chromatin. For this experiment, acetyl-CoA was added to a final concentration of 10 μM in all samples. Lys-CoA was added to samples at final concentrations of 10 μM (+) and 50 μM (++).
Figure Legend Snippet: KIX, SREBP-1a, and Lys-CoA inhibit Tax-activated transcription on chromatin templates lacking histone amino-terminal tails. Transcription reaction mixtures contained the p4TxRE-G-less template assembled into chromatin by using tailless histones, CEM nuclear extract, and Tax/CREB, as indicated. The recovery standard and full-length G-less transcripts are indicated. (A) The KIX polypeptide specifically inhibits Tax/CREB transcription on tailless chromatin. GST-KIX or GST-KIXmut was added at an equimolar concentration (+) or fivefold molar excess (++) relative to the Tax/CREB complex, as indicated. (B) SREBP-1a specifically inhibits Tax/CREB transcription on tailless chromatin. GST-SREBP-1a and GST were added at equimolar concentration (+) or fivefold molar excess (++) relative to the Tax/CREB complex, as indicated. (C) Exogenous p300 relieves KIX and SREBP-1a-mediated inhibition of Tax/CREB transcription on tailless chromatin. GST-KIX and GST-SREBP-1a were added to the indicated transcription reaction mixtures at fivefold molar excess (++) relative to the Tax/CREB complex. Recombinant p300 was added at 0.05 and 0.1 pmol. (D) Lys-CoA inhibits Tax/CREB transcription on tailless chromatin. For this experiment, acetyl-CoA was added to a final concentration of 10 μM in all samples. Lys-CoA was added to samples at final concentrations of 10 μM (+) and 50 μM (++).

Techniques Used: Concentration Assay, Inhibition, Recombinant

39) Product Images from "Use of the Pharmacological Inhibitor BX795 to Study the Regulation and Physiological Roles of TBK1 and I?B Kinase ?"

Article Title: Use of the Pharmacological Inhibitor BX795 to Study the Regulation and Physiological Roles of TBK1 and I?B Kinase ?

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.000414

The overexpression of TBK1 and IKKε leads to autophosphorylation and transphosphorylation of Ser-172. A , wild type ( WT ) and two different catalytically-inactive FLAG-tagged mutants of TBK1 (TBK1-(K38A) and TBK1[D157A]) were expressed in HEK293
Figure Legend Snippet: The overexpression of TBK1 and IKKε leads to autophosphorylation and transphosphorylation of Ser-172. A , wild type ( WT ) and two different catalytically-inactive FLAG-tagged mutants of TBK1 (TBK1-(K38A) and TBK1[D157A]) were expressed in HEK293

Techniques Used: Over Expression

40) Product Images from "Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans"

Article Title: Identification and characterization of a highly conserved calcineurin binding protein, CBP1/calcipressin, in Cryptococcus neoformans

Journal: The EMBO Journal

doi: 10.1093/emboj/19.14.3618

Fig. 5. CBP1 electrophoretic mobility is regulated by calcineurin. ( A ) Strains H99 ( CNA1 ) and Y5/AO4 ( Δcna1 ) expressing a CBP1–GFP fusion protein were incubated for 2 h in the presence (+) or absence (–) of 100 mM CaCl 2 or 1 µg/ml FK506. Total proteins were extracted, separated by 9% SDS–PAGE, transferred to nitrocellulose, and probed with anti-GFP antibody. ( B ) Strains expressing CBP1–GFP fusion proteins, either wild-type (CBP1) or mutant forms (CBP1 S68A,S142A , CBP1 S68A,S138A , CBP1 S138A,S142A ), were incubated for 2 h in the presence (+) or absence (–) of 100 mM CaCl 2 or 1 µg/ml FK506. Total proteins were extracted, separated by SDS–PAGE, transferred to nitrocellulose, and probed with anti-GFP antibody.
Figure Legend Snippet: Fig. 5. CBP1 electrophoretic mobility is regulated by calcineurin. ( A ) Strains H99 ( CNA1 ) and Y5/AO4 ( Δcna1 ) expressing a CBP1–GFP fusion protein were incubated for 2 h in the presence (+) or absence (–) of 100 mM CaCl 2 or 1 µg/ml FK506. Total proteins were extracted, separated by 9% SDS–PAGE, transferred to nitrocellulose, and probed with anti-GFP antibody. ( B ) Strains expressing CBP1–GFP fusion proteins, either wild-type (CBP1) or mutant forms (CBP1 S68A,S142A , CBP1 S68A,S138A , CBP1 S138A,S142A ), were incubated for 2 h in the presence (+) or absence (–) of 100 mM CaCl 2 or 1 µg/ml FK506. Total proteins were extracted, separated by SDS–PAGE, transferred to nitrocellulose, and probed with anti-GFP antibody.

Techniques Used: Expressing, Incubation, SDS Page, Mutagenesis

Fig. 3. CBP1 binds to calcineurin in vitro and in vivo . ( A ) Purified GST–CBP fusion protein bound to glutathione agarose was incu bated with bovine calcineurin in the presence (+) or absence (–) of calmodulin, FKBP12, FK506 or EGTA for 2 h at 4°C. Reactions were separated on 10% SDS–PAGE and transferred to PVDF membranes. Membranes were incubated with anti-calcineurin (bovine) antibody to detect binding of CBP to calcineurin or anti-GST antibody to detect the GST–CBP fusion protein. The arrow indicates the position of calcineurin. The panel on the right indicates the position of the GST–CBP fusion protein. ( B ) Wild-type strain H99 ( CNA1 ) and the isogenic Δcna1 mutant strain expressing wild-type CBP1 (H99, AO4) or a CBP1–GFP fusion protein (JMC4, JMC6) were grown over night in rich medium, cells were mechanically disrupted, and immuno precipitation experiments were conducted with total cell extracts for 1 h at 4°C in the presence (+) and absence (–) of anti-GFP antisera (α-GFP Ab). Proteins bound to the antibody were subsequently precipitated with protein A–Sepharose (PAS) and separated on 12% SDS–PAGE. The calcineurin A protein (CNA1) was detected by incubating the western blots with [ 125 I]calmodulin. The first two lanes of the right and left panels are total extract controls that were not incubated with the anti-GFP antisera.
Figure Legend Snippet: Fig. 3. CBP1 binds to calcineurin in vitro and in vivo . ( A ) Purified GST–CBP fusion protein bound to glutathione agarose was incu bated with bovine calcineurin in the presence (+) or absence (–) of calmodulin, FKBP12, FK506 or EGTA for 2 h at 4°C. Reactions were separated on 10% SDS–PAGE and transferred to PVDF membranes. Membranes were incubated with anti-calcineurin (bovine) antibody to detect binding of CBP to calcineurin or anti-GST antibody to detect the GST–CBP fusion protein. The arrow indicates the position of calcineurin. The panel on the right indicates the position of the GST–CBP fusion protein. ( B ) Wild-type strain H99 ( CNA1 ) and the isogenic Δcna1 mutant strain expressing wild-type CBP1 (H99, AO4) or a CBP1–GFP fusion protein (JMC4, JMC6) were grown over night in rich medium, cells were mechanically disrupted, and immuno precipitation experiments were conducted with total cell extracts for 1 h at 4°C in the presence (+) and absence (–) of anti-GFP antisera (α-GFP Ab). Proteins bound to the antibody were subsequently precipitated with protein A–Sepharose (PAS) and separated on 12% SDS–PAGE. The calcineurin A protein (CNA1) was detected by incubating the western blots with [ 125 I]calmodulin. The first two lanes of the right and left panels are total extract controls that were not incubated with the anti-GFP antisera.

Techniques Used: In Vitro, In Vivo, Purification, SDS Page, Incubation, Binding Assay, Mutagenesis, Expressing, Immunoprecipitation, Western Blot

Fig. 4. Effect of CBP1 peptides on calcineurin phosphatase activity. ( A ) Calcineurin phosphatase activity was determined by measurement of radiolabel released from [ 32 P]phospho-RII peptide at 30°C in the presence or absence (–CAM) of calmodulin, and with the addition of FKBP12, FKBP12–FK506, autoinhibitory peptide (AID), or of the DSCR1 or CBP1 peptides at 20, 100 or 200 µM. ( B ) Calcineurin phosphatase activity was determined by spectrophotometric determination at 405 nm of the reaction product released from pNPP at 25°C. Values shown are % activities relative to uninhibited calcineurin control reactions. Each sample was performed in duplicate.
Figure Legend Snippet: Fig. 4. Effect of CBP1 peptides on calcineurin phosphatase activity. ( A ) Calcineurin phosphatase activity was determined by measurement of radiolabel released from [ 32 P]phospho-RII peptide at 30°C in the presence or absence (–CAM) of calmodulin, and with the addition of FKBP12, FKBP12–FK506, autoinhibitory peptide (AID), or of the DSCR1 or CBP1 peptides at 20, 100 or 200 µM. ( B ) Calcineurin phosphatase activity was determined by spectrophotometric determination at 405 nm of the reaction product released from pNPP at 25°C. Values shown are % activities relative to uninhibited calcineurin control reactions. Each sample was performed in duplicate.

Techniques Used: Activity Assay, Chick Chorioallantoic Membrane Assay

Fig. 1. Calcineurin and CBP1 interact in the two-hybrid assay. ( A ) The C.neoformans calcineurin A protein (cCNA1) specifically interacts with CBP1 and this interaction is inhibited by FK506. β-galactosidase assays were conducted in the presence and absence of 1 µg/ml FK506 as indicated. ( B ) FK506 inhibition of calcineurin–CBP1 binding requires FKBP12. An S.cerevisiae strain lacking FKBP12 ( fpr1 strain SMY87-4) was transformed with plasmids expressing the GAL4 activation domain (AD) or DNA binding domain (BD) fused to the indicated proteins. To detect expression of the GAL-ADE2 reporter gene, cells were grown on synthetic medium minus leucine, tryptophan and adenine (SD-leu-trp-ade) for 7 days at 30°C. ‘y’ denotes S.cerevisiae proteins and ‘c’ denotes C.neoformans proteins. FK506 stimulated FKBP12 binding to calcineurin A as expected. FK506 did not inhibit CBP1 binding to calcineurin in these cells lacking endogenous FKBP12. Calcineurin A (cCNA1) binding to calcineurin B (yCNB) was not affected by FK506. ( C ) Saccharomyces cerevisiae calcineurin B interacts with CBP1 and is required for CBP1 binding to calcineurin A. Isogenic S.cerevisiae strains expressing (PJ69-4A) or lacking calcineurin B (ΔCnB, SMY3) were transformed with plasmids expressing the GAL4 AD or BD fused to the indicated proteins. Cells were grown on medium lacking adenine (SD-leu-trp-ade) to detect expression of the GAL-ADE2 reporter gene, and on medium lacking histidine (SD-leu-trp-his + 5 mM 3-AT) to detect expression of the GAL-HIS3 reporter gene. ( D ) Two-hybrid analysis of the binding of truncated forms of CBP1 to calcineurin A. The black box represents the highly conserved region of CBP1. Fragment length is indicated in amino acid residues. * indicates the GST–CBP1 fusion protein.
Figure Legend Snippet: Fig. 1. Calcineurin and CBP1 interact in the two-hybrid assay. ( A ) The C.neoformans calcineurin A protein (cCNA1) specifically interacts with CBP1 and this interaction is inhibited by FK506. β-galactosidase assays were conducted in the presence and absence of 1 µg/ml FK506 as indicated. ( B ) FK506 inhibition of calcineurin–CBP1 binding requires FKBP12. An S.cerevisiae strain lacking FKBP12 ( fpr1 strain SMY87-4) was transformed with plasmids expressing the GAL4 activation domain (AD) or DNA binding domain (BD) fused to the indicated proteins. To detect expression of the GAL-ADE2 reporter gene, cells were grown on synthetic medium minus leucine, tryptophan and adenine (SD-leu-trp-ade) for 7 days at 30°C. ‘y’ denotes S.cerevisiae proteins and ‘c’ denotes C.neoformans proteins. FK506 stimulated FKBP12 binding to calcineurin A as expected. FK506 did not inhibit CBP1 binding to calcineurin in these cells lacking endogenous FKBP12. Calcineurin A (cCNA1) binding to calcineurin B (yCNB) was not affected by FK506. ( C ) Saccharomyces cerevisiae calcineurin B interacts with CBP1 and is required for CBP1 binding to calcineurin A. Isogenic S.cerevisiae strains expressing (PJ69-4A) or lacking calcineurin B (ΔCnB, SMY3) were transformed with plasmids expressing the GAL4 AD or BD fused to the indicated proteins. Cells were grown on medium lacking adenine (SD-leu-trp-ade) to detect expression of the GAL-ADE2 reporter gene, and on medium lacking histidine (SD-leu-trp-his + 5 mM 3-AT) to detect expression of the GAL-HIS3 reporter gene. ( D ) Two-hybrid analysis of the binding of truncated forms of CBP1 to calcineurin A. The black box represents the highly conserved region of CBP1. Fragment length is indicated in amino acid residues. * indicates the GST–CBP1 fusion protein.

Techniques Used: Two Hybrid Assay, Inhibition, Binding Assay, Transformation Assay, Expressing, Activation Assay

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  • 99
    Millipore raf1 ras binding domain glutathione agarose
    ERM proteins are necessary for PDGFR signaling. A , Down-regulation of ERM proteins reduces PDGF-dependent Erk phosphorylation. NIH 3T3 cells plated at low density were treated with a combination of siRNA SMARTpools against mouse ERM proteins for 24 hours. For exogenous reconstitution of human ERMs, cells were transfected with plasmid DNA coding for human ezrin-VSVg, radixin-Flag and untagged moesin or ezrin-VSVg alone. Cells were serum starved overnight prior to treatment with PDGF for 5 min. Lysates were immunoblotted as indicated. B , Schematic representation of the architecture of ezrin mutants. C , Ezrin mutants inhibit PDGF-dependent Erk phosphorylation. RT4 cells at low density were transfected with either empty vector (control) or ezrin mutants (ezrinNterm-GFP or ezrin deleted in the Actin-Binding-Domain ezrinΔABD-GFP) (left panel) ezrin wild type or ezrin mutant R579A (right panel). Cells with high GFP expression were sorted by FACS, replated at low cell density and serum starved overnight prior to induction with PDGF for 5 min. Lysates were immunoblotted as indicated. D , NIH 3T3 cells were plated at a low density, co-transfected in a 5∶1 ratio with constructs encoding either ezrin wild type-GFP or ezrin mutant-GFP and with a hygromycin resistance construct, selected by hygromycin for 1 day, and serum starved overnight prior to treatment with PDGF or EGF for 3 min. For Ras-GTP levels, lysates were treated with <t>GST-Raf1-RBD</t> (Ras-binding domain, RBD). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates were immunoblotted as indicated. E , Ezrin R579A also inhibits PDGF-dependent Ras activation in RT4 cells. Experiment as in D, except ezrin constructs encoding either ezrin wild type-VSVg or ezrin mutant-VSVg were used. F , Down-regulation of individual ERM proteins using a cocktail of specific siRNAs reduces PDGF-dependent Ras activation in NIH 3T3 cells. Ras activity was determined as in D . G , Ezrin mutant, but not wild type ezrin, inhibits agar colony formation in RT4 cells. Dox-inducible ezrin wild type- or mutant-expressing cells were generated and placed in soft agar (− and + dox). Results represent mean absolute colony number ± s.d. of at least three independent experiments, ** P
    Raf1 Ras Binding Domain Glutathione Agarose, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore his tagged hrv 3c protease
    . (B) SDS-PAGE separation of buffer-exchanged Nup84 complex obtained from affinity isolation and elution by <t>HRV</t> 3C protease.
    His Tagged Hrv 3c Protease, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore glutathione monoethyl ester gsh mee
    De novo synthesis of <t>GSH</t> is required for EGCG to inhibit HSC growth and to regulate the expression of genes relevant to cell proliferation. HSCs were treated for 24 h with EGCG (50 μ M), NAC (5 mM), or <t>GSH-MEE</t> (2 mM) with or without the pre-exposure to BSO (0.25 mM) for 1 h. Values are means ± S.D. ( n ≥ 3). *, p
    Glutathione Monoethyl Ester Gsh Mee, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ERM proteins are necessary for PDGFR signaling. A , Down-regulation of ERM proteins reduces PDGF-dependent Erk phosphorylation. NIH 3T3 cells plated at low density were treated with a combination of siRNA SMARTpools against mouse ERM proteins for 24 hours. For exogenous reconstitution of human ERMs, cells were transfected with plasmid DNA coding for human ezrin-VSVg, radixin-Flag and untagged moesin or ezrin-VSVg alone. Cells were serum starved overnight prior to treatment with PDGF for 5 min. Lysates were immunoblotted as indicated. B , Schematic representation of the architecture of ezrin mutants. C , Ezrin mutants inhibit PDGF-dependent Erk phosphorylation. RT4 cells at low density were transfected with either empty vector (control) or ezrin mutants (ezrinNterm-GFP or ezrin deleted in the Actin-Binding-Domain ezrinΔABD-GFP) (left panel) ezrin wild type or ezrin mutant R579A (right panel). Cells with high GFP expression were sorted by FACS, replated at low cell density and serum starved overnight prior to induction with PDGF for 5 min. Lysates were immunoblotted as indicated. D , NIH 3T3 cells were plated at a low density, co-transfected in a 5∶1 ratio with constructs encoding either ezrin wild type-GFP or ezrin mutant-GFP and with a hygromycin resistance construct, selected by hygromycin for 1 day, and serum starved overnight prior to treatment with PDGF or EGF for 3 min. For Ras-GTP levels, lysates were treated with GST-Raf1-RBD (Ras-binding domain, RBD). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates were immunoblotted as indicated. E , Ezrin R579A also inhibits PDGF-dependent Ras activation in RT4 cells. Experiment as in D, except ezrin constructs encoding either ezrin wild type-VSVg or ezrin mutant-VSVg were used. F , Down-regulation of individual ERM proteins using a cocktail of specific siRNAs reduces PDGF-dependent Ras activation in NIH 3T3 cells. Ras activity was determined as in D . G , Ezrin mutant, but not wild type ezrin, inhibits agar colony formation in RT4 cells. Dox-inducible ezrin wild type- or mutant-expressing cells were generated and placed in soft agar (− and + dox). Results represent mean absolute colony number ± s.d. of at least three independent experiments, ** P

    Journal: PLoS ONE

    Article Title: Activation of Ras Requires the ERM-Dependent Link of Actin to the Plasma Membrane

    doi: 10.1371/journal.pone.0027511

    Figure Lengend Snippet: ERM proteins are necessary for PDGFR signaling. A , Down-regulation of ERM proteins reduces PDGF-dependent Erk phosphorylation. NIH 3T3 cells plated at low density were treated with a combination of siRNA SMARTpools against mouse ERM proteins for 24 hours. For exogenous reconstitution of human ERMs, cells were transfected with plasmid DNA coding for human ezrin-VSVg, radixin-Flag and untagged moesin or ezrin-VSVg alone. Cells were serum starved overnight prior to treatment with PDGF for 5 min. Lysates were immunoblotted as indicated. B , Schematic representation of the architecture of ezrin mutants. C , Ezrin mutants inhibit PDGF-dependent Erk phosphorylation. RT4 cells at low density were transfected with either empty vector (control) or ezrin mutants (ezrinNterm-GFP or ezrin deleted in the Actin-Binding-Domain ezrinΔABD-GFP) (left panel) ezrin wild type or ezrin mutant R579A (right panel). Cells with high GFP expression were sorted by FACS, replated at low cell density and serum starved overnight prior to induction with PDGF for 5 min. Lysates were immunoblotted as indicated. D , NIH 3T3 cells were plated at a low density, co-transfected in a 5∶1 ratio with constructs encoding either ezrin wild type-GFP or ezrin mutant-GFP and with a hygromycin resistance construct, selected by hygromycin for 1 day, and serum starved overnight prior to treatment with PDGF or EGF for 3 min. For Ras-GTP levels, lysates were treated with GST-Raf1-RBD (Ras-binding domain, RBD). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates were immunoblotted as indicated. E , Ezrin R579A also inhibits PDGF-dependent Ras activation in RT4 cells. Experiment as in D, except ezrin constructs encoding either ezrin wild type-VSVg or ezrin mutant-VSVg were used. F , Down-regulation of individual ERM proteins using a cocktail of specific siRNAs reduces PDGF-dependent Ras activation in NIH 3T3 cells. Ras activity was determined as in D . G , Ezrin mutant, but not wild type ezrin, inhibits agar colony formation in RT4 cells. Dox-inducible ezrin wild type- or mutant-expressing cells were generated and placed in soft agar (− and + dox). Results represent mean absolute colony number ± s.d. of at least three independent experiments, ** P

    Article Snippet: Growth factors, antibodies and reagents Recombinant human platelet-derived growth factor BB (PDGF) (Biomol); recombinant human interleukin-6 (IL-6), epidermal growth factor (EGF), lysophosphatidic acid (LPA), 12-o-tetradecanoyl-phorbol-13-acetate (TPA), Igepal CA-630, Triton-X-100, GDP, GTPγS and doxycycline (dox; Sigma); mantGDP, mantGTP and GST protein (Jena Bioscience); Lubrol 17A17 (Uniqema); ATP (Roche); GST-Grb2 glutathione agarose (GST-Grb2), Raf1-Ras-binding domain glutathione agarose (GST-Raf1-RBD), GST-Ras- and GST-agarose (Upstate); latrunculin B (Calbiochem); glutathione agarose (Santa Cruz).

    Techniques: Transfection, Plasmid Preparation, Binding Assay, Mutagenesis, Expressing, FACS, Construct, Activation Assay, Activity Assay, Generated

    Latrunculin B mimics the ezrin mutants. A Reduction in actin filaments by treatment with latrunculin B. The parental schwannoma cells RT4 were plated at low density and treated with latrunculin B (1.25 µM, 10 min). Cells were processed as described in material and methods (scale bar 10 µm). B Latrunculin B inhibits signaling. RT4 cells at low density were serum starved overnight, then treated with latrunculin B (1.25 µM, 5 min) prior to treatment with PDGF (10 ng/ml, 5 min). Lysates were treated with GST-Raf1-RBD (to pulldown Ras-GTP). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates immunoblotted as indicated. C , D , E Specific interference with signaling by latrunculin B. RT4 cells prepared and treated with latrunculin B as in panel A , then stimulated with LPA (20 µM, 5 min, panel B ), IL-6 (1 ng/ml, 5 min, panel C ) or TPA (100 ng/ml, 5 min, panel D ). Lysates immunoblotted as indicated. The results are representative of at least three independent assays and each panel represents experiments from the same blot and the same exposure.

    Journal: PLoS ONE

    Article Title: Activation of Ras Requires the ERM-Dependent Link of Actin to the Plasma Membrane

    doi: 10.1371/journal.pone.0027511

    Figure Lengend Snippet: Latrunculin B mimics the ezrin mutants. A Reduction in actin filaments by treatment with latrunculin B. The parental schwannoma cells RT4 were plated at low density and treated with latrunculin B (1.25 µM, 10 min). Cells were processed as described in material and methods (scale bar 10 µm). B Latrunculin B inhibits signaling. RT4 cells at low density were serum starved overnight, then treated with latrunculin B (1.25 µM, 5 min) prior to treatment with PDGF (10 ng/ml, 5 min). Lysates were treated with GST-Raf1-RBD (to pulldown Ras-GTP). Co-precipitated proteins were immunoblotted with antibodies against Ras. Lysates immunoblotted as indicated. C , D , E Specific interference with signaling by latrunculin B. RT4 cells prepared and treated with latrunculin B as in panel A , then stimulated with LPA (20 µM, 5 min, panel B ), IL-6 (1 ng/ml, 5 min, panel C ) or TPA (100 ng/ml, 5 min, panel D ). Lysates immunoblotted as indicated. The results are representative of at least three independent assays and each panel represents experiments from the same blot and the same exposure.

    Article Snippet: Growth factors, antibodies and reagents Recombinant human platelet-derived growth factor BB (PDGF) (Biomol); recombinant human interleukin-6 (IL-6), epidermal growth factor (EGF), lysophosphatidic acid (LPA), 12-o-tetradecanoyl-phorbol-13-acetate (TPA), Igepal CA-630, Triton-X-100, GDP, GTPγS and doxycycline (dox; Sigma); mantGDP, mantGTP and GST protein (Jena Bioscience); Lubrol 17A17 (Uniqema); ATP (Roche); GST-Grb2 glutathione agarose (GST-Grb2), Raf1-Ras-binding domain glutathione agarose (GST-Raf1-RBD), GST-Ras- and GST-agarose (Upstate); latrunculin B (Calbiochem); glutathione agarose (Santa Cruz).

    Techniques:

    . (B) SDS-PAGE separation of buffer-exchanged Nup84 complex obtained from affinity isolation and elution by HRV 3C protease.

    Journal: Analytical chemistry

    Article Title: A robust workflow for native mass spectrometric analysis of affinity-isolated endogenous protein assemblies

    doi: 10.1021/acs.analchem.5b04477

    Figure Lengend Snippet: . (B) SDS-PAGE separation of buffer-exchanged Nup84 complex obtained from affinity isolation and elution by HRV 3C protease.

    Article Snippet: Depending on the engineered cleavage site available, either the His-tagged HRV 3C protease (1 μg/μL stock; EMD Biosciences) or the His-tagged AcTEV protease (1 μg/μL stock; Life Technologies) was used.

    Techniques: SDS Page, Isolation

    Elution with HRV 3C protease

    Journal: Analytical chemistry

    Article Title: A robust workflow for native mass spectrometric analysis of affinity-isolated endogenous protein assemblies

    doi: 10.1021/acs.analchem.5b04477

    Figure Lengend Snippet: Elution with HRV 3C protease

    Article Snippet: Depending on the engineered cleavage site available, either the His-tagged HRV 3C protease (1 μg/μL stock; EMD Biosciences) or the His-tagged AcTEV protease (1 μg/μL stock; Life Technologies) was used.

    Techniques:

    De novo synthesis of GSH is required for EGCG to inhibit HSC growth and to regulate the expression of genes relevant to cell proliferation. HSCs were treated for 24 h with EGCG (50 μ M), NAC (5 mM), or GSH-MEE (2 mM) with or without the pre-exposure to BSO (0.25 mM) for 1 h. Values are means ± S.D. ( n ≥ 3). *, p

    Journal: Molecular pharmacology

    Article Title: Epigallocatechin-3-gallate Inhibits Growth of Activated Hepatic Stellate Cells by Enhancing the Capacity of Glutathione Synthesis

    doi: 10.1124/mol.107.040634

    Figure Lengend Snippet: De novo synthesis of GSH is required for EGCG to inhibit HSC growth and to regulate the expression of genes relevant to cell proliferation. HSCs were treated for 24 h with EGCG (50 μ M), NAC (5 mM), or GSH-MEE (2 mM) with or without the pre-exposure to BSO (0.25 mM) for 1 h. Values are means ± S.D. ( n ≥ 3). *, p

    Article Snippet: Glutathione monoethyl ester (GSH-MEE) was purchased from Calbiochem (San Diego, CA).

    Techniques: Expressing

    (A–B) Levels of serum SOD ( A ) and GSH ( B ) in control, TAA, basil leaves extract plus TAA and basil leaves extract treated rats after six weeks. * Indicates a significant difference between control and treated groups. ** Indicates a significant difference between rats treated with TAA and basil leaves extract plus TAA and basil leaves extract. ***indicates a significant difference between rats treated with basil leaves extract plus TAA and basil leaves extract.

    Journal: Saudi Journal of Biological Sciences

    Article Title: Physiological and histopathological study on the influence of Ocimum basilicum leaves extract on thioacetamide-induced nephrotoxicity in male rats

    doi: 10.1016/j.sjbs.2020.05.034

    Figure Lengend Snippet: (A–B) Levels of serum SOD ( A ) and GSH ( B ) in control, TAA, basil leaves extract plus TAA and basil leaves extract treated rats after six weeks. * Indicates a significant difference between control and treated groups. ** Indicates a significant difference between rats treated with TAA and basil leaves extract plus TAA and basil leaves extract. ***indicates a significant difference between rats treated with basil leaves extract plus TAA and basil leaves extract.

    Article Snippet: The methods of , were used to evaluate the levels of serum superoxide dismutase (SOD) and glutathione (GSH) respectively.

    Techniques: