Structured Review

GE Healthcare glutathione sepharose 4b
Expression and purification of K9-K39-K26 chimera. Lane M: molecular weight marker; lane 1: total cell lysate of E. coli expressing GST-K9-K39-K26 fusion protein; lane 2: K9-K39-K26 chimera after <t>Sepharose</t> 4 B purification and cleavage of the GST carrier
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Images

1) Product Images from "Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes of Leishmania infantum for Serodiagnosis of Visceral Leishmaniasis"

Article Title: Development of Recombinant Chimeric Antigen Expressing Immunodominant B Epitopes of Leishmania infantum for Serodiagnosis of Visceral Leishmaniasis

Journal:

doi: 10.1128/CDLI.12.5.647-653.2005

Expression and purification of K9-K39-K26 chimera. Lane M: molecular weight marker; lane 1: total cell lysate of E. coli expressing GST-K9-K39-K26 fusion protein; lane 2: K9-K39-K26 chimera after Sepharose 4 B purification and cleavage of the GST carrier
Figure Legend Snippet: Expression and purification of K9-K39-K26 chimera. Lane M: molecular weight marker; lane 1: total cell lysate of E. coli expressing GST-K9-K39-K26 fusion protein; lane 2: K9-K39-K26 chimera after Sepharose 4 B purification and cleavage of the GST carrier

Techniques Used: Expressing, Purification, Molecular Weight, Marker

2) Product Images from "Activation of the Small G Protein Arf6 by Dynamin2 through Guanine Nucleotide Exchange Factors in Endocytosis"

Article Title: Activation of the Small G Protein Arf6 by Dynamin2 through Guanine Nucleotide Exchange Factors in Endocytosis

Journal: Scientific Reports

doi: 10.1038/srep14919

Dyn2 activates Arf6 in a manner dependent on its GTPase activity. ( A ) HA-tagged wild type of Dyn2 or its GTPase-deficient mutant K44A was coexpressed with Arf6-Flag in HeLa cells. After 24 hr, the active GTP-Arf6 was pulled down with glutathione-Sepharose beads conjugated with glutathione S -transferase (GST)-tagged leucine zipper region II (LZII) (amino acids 398–455) of JNK-interacting protein (JIP), which specifically binds to the active form of Arf6, and immunoblotted with anti-Flag antibody (left panel). Total Arf6 and Dyn2 expressed in the cell were also immunoblotted with anti-Flag and -HA antibodies, respectively. Right panel shows the means ± SEM of the levels of GTP-Arf6 from eight independent experiments. Statistical significance was calculated using Tukey multiple comparison test; ** P
Figure Legend Snippet: Dyn2 activates Arf6 in a manner dependent on its GTPase activity. ( A ) HA-tagged wild type of Dyn2 or its GTPase-deficient mutant K44A was coexpressed with Arf6-Flag in HeLa cells. After 24 hr, the active GTP-Arf6 was pulled down with glutathione-Sepharose beads conjugated with glutathione S -transferase (GST)-tagged leucine zipper region II (LZII) (amino acids 398–455) of JNK-interacting protein (JIP), which specifically binds to the active form of Arf6, and immunoblotted with anti-Flag antibody (left panel). Total Arf6 and Dyn2 expressed in the cell were also immunoblotted with anti-Flag and -HA antibodies, respectively. Right panel shows the means ± SEM of the levels of GTP-Arf6 from eight independent experiments. Statistical significance was calculated using Tukey multiple comparison test; ** P

Techniques Used: Activity Assay, Mutagenesis

3) Product Images from "Importance of Protein-tyrosine Phosphatase-? Catalytic Domains for Interactions with SHP-2 and Interleukin-1-induced Matrix Metalloproteinase-3 Expression *"

Article Title: Importance of Protein-tyrosine Phosphatase-? Catalytic Domains for Interactions with SHP-2 and Interleukin-1-induced Matrix Metalloproteinase-3 Expression *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.102426

Pulldown assays of PTPα and SHP-2. A , left panel , association between PTPα and SHP-2 was assessed by pulldown assays using glutathione-Sepharose bead-bound bacterial fusion proteins: GST-cPTPα, GST-PTPα D1, and GST-PTPα
Figure Legend Snippet: Pulldown assays of PTPα and SHP-2. A , left panel , association between PTPα and SHP-2 was assessed by pulldown assays using glutathione-Sepharose bead-bound bacterial fusion proteins: GST-cPTPα, GST-PTPα D1, and GST-PTPα

Techniques Used:

4) Product Images from "The Kinesin-associated Protein UNC-76 Is Required for Axonal Transport in the Drosophila Nervous System"

Article Title: The Kinesin-associated Protein UNC-76 Is Required for Axonal Transport in the Drosophila Nervous System

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-12-0800

UNC-76 interacts with the KHC tail domain in the yeast two-hybrid assay and in copurification assays. (A) Yeast cells containing a lacZ reporter gene and various combinations of LexA DNA binding domain (baits, left column) and B42 activation domain (preys, right column) fusion proteins were grown on CM Gal/Raff Xgal plates. Colonies in which reporter gene activation is enhanced by specific bait-prey interactions are blue, whereas colonies in which a bait-prey interaction do not occur are white. Colonies that contain the KHC stalk and tail domains (aa 675-976) or the KHC tail domain (aa 850-975) bait fusions and the UNC-76 prey fusion enhance reporter gene transcription, but colonies containing the KHC stalk domain (aa 675-850) and UNC-76 do not. Control, LexA DNA binding domain bait or B42 activation domain prey. (B) Western analysis of protein fractions from UNC-76 copurification assay. 6xHIS-tagged full-length UNC-76 was bound to Ni 2 + -NTA agarose beads and incubated with detergent-soluble extracts of adult flies containing a transgenic copy of myc epitope-tagged KLC. KHC and KLC copurify with UNC-76 beads (lane U), but not with Ni 2 + -NTA agarose beads alone (lane B). KHC, blot probed with anti-KHC antibody; KLC, blot probed with anti-myc antibody to detect transgenic KLC; L, detergent-soluble lysate; B, proteins that copurify with Ni 2 + -NTA beads; S, supernatant from UNC-76 copurification assay; U, proteins that copurify with 6xHIS-UNC-76 beads.
Figure Legend Snippet: UNC-76 interacts with the KHC tail domain in the yeast two-hybrid assay and in copurification assays. (A) Yeast cells containing a lacZ reporter gene and various combinations of LexA DNA binding domain (baits, left column) and B42 activation domain (preys, right column) fusion proteins were grown on CM Gal/Raff Xgal plates. Colonies in which reporter gene activation is enhanced by specific bait-prey interactions are blue, whereas colonies in which a bait-prey interaction do not occur are white. Colonies that contain the KHC stalk and tail domains (aa 675-976) or the KHC tail domain (aa 850-975) bait fusions and the UNC-76 prey fusion enhance reporter gene transcription, but colonies containing the KHC stalk domain (aa 675-850) and UNC-76 do not. Control, LexA DNA binding domain bait or B42 activation domain prey. (B) Western analysis of protein fractions from UNC-76 copurification assay. 6xHIS-tagged full-length UNC-76 was bound to Ni 2 + -NTA agarose beads and incubated with detergent-soluble extracts of adult flies containing a transgenic copy of myc epitope-tagged KLC. KHC and KLC copurify with UNC-76 beads (lane U), but not with Ni 2 + -NTA agarose beads alone (lane B). KHC, blot probed with anti-KHC antibody; KLC, blot probed with anti-myc antibody to detect transgenic KLC; L, detergent-soluble lysate; B, proteins that copurify with Ni 2 + -NTA beads; S, supernatant from UNC-76 copurification assay; U, proteins that copurify with 6xHIS-UNC-76 beads.

Techniques Used: Y2H Assay, Copurification, Binding Assay, Activation Assay, Western Blot, Incubation, Transgenic Assay

5) Product Images from "Protein-tyrosine Phosphatase-? and Src Functionally Link Focal Adhesions to the Endoplasmic Reticulum to Mediate Interleukin-1-induced Ca2+ Signaling *"

Article Title: Protein-tyrosine Phosphatase-? and Src Functionally Link Focal Adhesions to the Endoplasmic Reticulum to Mediate Interleukin-1-induced Ca2+ Signaling *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M808828200

PTPα associates with IP 3 R1. GST pulldown experiments to show association between PTPα and IP 3 R1 in vitro are shown. A , glutathione-agarose (GST) beads bound to bacterially expressed cPTPα, cPTPαD1, or cPTPαD2 fusion
Figure Legend Snippet: PTPα associates with IP 3 R1. GST pulldown experiments to show association between PTPα and IP 3 R1 in vitro are shown. A , glutathione-agarose (GST) beads bound to bacterially expressed cPTPα, cPTPαD1, or cPTPαD2 fusion

Techniques Used: In Vitro

6) Product Images from "IGF-1-induced phosphorylation and altered distribution of TSC1/TSC2 in C2C12 myotubes"

Article Title: IGF-1-induced phosphorylation and altered distribution of TSC1/TSC2 in C2C12 myotubes

Journal: The FEBS journal

doi: 10.1111/j.1742-4658.2010.07635.x

Protein interaction between TSC2 and 14-3-3 protein is mediated thorough phosphorylation of TSC2 at S939 site C2C12 myoblasts were transfected and then differentiated for totally 4 days. A) in vitro GST-14-3-3 pull-down assay. Total cell lysates containing myc-TSC1 (wild type) and Flag-TSC2 (wild-type, S939A or T1462A) proteins were pulled-down with batch-purified GST-14-3-3-beta. Affinity-purified protein complex with Glutathione sepharose beads were then subjected to SDS-PAGE. The interaction between TSC2 mutant S939A and 14-3-3 was clearly decreased compared to wild-type TSC2 or mutant TSC2-T1462A. B) Unphosphorylatable mutant of TSC2 (S939A) showed no increase in the cytosolic relative adundance following the IGF-1 stimulation. Cleared protein lysates were fractionated into the membrane and the cytosolic fraction by ultra-centrifugation. Pan-cadherin antibody was used for the membrane fraction control, and S6K1 was used as cytosolic fraction control. Image-J software (National Institute of Health) was used for the quantification. Protein contents in IGF-1 stimulated sample were quantified as relative amount compared to the simultaneously transfected control. n = 4 per condition. Each value is means ± SE. Amounts of myc-tagged TSC1 and Flag-tagged TSC2 (wild-type) in the cytosolic fraction were increased by IGF-1 stimulation compared to control. In contrast, no changes in the cytosolic pool of myc-TSC1 and Flag-TSC2 (S939A) were observed after IGF-1 stimulation. We could not see significant alterations in the distributions of myc-TSC1 or Flag-TSC2 in the membrane fraction.
Figure Legend Snippet: Protein interaction between TSC2 and 14-3-3 protein is mediated thorough phosphorylation of TSC2 at S939 site C2C12 myoblasts were transfected and then differentiated for totally 4 days. A) in vitro GST-14-3-3 pull-down assay. Total cell lysates containing myc-TSC1 (wild type) and Flag-TSC2 (wild-type, S939A or T1462A) proteins were pulled-down with batch-purified GST-14-3-3-beta. Affinity-purified protein complex with Glutathione sepharose beads were then subjected to SDS-PAGE. The interaction between TSC2 mutant S939A and 14-3-3 was clearly decreased compared to wild-type TSC2 or mutant TSC2-T1462A. B) Unphosphorylatable mutant of TSC2 (S939A) showed no increase in the cytosolic relative adundance following the IGF-1 stimulation. Cleared protein lysates were fractionated into the membrane and the cytosolic fraction by ultra-centrifugation. Pan-cadherin antibody was used for the membrane fraction control, and S6K1 was used as cytosolic fraction control. Image-J software (National Institute of Health) was used for the quantification. Protein contents in IGF-1 stimulated sample were quantified as relative amount compared to the simultaneously transfected control. n = 4 per condition. Each value is means ± SE. Amounts of myc-tagged TSC1 and Flag-tagged TSC2 (wild-type) in the cytosolic fraction were increased by IGF-1 stimulation compared to control. In contrast, no changes in the cytosolic pool of myc-TSC1 and Flag-TSC2 (S939A) were observed after IGF-1 stimulation. We could not see significant alterations in the distributions of myc-TSC1 or Flag-TSC2 in the membrane fraction.

Techniques Used: Transfection, In Vitro, Pull Down Assay, Purification, Affinity Purification, SDS Page, Mutagenesis, Centrifugation, Software

7) Product Images from "The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells"

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201208150

Morg1 and Cdc42 interact with Par6 in a mutually exclusive manner. (A) Schematic structure of Par6β and its truncated proteins used in the present study. (B, C, E, and H) Proteins in the lysate of COS-7 cells expressing indicated proteins (Cell lysate) were immunoprecipitated (IP) and then analyzed by immunoblot (Blot) with the indicated antibodies. (D) GST–Par6β (126–150, 153–254, or 126–254) or GST alone was incubated with MBP–Morg1 or MBP alone, and pulled down with glutathione-Sepharose 4B beads, followed by SDS-PAGE analysis with Coomassie brilliant blue (CBB) staining or immunoblot with anti-MBP antibodies. (F) GST–Crb3-(84–120) or GST–Crb3-(84–116) was incubated with MBP–Par6β-(126–254), and analyzed as in D. (G) GST–Par6β-(126–254) or GST alone was incubated with MBP–Morg1 in the presence of a two- or sixfold molar excess of Cdc42 (Q61L) or Cdc42 (T17N) relative to Morg1 (Ratio). Proteins pulled down with glutathione-Sepharose 4B beads were subjected to SDS-PAGE and stained with CBB.
Figure Legend Snippet: Morg1 and Cdc42 interact with Par6 in a mutually exclusive manner. (A) Schematic structure of Par6β and its truncated proteins used in the present study. (B, C, E, and H) Proteins in the lysate of COS-7 cells expressing indicated proteins (Cell lysate) were immunoprecipitated (IP) and then analyzed by immunoblot (Blot) with the indicated antibodies. (D) GST–Par6β (126–150, 153–254, or 126–254) or GST alone was incubated with MBP–Morg1 or MBP alone, and pulled down with glutathione-Sepharose 4B beads, followed by SDS-PAGE analysis with Coomassie brilliant blue (CBB) staining or immunoblot with anti-MBP antibodies. (F) GST–Crb3-(84–120) or GST–Crb3-(84–116) was incubated with MBP–Par6β-(126–254), and analyzed as in D. (G) GST–Par6β-(126–254) or GST alone was incubated with MBP–Morg1 in the presence of a two- or sixfold molar excess of Cdc42 (Q61L) or Cdc42 (T17N) relative to Morg1 (Ratio). Proteins pulled down with glutathione-Sepharose 4B beads were subjected to SDS-PAGE and stained with CBB.

Techniques Used: Expressing, Immunoprecipitation, Incubation, SDS Page, Staining

Cdc42 facilitates Par6 binding to Crb3. (A and C) Proteins in the lysate of COS-7 cells expressing indicated proteins (Cell lysate) were immunoprecipitated (IP) and then analyzed by immunoblot (Blot) with the indicated antibodies. In C, the arrow and arrowhead indicate the positions of FLAG–Crb3 and Myc–Par6, respectively; Single and double asterisks denote the heavy and light chains of IgG, respectively. (B) GST–Crb3-(84–120 or 84–116) was incubated with MBP–Par6β-(126–254) in the presence of Cdc42 (Q61L or T17N), and pulled down with glutathione-Sepharose 4B beads, followed by SDS-PAGE analysis with CBB staining.
Figure Legend Snippet: Cdc42 facilitates Par6 binding to Crb3. (A and C) Proteins in the lysate of COS-7 cells expressing indicated proteins (Cell lysate) were immunoprecipitated (IP) and then analyzed by immunoblot (Blot) with the indicated antibodies. In C, the arrow and arrowhead indicate the positions of FLAG–Crb3 and Myc–Par6, respectively; Single and double asterisks denote the heavy and light chains of IgG, respectively. (B) GST–Crb3-(84–120 or 84–116) was incubated with MBP–Par6β-(126–254) in the presence of Cdc42 (Q61L or T17N), and pulled down with glutathione-Sepharose 4B beads, followed by SDS-PAGE analysis with CBB staining.

Techniques Used: Binding Assay, Expressing, Immunoprecipitation, Incubation, SDS Page, Staining

8) Product Images from "Intrasteric control of AMPK via the ?1 subunit AMP allosteric regulatory site"

Article Title: Intrasteric control of AMPK via the ?1 subunit AMP allosteric regulatory site

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.03340004

Effect of Arg mutations on AMP activation. Wild-type γ 1 or mutant γ 1 was cotransfected with α 1 and β 1 in COS-7 cells and AMPK αβγ holoenzyme was purified by Glutathione Sepharose chromatograph. AMPK activity was determined using the SAMS peptide (see Materials and Methods). The activity measured in the absence of AMP was subtracted from plus AMP values of the wild-type and Phe mutants and fitted to the one-site binding Michaelis-Menten Equation curve. The activity of the Arg mutants was AMP-independent.
Figure Legend Snippet: Effect of Arg mutations on AMP activation. Wild-type γ 1 or mutant γ 1 was cotransfected with α 1 and β 1 in COS-7 cells and AMPK αβγ holoenzyme was purified by Glutathione Sepharose chromatograph. AMPK activity was determined using the SAMS peptide (see Materials and Methods). The activity measured in the absence of AMP was subtracted from plus AMP values of the wild-type and Phe mutants and fitted to the one-site binding Michaelis-Menten Equation curve. The activity of the Arg mutants was AMP-independent.

Techniques Used: Activation Assay, Mutagenesis, Purification, Activity Assay, Binding Assay

9) Product Images from "Complexes of D-type cyclins with CDKs during maize germination"

Article Title: Complexes of D-type cyclins with CDKs during maize germination

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/ert340

Method of sequential immunoprecipitation. Left-hand panel: (A) immunoprecipitation of D-type cyclins, (B) incubation of immunoprecipitates at 65 °C for 3h and centrifugation, (C) D-type cyclin–CDK complex removal, (D) immunoprecipitation with anti-CDKB1;1 antibody, (E) immunoprecipitation of resulting supernatant after step (C) with anti-CDKA antibody, and (F) kinase activity in immunoprecipitates. Right-hand panel: detection of target proteins after each immunoprecipitation. (C) Recognition of CycD2;2 in immunoprecipitates with anti-CycD2;2 antibody, (D) recognition of CycD2;2 and CDKB1;1 in immunoprecipitates using anti-CDKB1;1 antibody, and (E) recognition of CycD2;2 and CDKA in immunoprecipitates using the anti-CDKA antibody. C(+), Protein extract from 12 h-imbibed maize axes; C(−), protein A–agarose+antibody (no protein extract).
Figure Legend Snippet: Method of sequential immunoprecipitation. Left-hand panel: (A) immunoprecipitation of D-type cyclins, (B) incubation of immunoprecipitates at 65 °C for 3h and centrifugation, (C) D-type cyclin–CDK complex removal, (D) immunoprecipitation with anti-CDKB1;1 antibody, (E) immunoprecipitation of resulting supernatant after step (C) with anti-CDKA antibody, and (F) kinase activity in immunoprecipitates. Right-hand panel: detection of target proteins after each immunoprecipitation. (C) Recognition of CycD2;2 in immunoprecipitates with anti-CycD2;2 antibody, (D) recognition of CycD2;2 and CDKB1;1 in immunoprecipitates using anti-CDKB1;1 antibody, and (E) recognition of CycD2;2 and CDKA in immunoprecipitates using the anti-CDKA antibody. C(+), Protein extract from 12 h-imbibed maize axes; C(−), protein A–agarose+antibody (no protein extract).

Techniques Used: Immunoprecipitation, Incubation, Centrifugation, Activity Assay

10) Product Images from "The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation"

Article Title: The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.776195

Kindlin-2 interacts with K2 C-terminal peptide. Lysates of αIIbβ3-CHO cells transfected with PSGL-1 alone, PSGL-1–K2CT, PSGL-1–K2CTΔ679, or PSGL-1–K2CT double mutant were used for co-IP assays. After incubating with A/G-agarose and PSGL-1 antibody, full-length K2 bound to PSGL-1 constructs was evaluated by SDS-PAGE and Western blotting using anti-K2 (Cell Signaling), which does not detect the C-terminal end of K2. PSGL-1 and K2 levels in total lysates ( TL ) are also shown, with actin used as a loading control.
Figure Legend Snippet: Kindlin-2 interacts with K2 C-terminal peptide. Lysates of αIIbβ3-CHO cells transfected with PSGL-1 alone, PSGL-1–K2CT, PSGL-1–K2CTΔ679, or PSGL-1–K2CT double mutant were used for co-IP assays. After incubating with A/G-agarose and PSGL-1 antibody, full-length K2 bound to PSGL-1 constructs was evaluated by SDS-PAGE and Western blotting using anti-K2 (Cell Signaling), which does not detect the C-terminal end of K2. PSGL-1 and K2 levels in total lysates ( TL ) are also shown, with actin used as a loading control.

Techniques Used: Transfection, Mutagenesis, Co-Immunoprecipitation Assay, Construct, SDS Page, Western Blot

11) Product Images from "Antagonistic Interplay between Necdin and Bmi1 Controls Proliferation of Neural Precursor Cells in the Embryonic Mouse Neocortex"

Article Title: Antagonistic Interplay between Necdin and Bmi1 Controls Proliferation of Neural Precursor Cells in the Embryonic Mouse Neocortex

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084460

Necdin interacts with Bmi1 in vivo and in vitro . ( A ) Bmi1 deletion mutants. Bmi1 full-length (FL), C-terminal deletion (ΔCT), and mutants containing the Ring finger (RF), helix-turn-helix (HTH) and proline/serine-rich (PS) domains are schematically shown. ( B ) Co-immunoprecipitation assay. HEK293A cells were transfected with expression vectors for necdin and Myc-tagged FL, RF, HTH, p53 (positive control), and p53ΔN (negative control). Cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with anti-Myc (Myc) and anti-necdin (Necdin) antibodies. ( C ) In vitro binding assay. GST-Bmi1 mutants immobilized on glutathione-agarose were incubated with His-tagged necdin (His-necdin), and bound His-necdin was detected by immunoblotting with anti-necdin antibody (upper panel). GST-Bmi1 deletion mutants were stained with Coomassie Brilliant Blue (lower panel). Arrows indicate the predicted protein positions ( B , C ). ( D ) Co-immunoprecipitation assay for endogenous complex containing necdin and Bmi1 in primary NPCs. Lysates of NPCs prepared from E14.5 wild-type (WT) and necdin-null (KO) mice were immunoprecipitated with anti-necdin IgG (Nec) or control preimmune IgG (Pre). Bmi1, PCNA (negative control), and necdin were detected by Western blotting. Lysate, tissue lysate (10 µg).
Figure Legend Snippet: Necdin interacts with Bmi1 in vivo and in vitro . ( A ) Bmi1 deletion mutants. Bmi1 full-length (FL), C-terminal deletion (ΔCT), and mutants containing the Ring finger (RF), helix-turn-helix (HTH) and proline/serine-rich (PS) domains are schematically shown. ( B ) Co-immunoprecipitation assay. HEK293A cells were transfected with expression vectors for necdin and Myc-tagged FL, RF, HTH, p53 (positive control), and p53ΔN (negative control). Cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with anti-Myc (Myc) and anti-necdin (Necdin) antibodies. ( C ) In vitro binding assay. GST-Bmi1 mutants immobilized on glutathione-agarose were incubated with His-tagged necdin (His-necdin), and bound His-necdin was detected by immunoblotting with anti-necdin antibody (upper panel). GST-Bmi1 deletion mutants were stained with Coomassie Brilliant Blue (lower panel). Arrows indicate the predicted protein positions ( B , C ). ( D ) Co-immunoprecipitation assay for endogenous complex containing necdin and Bmi1 in primary NPCs. Lysates of NPCs prepared from E14.5 wild-type (WT) and necdin-null (KO) mice were immunoprecipitated with anti-necdin IgG (Nec) or control preimmune IgG (Pre). Bmi1, PCNA (negative control), and necdin were detected by Western blotting. Lysate, tissue lysate (10 µg).

Techniques Used: In Vivo, In Vitro, Co-Immunoprecipitation Assay, Transfection, Expressing, Positive Control, Negative Control, Immunoprecipitation, Binding Assay, Incubation, Staining, Mouse Assay, Western Blot

12) Product Images from "The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes"

Article Title: The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes

Journal: Cell Research

doi: 10.1038/cr.2014.99

HER2 ubiquitination is regulated by the PTPN18/β-Trcp complex in breast cancer cell lines and the HER2/PTPN18 ratio (expression ratio) is correlated with breast cancer stage. (A) Protein expression levels of PTPN18 and HER2 in seven breast cancer cell lines and HepG2. (B) Endogenous interaction between PTPN18 and β-Trcp was revealed by co-immunoprecipitation in MCF-7 cells. Left: PTPN18 was immunoprecipitated with protein A+G agarose beads and PTPN18 antibody with or without 100 ng/ml EGF stimulation for 30 min. PTPN18-associated β-Trcp was detected with a specific β-Trcp antibody. Right: reverse immunoprecipitation of PTPN18 with the β-Trcp antibody. (C) MCF-7 cells were transfected with HA-ubiquitin and PTPN18 siRNA. After 48 h, cells were stimulated with 100 ng/ml EGF for 4 h. Ubiquitinated HER2 was immunoprecipitated by anti-HA resin and detected with an anti-HER2 antibody. (D) Coordinated regulation of EGF-induced HER2 ubiquitination by β-Trcp and c-Cbl. MCF-7 cells were transfected with HA-ubiquitin and siRNAs directed against β-Trcp or c-Cbl. Ubiquitinated HER2 was examined with an anti-HER2 antibody as in C . (E) The relative ubiquitinated HER2 level in D was quantified and shown as a bar graph. * P
Figure Legend Snippet: HER2 ubiquitination is regulated by the PTPN18/β-Trcp complex in breast cancer cell lines and the HER2/PTPN18 ratio (expression ratio) is correlated with breast cancer stage. (A) Protein expression levels of PTPN18 and HER2 in seven breast cancer cell lines and HepG2. (B) Endogenous interaction between PTPN18 and β-Trcp was revealed by co-immunoprecipitation in MCF-7 cells. Left: PTPN18 was immunoprecipitated with protein A+G agarose beads and PTPN18 antibody with or without 100 ng/ml EGF stimulation for 30 min. PTPN18-associated β-Trcp was detected with a specific β-Trcp antibody. Right: reverse immunoprecipitation of PTPN18 with the β-Trcp antibody. (C) MCF-7 cells were transfected with HA-ubiquitin and PTPN18 siRNA. After 48 h, cells were stimulated with 100 ng/ml EGF for 4 h. Ubiquitinated HER2 was immunoprecipitated by anti-HA resin and detected with an anti-HER2 antibody. (D) Coordinated regulation of EGF-induced HER2 ubiquitination by β-Trcp and c-Cbl. MCF-7 cells were transfected with HA-ubiquitin and siRNAs directed against β-Trcp or c-Cbl. Ubiquitinated HER2 was examined with an anti-HER2 antibody as in C . (E) The relative ubiquitinated HER2 level in D was quantified and shown as a bar graph. * P

Techniques Used: Expressing, Immunoprecipitation, Transfection

13) Product Images from "Annexin A2 Binds RNA and Reduces the Frameshifting Efficiency of Infectious Bronchitis Virus"

Article Title: Annexin A2 Binds RNA and Reduces the Frameshifting Efficiency of Infectious Bronchitis Virus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024067

ANXA2 specifically binds to pseudoknot RNA. (A) GST pull-down analysis of IBV RNA. Wild-type (WT) and mutant (MT) IBV pseudoknot RNA were labeled with [γ- 32 P] ATP and mixed with GST or GST-ANXA2 protein. Each sample was pulled down with Glutathione 4B Sepharose and pelleted radioactivity was measured with a scintillation counter. Three independent experiments were performed and statistical analysis was done. All experiments were performed in triplicate and mean ± s.d. are shown. ***: p
Figure Legend Snippet: ANXA2 specifically binds to pseudoknot RNA. (A) GST pull-down analysis of IBV RNA. Wild-type (WT) and mutant (MT) IBV pseudoknot RNA were labeled with [γ- 32 P] ATP and mixed with GST or GST-ANXA2 protein. Each sample was pulled down with Glutathione 4B Sepharose and pelleted radioactivity was measured with a scintillation counter. Three independent experiments were performed and statistical analysis was done. All experiments were performed in triplicate and mean ± s.d. are shown. ***: p

Techniques Used: Mutagenesis, Labeling, Radioactivity

14) Product Images from "The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿"

Article Title: The CRL4Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01135-09

Xic1 interacts with DDB1 and XCdt2. (A) Schematic representation of XCdt2. XCdt2 contains two WDXR motifs (gray boxes), six WD40 domains (white boxes), and a conserved arginine residue (R247) essential for DDB1 binding. (B) GST pulldown assay. GST or GST-Xic1 immobilized on glutathione-Sepharose beads was incubated with Xenopus interphase egg extract and immunoblotted with antibody against Xenopus DDB1 and PCNA (Western blot). GST and GST-Xic1 proteins (20% of Western blot reaction) were stained with Coomassie brilliant blue. The input (4%) is shown in lane 1. α, anti; *, nonspecific bacterial protein. (C) Coimmunoprecipitation assay. Immunoprecipitated DDB1 (IP) from the egg extract was bound to protein A beads and incubated with 35 S-labeled wild-type XCdt2 (WT), XCdt2 R247A (R247A), XCdt2 1-400 (1-400), or XCdt2 401-710 (401-710). As a control, nonspecific normal rabbit serum (NRS) was used in the place of DDB1 antiserum. Efficient immunoprecipitation of XDDB1 was confirmed by immunoblotting with anti-DDB1 antibody (top). Binding of XCdt2 proteins ( 35 S-Cdt2) was analyzed by SDS-PAGE and phosphorimaging, and 5% of the input proteins is shown (5% input). (D) Coimmunoprecipitation assay. Immunoprecipitated XCdt2 (anti-CDT2, IP) from the egg extract was incubated with 35 S-labeled Xic1 and subjected to SDS-PAGE and phosphorimager analysis. Efficient immunoprecipitation of XCdt2 was confirmed by immunoblotting with anti-Cdt2 antibody (top). Immunoprecipitation with normal rabbit serum (NRS) was included as a control, and input samples are indicated. (E) GST pulldown assay. Bacterially expressed GST or GST-Xic1 (5 μg) was immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled XCdt2 proteins, as indicated. A total of 5% of the input reaction is shown. The percentage of Cdt2 bound by all GST-Xic1 proteins (% binding) is an average value obtained from 2 independent experiments.
Figure Legend Snippet: Xic1 interacts with DDB1 and XCdt2. (A) Schematic representation of XCdt2. XCdt2 contains two WDXR motifs (gray boxes), six WD40 domains (white boxes), and a conserved arginine residue (R247) essential for DDB1 binding. (B) GST pulldown assay. GST or GST-Xic1 immobilized on glutathione-Sepharose beads was incubated with Xenopus interphase egg extract and immunoblotted with antibody against Xenopus DDB1 and PCNA (Western blot). GST and GST-Xic1 proteins (20% of Western blot reaction) were stained with Coomassie brilliant blue. The input (4%) is shown in lane 1. α, anti; *, nonspecific bacterial protein. (C) Coimmunoprecipitation assay. Immunoprecipitated DDB1 (IP) from the egg extract was bound to protein A beads and incubated with 35 S-labeled wild-type XCdt2 (WT), XCdt2 R247A (R247A), XCdt2 1-400 (1-400), or XCdt2 401-710 (401-710). As a control, nonspecific normal rabbit serum (NRS) was used in the place of DDB1 antiserum. Efficient immunoprecipitation of XDDB1 was confirmed by immunoblotting with anti-DDB1 antibody (top). Binding of XCdt2 proteins ( 35 S-Cdt2) was analyzed by SDS-PAGE and phosphorimaging, and 5% of the input proteins is shown (5% input). (D) Coimmunoprecipitation assay. Immunoprecipitated XCdt2 (anti-CDT2, IP) from the egg extract was incubated with 35 S-labeled Xic1 and subjected to SDS-PAGE and phosphorimager analysis. Efficient immunoprecipitation of XCdt2 was confirmed by immunoblotting with anti-Cdt2 antibody (top). Immunoprecipitation with normal rabbit serum (NRS) was included as a control, and input samples are indicated. (E) GST pulldown assay. Bacterially expressed GST or GST-Xic1 (5 μg) was immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled XCdt2 proteins, as indicated. A total of 5% of the input reaction is shown. The percentage of Cdt2 bound by all GST-Xic1 proteins (% binding) is an average value obtained from 2 independent experiments.

Techniques Used: Binding Assay, GST Pulldown Assay, Incubation, Western Blot, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Labeling, SDS Page

Xic1 turnover does not require the tandem arrangement of PCNA and Cdt2 binding domains. (A) Amino acid sequence alignment of p21 (p21Cip1) and Xic1 (p27Xic1). Cdt2 binding regions indicated by italicized amino acid residues and bold lines, the PCNA binding element (PIP box) indicated by gray box, and critical lysine residues of Xic1 indicated by underlining, italicizing, and boldfacing of amino acid residues. (B) Schematic representation of mutant Xic1 proteins. CDK2-cyclin and wild-type PCNA binding domains are indicated by dark gray shading, while the I174A PCNA binding mutant is indicated by a white box. Xic1 residue numbers are indicated below each schematic. The NPIP1 and NPIP2 domains are fused to the N terminus of wild-type Xic1 (WT-NPIP), Xic1-I174A (I174A-NPIP), or amino acids 1 to 160 of Xic1 (N160-NPIP) as indicated and includes Xic1 amino acids 171 to 186 (TTPITDYFPKRKKILS) for NPIP1 and p21 residues 135 to 164 with an internal deletion of residues 156 to 161 for NPIP2. The NPIP2 domain serves solely as a PCNA binding domain and does not retain the ability to efficiently bind Cdt2. (C) GST pulldown assay. GST or GST-Xic1 wild-type and mutant proteins (top, NPIP1; bottom, NPIP2) were immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled Xenopus Cdt2 ( 35 S-XCDT2). The 35 S-XCdt2 input control (5% input) is shown in lane 1. (D) Xic1 degradation assay. (Top and middle) 35 S-labeled Xic1 wild-type (WT) and mutant proteins (WT-NPIP2, I174A, I174A-NPIP2, and N160-NPIP2) as indicated were incubated in HSS with (+) or without (−) single-stranded DNA for the indicated times, followed by SDS-PAGE and phosphorimager analysis. Asterisks indicate internal initiation translation products. (Bottom) Quantitation of Xic1 degradation. The mean percentage of Xic1 remaining from two (WT, WT-NPIP1, I174A-NPIP1, and N160-NPIP1) or three (WT-NPIP2, I174A, I174A-NPIP2, and N160-NPIP2) independent experiments as described above is shown, where the 0-h time point was normalized to 100% of Xic1 remaining for each sample. SEMs are shown as error bars. (E) Quantitation of Xic1 binding to PCNA. GST or GST-PCNA proteins were immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled Xic1 wild-type (WT) or mutant proteins (I174A, WT-NPIP1, I174A-NPIP1, N160-NPIP1, WT-NPIP2, I174A-NPIP2, and N160-NPIP2). The average percentage of Xic1 bound by GST-PCNA (% PCNA binding) is shown, where values for WT Xic1 and I174A are averages of results from 4 independent experiments, and the values of the NPIP mutants (WT-NPIP1, I174A-NPIP1, N160-NPIP1, WT-NPIP2, I174A-NPIP2, and N160-NPIP2) are averages of results from 2 independent experiments. SEMs are shown as error bars.
Figure Legend Snippet: Xic1 turnover does not require the tandem arrangement of PCNA and Cdt2 binding domains. (A) Amino acid sequence alignment of p21 (p21Cip1) and Xic1 (p27Xic1). Cdt2 binding regions indicated by italicized amino acid residues and bold lines, the PCNA binding element (PIP box) indicated by gray box, and critical lysine residues of Xic1 indicated by underlining, italicizing, and boldfacing of amino acid residues. (B) Schematic representation of mutant Xic1 proteins. CDK2-cyclin and wild-type PCNA binding domains are indicated by dark gray shading, while the I174A PCNA binding mutant is indicated by a white box. Xic1 residue numbers are indicated below each schematic. The NPIP1 and NPIP2 domains are fused to the N terminus of wild-type Xic1 (WT-NPIP), Xic1-I174A (I174A-NPIP), or amino acids 1 to 160 of Xic1 (N160-NPIP) as indicated and includes Xic1 amino acids 171 to 186 (TTPITDYFPKRKKILS) for NPIP1 and p21 residues 135 to 164 with an internal deletion of residues 156 to 161 for NPIP2. The NPIP2 domain serves solely as a PCNA binding domain and does not retain the ability to efficiently bind Cdt2. (C) GST pulldown assay. GST or GST-Xic1 wild-type and mutant proteins (top, NPIP1; bottom, NPIP2) were immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled Xenopus Cdt2 ( 35 S-XCDT2). The 35 S-XCdt2 input control (5% input) is shown in lane 1. (D) Xic1 degradation assay. (Top and middle) 35 S-labeled Xic1 wild-type (WT) and mutant proteins (WT-NPIP2, I174A, I174A-NPIP2, and N160-NPIP2) as indicated were incubated in HSS with (+) or without (−) single-stranded DNA for the indicated times, followed by SDS-PAGE and phosphorimager analysis. Asterisks indicate internal initiation translation products. (Bottom) Quantitation of Xic1 degradation. The mean percentage of Xic1 remaining from two (WT, WT-NPIP1, I174A-NPIP1, and N160-NPIP1) or three (WT-NPIP2, I174A, I174A-NPIP2, and N160-NPIP2) independent experiments as described above is shown, where the 0-h time point was normalized to 100% of Xic1 remaining for each sample. SEMs are shown as error bars. (E) Quantitation of Xic1 binding to PCNA. GST or GST-PCNA proteins were immobilized on glutathione-Sepharose beads and incubated with 35 S-labeled Xic1 wild-type (WT) or mutant proteins (I174A, WT-NPIP1, I174A-NPIP1, N160-NPIP1, WT-NPIP2, I174A-NPIP2, and N160-NPIP2). The average percentage of Xic1 bound by GST-PCNA (% PCNA binding) is shown, where values for WT Xic1 and I174A are averages of results from 4 independent experiments, and the values of the NPIP mutants (WT-NPIP1, I174A-NPIP1, N160-NPIP1, WT-NPIP2, I174A-NPIP2, and N160-NPIP2) are averages of results from 2 independent experiments. SEMs are shown as error bars.

Techniques Used: Binding Assay, Sequencing, Mutagenesis, GST Pulldown Assay, Incubation, Labeling, Degradation Assay, SDS Page, Quantitation Assay

PCNA directly interacts with the C-terminal domain of XCdt2. (A) GST pulldown assay. Bacterially expressed GST, GST-XCdt2 1-400 , or GST-XCdt2 401-710 was bound to glutathione-Sepharose and incubated with purified XPCNA (0, 5, 25, and 50 μg) or bovine serum albumin (BSA; 0 and 50 μg) (left) as indicated and MBP-Xic1 (0, 5, 25, and 50 μg) (right), followed by staining with Coomassie blue. Protein bands were identified by mass spectrometry and are labeled accordingly. Several bacterial contaminants were identified. “+” was identified as the bacterial DnaK protein, and “*” was identified as the bacterial GroEL protein. (B) GST pulldown and competitive binding assay. Bacterially expressed GST or GST-PCNA (5 μg) was bound to glutathione-Sepharose beads and incubated with 0 to 50 μg of purified MBP-Xic1 or GST as indicated and 35 S-labeled wild-type XCdt2. (C) GST pulldown and competition study. GST or GST-PCNA was bound to glutathione-Sepharose beads and incubated with 0 to 50 μg of purified MBP-Xic1 as indicated. Following a washing step, samples were incubated with 35 S-labeled XCdt2. (D) GST pulldown assay and competitive binding assay. GST, GST-Xic1 WT , or GST-Xic1 I174A bound to glutathione-Sepharose beads was incubated with 0 to 50 μg of purified XPCNA and 35 S-labeled wild-type XCdt2. (B to D) Samples were analyzed by Coomassie blue staining and phosphorimaging. (Left) Schematic representation of proteins analyzed in binding assays. (Right) The average relative Cdt2 binding values [relative Cdt2 binding (%)] of results from at least 2 independent experiments are shown, where the “zero competitor” value was normalized to 100%.
Figure Legend Snippet: PCNA directly interacts with the C-terminal domain of XCdt2. (A) GST pulldown assay. Bacterially expressed GST, GST-XCdt2 1-400 , or GST-XCdt2 401-710 was bound to glutathione-Sepharose and incubated with purified XPCNA (0, 5, 25, and 50 μg) or bovine serum albumin (BSA; 0 and 50 μg) (left) as indicated and MBP-Xic1 (0, 5, 25, and 50 μg) (right), followed by staining with Coomassie blue. Protein bands were identified by mass spectrometry and are labeled accordingly. Several bacterial contaminants were identified. “+” was identified as the bacterial DnaK protein, and “*” was identified as the bacterial GroEL protein. (B) GST pulldown and competitive binding assay. Bacterially expressed GST or GST-PCNA (5 μg) was bound to glutathione-Sepharose beads and incubated with 0 to 50 μg of purified MBP-Xic1 or GST as indicated and 35 S-labeled wild-type XCdt2. (C) GST pulldown and competition study. GST or GST-PCNA was bound to glutathione-Sepharose beads and incubated with 0 to 50 μg of purified MBP-Xic1 as indicated. Following a washing step, samples were incubated with 35 S-labeled XCdt2. (D) GST pulldown assay and competitive binding assay. GST, GST-Xic1 WT , or GST-Xic1 I174A bound to glutathione-Sepharose beads was incubated with 0 to 50 μg of purified XPCNA and 35 S-labeled wild-type XCdt2. (B to D) Samples were analyzed by Coomassie blue staining and phosphorimaging. (Left) Schematic representation of proteins analyzed in binding assays. (Right) The average relative Cdt2 binding values [relative Cdt2 binding (%)] of results from at least 2 independent experiments are shown, where the “zero competitor” value was normalized to 100%.

Techniques Used: GST Pulldown Assay, Incubation, Purification, Staining, Mass Spectrometry, Labeling, Competitive Binding Assay, Binding Assay

p21 is ubiquitinated during the events of DNA polymerase switching/elongation in the Xenopus egg extract. (A) Amino acid sequence similarity between Xenopus and human Cul4a, Cul4b, DDB1, Cdt2, and PCNA. Xenopus residue numbers are indicated at the bottom of the sequence alignments, and the percentages of similarity (S) and identity (I) between the Xenopus and human proteins are shown on the right. Xenopus Cul4a, the MGC115611 protein (gi 71679818), contains 200 additional residues in the N terminus compared to human Cul4a, so only residues 200 to 858 of Xenopus Cul4a were compared in the alignment. (B) GST pulldown assay. GST, GST-p21, or GST-p27 proteins were immobilized on glutathione-Sepharose beads and incubated with 35 S-hCDT2. A total of 5% of the input hCdt2 is shown (5% input). (C) Schematic representation of p21 mutants. CDK-cyclin and PCNA binding domains for untagged and GST-tagged p21 mutants are indicated. In the p21 point mutant F150A, phenylalanine is replaced by alanine at residue 150. Mutant Δ156-161 contains a deletion of residues 156 to 161, while other deletion mutants are named by the remaining residues of p21. (D) GST pulldown assay. (Top) GST or GST-p21 wild-type or mutant proteins were bound to glutathione-Sepharose beads, followed by incubation with 10 μl of HSS in NETN buffer. The bead fraction was analyzed by immunoblotting with anti-hPCNA antibody (Santa Cruz), and 0.5 μl HSS was included as an input control (5% input). (Bottom) GST or GST-p21 wild-type or mutant proteins were immobilized onto glutathione-Sepharose beads, followed by incubation with 35 S-hCDT2 and analysis by SDS-PAGE and phosphorimaging. The average percentage of hCdt2 bound (ave % CDT2 binding) was calculated using results from 2 independent experiments and was normalized to the level of hCdt2 binding to wild-type p21, which was set at 100%. (E) p21 ubiquitination and degradation assay. 35 S-labeled wild-type p21 was incubated in HSS supplemented with 2.5 μl XB − buffer, unprogrammed reticulocyte lysate (unprog; lysate programmed with vector DNA), or in vitro -translated hCdt2 with (+) or without (−) single-stranded DNA (ssDNA). Samples were analyzed at time points between 0 and 180 min as indicated. Ubiquitinated p21 species (Ub n ) are shown on the right, and molecular mass markers are shown in kilodaltons on the left. The percentage of p21 remaining at each time point was calculated as a percentage of the amount of p21 at the zero time point, which was normalized to 100%. (F) p21 ubiquitination assay. 35 S-labeled wild-type p21 (WT), the p21 F150A point mutant (F150A), or the p21 Δ156-161 deletion mutant (Δ156-161) was incubated in HSS supplemented with 2.5 μl in vitro -translated Xenopus Cdt2 (XCdt2) or human Cdt2 (hCdt2) as indicated in the presence (+) or absence (−) of single-stranded DNA (ssDNA), followed by analysis at 0 and 120 min. Ubiquitinated p21 species (Ub n ) are shown on the left, and molecular mass markers are shown in kilodaltons on the right.
Figure Legend Snippet: p21 is ubiquitinated during the events of DNA polymerase switching/elongation in the Xenopus egg extract. (A) Amino acid sequence similarity between Xenopus and human Cul4a, Cul4b, DDB1, Cdt2, and PCNA. Xenopus residue numbers are indicated at the bottom of the sequence alignments, and the percentages of similarity (S) and identity (I) between the Xenopus and human proteins are shown on the right. Xenopus Cul4a, the MGC115611 protein (gi 71679818), contains 200 additional residues in the N terminus compared to human Cul4a, so only residues 200 to 858 of Xenopus Cul4a were compared in the alignment. (B) GST pulldown assay. GST, GST-p21, or GST-p27 proteins were immobilized on glutathione-Sepharose beads and incubated with 35 S-hCDT2. A total of 5% of the input hCdt2 is shown (5% input). (C) Schematic representation of p21 mutants. CDK-cyclin and PCNA binding domains for untagged and GST-tagged p21 mutants are indicated. In the p21 point mutant F150A, phenylalanine is replaced by alanine at residue 150. Mutant Δ156-161 contains a deletion of residues 156 to 161, while other deletion mutants are named by the remaining residues of p21. (D) GST pulldown assay. (Top) GST or GST-p21 wild-type or mutant proteins were bound to glutathione-Sepharose beads, followed by incubation with 10 μl of HSS in NETN buffer. The bead fraction was analyzed by immunoblotting with anti-hPCNA antibody (Santa Cruz), and 0.5 μl HSS was included as an input control (5% input). (Bottom) GST or GST-p21 wild-type or mutant proteins were immobilized onto glutathione-Sepharose beads, followed by incubation with 35 S-hCDT2 and analysis by SDS-PAGE and phosphorimaging. The average percentage of hCdt2 bound (ave % CDT2 binding) was calculated using results from 2 independent experiments and was normalized to the level of hCdt2 binding to wild-type p21, which was set at 100%. (E) p21 ubiquitination and degradation assay. 35 S-labeled wild-type p21 was incubated in HSS supplemented with 2.5 μl XB − buffer, unprogrammed reticulocyte lysate (unprog; lysate programmed with vector DNA), or in vitro -translated hCdt2 with (+) or without (−) single-stranded DNA (ssDNA). Samples were analyzed at time points between 0 and 180 min as indicated. Ubiquitinated p21 species (Ub n ) are shown on the right, and molecular mass markers are shown in kilodaltons on the left. The percentage of p21 remaining at each time point was calculated as a percentage of the amount of p21 at the zero time point, which was normalized to 100%. (F) p21 ubiquitination assay. 35 S-labeled wild-type p21 (WT), the p21 F150A point mutant (F150A), or the p21 Δ156-161 deletion mutant (Δ156-161) was incubated in HSS supplemented with 2.5 μl in vitro -translated Xenopus Cdt2 (XCdt2) or human Cdt2 (hCdt2) as indicated in the presence (+) or absence (−) of single-stranded DNA (ssDNA), followed by analysis at 0 and 120 min. Ubiquitinated p21 species (Ub n ) are shown on the left, and molecular mass markers are shown in kilodaltons on the right.

Techniques Used: Sequencing, GST Pulldown Assay, Incubation, Binding Assay, Mutagenesis, SDS Page, Degradation Assay, Labeling, Plasmid Preparation, In Vitro, Ubiquitin Assay

Xic1 residues immediately upstream and downstream of its PCNA binding domain are important for Cdt2 binding. (A) Schematic representation of full-length Xic1 and Xic1 deletion mutants, with CDK/cyclin and PCNA binding domains indicated. Amino- or carboxy-terminal serial deletion mutants of Xic1 were in vitro -translated ( 35 S-Xic1) or bacterially expressed as GST-Xic1 fusion proteins (GST-Xic1). The Xic1 wild type (WT), point mutant I174A deficient for PCNA binding (I174A), CK − mutant deficient for CDK2-cyclin binding (CK − ), or Xic1 deletion mutants indicated by the residues contained within the mutant or deleted (Δ) within the mutant are shown. (B) Coimmunoprecipitation assay. Immunoprecipitated XCdt2 (anti-CDT2, IP) from the egg extract was incubated with the 35 S-Xic1 wild type (WT) or mutants as indicated. Equivalent immunoprecipitation of XCdt2 for each sample was confirmed by immunoblotting with anti-Cdt2 antibody (data not shown). Immunoprecipitation with normal rabbit serum (NRS) was conducted as a control, and 5% of the input 35 S-Xic1 is shown (5% input). (C) GST pulldown assay. GST or GST-Xic1 wild-type or mutant proteins as indicated were immobilized on glutathione-Sepharose beads and incubated with 35 S-CDT2. A total of 5% of the input XCdt2 for each reaction is shown (5% input). (D) Quantitation of the results shown in panels B and C. The relative XCdt2 binding value (% relative Cdt2 binding) for each Xic1 mutant is shown, where wild-type Xic1 (WT) binding was normalized to 100% for each experiment. Each sample was tested at least 2 or 3 times, and the standard error of the mean (SEM) is shown as an error bar for samples tested at least three times. IVT, in vitro transcribed.
Figure Legend Snippet: Xic1 residues immediately upstream and downstream of its PCNA binding domain are important for Cdt2 binding. (A) Schematic representation of full-length Xic1 and Xic1 deletion mutants, with CDK/cyclin and PCNA binding domains indicated. Amino- or carboxy-terminal serial deletion mutants of Xic1 were in vitro -translated ( 35 S-Xic1) or bacterially expressed as GST-Xic1 fusion proteins (GST-Xic1). The Xic1 wild type (WT), point mutant I174A deficient for PCNA binding (I174A), CK − mutant deficient for CDK2-cyclin binding (CK − ), or Xic1 deletion mutants indicated by the residues contained within the mutant or deleted (Δ) within the mutant are shown. (B) Coimmunoprecipitation assay. Immunoprecipitated XCdt2 (anti-CDT2, IP) from the egg extract was incubated with the 35 S-Xic1 wild type (WT) or mutants as indicated. Equivalent immunoprecipitation of XCdt2 for each sample was confirmed by immunoblotting with anti-Cdt2 antibody (data not shown). Immunoprecipitation with normal rabbit serum (NRS) was conducted as a control, and 5% of the input 35 S-Xic1 is shown (5% input). (C) GST pulldown assay. GST or GST-Xic1 wild-type or mutant proteins as indicated were immobilized on glutathione-Sepharose beads and incubated with 35 S-CDT2. A total of 5% of the input XCdt2 for each reaction is shown (5% input). (D) Quantitation of the results shown in panels B and C. The relative XCdt2 binding value (% relative Cdt2 binding) for each Xic1 mutant is shown, where wild-type Xic1 (WT) binding was normalized to 100% for each experiment. Each sample was tested at least 2 or 3 times, and the standard error of the mean (SEM) is shown as an error bar for samples tested at least three times. IVT, in vitro transcribed.

Techniques Used: Binding Assay, In Vitro, Mutagenesis, Co-Immunoprecipitation Assay, Immunoprecipitation, Incubation, GST Pulldown Assay, Quantitation Assay

15) Product Images from "MoImd4 mediates crosstalk between MoPdeH‐cAMP signalling and purine metabolism to govern growth and pathogenicity in Magnaporthe oryzae"

Article Title: MoImd4 mediates crosstalk between MoPdeH‐cAMP signalling and purine metabolism to govern growth and pathogenicity in Magnaporthe oryzae

Journal: Molecular Plant Pathology

doi: 10.1111/mpp.12770

T268, R269, D380, G382, R458, Y459, G403 and Y428 of MoImd4 are important for the promotion of MoPdeH enzymatic activity. (A) Glutathione‐ S ‐transferase (GST) pull‐down assays for interactions between GST‐MoPdeH and His‐MoImd4, His‐T268AR269A, His‐D380AG382A, His‐R458AY459A, His‐G403A and His‐Y428A alleles of MoImd4. Equal amounts of MoPdeH‐GST or GST protein were incubated with Glutathione Sepharose beads for 3 h at 4 °C prior to mixing with His infusion protein lysates for another 3 h at 4 °C. Equal His‐tag infusion proteins from input were used as controls. The eluted proteins were detected by western blot analysis with anti‐His and anti‐GST antibodies. The number represents the intensity of eluted proteins detected by the anti‐His antibody (top panel, elution protein; bottom panel, elution protein after the same dilution). The intensity of the elutions from wild‐type MoImd4 was set to 1.0. (B) Purified His‐fusion expression proteins affecting the enzyme activity of MoPdeH. Equal amounts of His‐fusion proteins and GST‐MoPdeH were mixed to measure the enzymatic activities. Fluorescence was read by a 10‐min kinetic reaction with excitation at 420 nm and emission at 450 nm. Experiments were repeated three times with similar results. The error bars indicate the standard deviations of three replicates. Different letters indicate statistically significant differences (Duncan’s new multiple range test, P
Figure Legend Snippet: T268, R269, D380, G382, R458, Y459, G403 and Y428 of MoImd4 are important for the promotion of MoPdeH enzymatic activity. (A) Glutathione‐ S ‐transferase (GST) pull‐down assays for interactions between GST‐MoPdeH and His‐MoImd4, His‐T268AR269A, His‐D380AG382A, His‐R458AY459A, His‐G403A and His‐Y428A alleles of MoImd4. Equal amounts of MoPdeH‐GST or GST protein were incubated with Glutathione Sepharose beads for 3 h at 4 °C prior to mixing with His infusion protein lysates for another 3 h at 4 °C. Equal His‐tag infusion proteins from input were used as controls. The eluted proteins were detected by western blot analysis with anti‐His and anti‐GST antibodies. The number represents the intensity of eluted proteins detected by the anti‐His antibody (top panel, elution protein; bottom panel, elution protein after the same dilution). The intensity of the elutions from wild‐type MoImd4 was set to 1.0. (B) Purified His‐fusion expression proteins affecting the enzyme activity of MoPdeH. Equal amounts of His‐fusion proteins and GST‐MoPdeH were mixed to measure the enzymatic activities. Fluorescence was read by a 10‐min kinetic reaction with excitation at 420 nm and emission at 450 nm. Experiments were repeated three times with similar results. The error bars indicate the standard deviations of three replicates. Different letters indicate statistically significant differences (Duncan’s new multiple range test, P

Techniques Used: Activity Assay, Incubation, Western Blot, Purification, Expressing, Fluorescence

16) Product Images from "Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers"

Article Title: Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers

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

doi: 10.1073/pnas.1820980116

( A ) BiFC assay in 5-d differentiated rat primary myotubes and HeLa cells. BiFC assay was performed on 5-d differentiated myotubes ( a – c ) or HeLa cells ( d – f ) expressing either pBiFC-VN173-JPH1TMD and pBiFC-VC155-JPH1TMD ( a and d ) or pBiFC-VN173-JPH1TMD and pBiFC-VC155-JPH4TMD ( b and e ) or pBiFC-VN173-JPH4TMD and pBiFC-VC155-JPH4TMD ( c and f ). Scale bar, 15 µm. ( B ) FRAP analysis on 12-d differentiated rat primary myotubes expressing GFP-JPH1 and GFP-JPH1 deletion mutants. FRAP analysis was performed on 12-d differentiated myotubes expressing either GFP-JPH1 or GFP-JPH1 deletion mutants. Data are expressed as percentage of mobile fraction ± SEM; n values are as follows: GFP-JPH1 ( n = 20); GFP-JPH1ΔMORN I-VIII ( n = 12), GFP-JPH1ΔTMD ( n = 7), and GFP-TMD-JPH1 ( n = 10). Asterisks indicate statistical significance compared with the mobile fraction of GFP-JPH1, as evaluated by the Kruskal–Wallis multiple comparisons test (** P ≤ 0.05; *** P ≤ 0.01). ( C ) Immunoprecipitation experiments on HEK293T cells coexpressing myc-JPH1 and GFP-JPH1 or myc-JPH1 and GFP-JPH2, or myc-JPH2 and GFP-JPH2. Total lysates from HEK293T cells were immunoprecipitated with anti-myc conjugated agarose beads. Immunocomplexes were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected by mouse monoclonal anti-myc or anti-GFP antibodies. The vertical black line at Bottom indicates that an unrelated lane was eliminated from the figure. ( D ) GST pull-down experiments on the microsomal fraction of mouse skeletal muscles and of HEK293T cells. A total of 500 µg of the microsomal fraction from mouse skeletal muscles or HEK293T cells expressing either GFP-JPH1 or GFP-JPH2 was used in GST pull-down experiments using GST-joining JPH1 or GST-joining JPH2 fusion proteins. Proteins were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected by specific antibodies. A total of 30 µg of solubilized microsomes was loaded as control.
Figure Legend Snippet: ( A ) BiFC assay in 5-d differentiated rat primary myotubes and HeLa cells. BiFC assay was performed on 5-d differentiated myotubes ( a – c ) or HeLa cells ( d – f ) expressing either pBiFC-VN173-JPH1TMD and pBiFC-VC155-JPH1TMD ( a and d ) or pBiFC-VN173-JPH1TMD and pBiFC-VC155-JPH4TMD ( b and e ) or pBiFC-VN173-JPH4TMD and pBiFC-VC155-JPH4TMD ( c and f ). Scale bar, 15 µm. ( B ) FRAP analysis on 12-d differentiated rat primary myotubes expressing GFP-JPH1 and GFP-JPH1 deletion mutants. FRAP analysis was performed on 12-d differentiated myotubes expressing either GFP-JPH1 or GFP-JPH1 deletion mutants. Data are expressed as percentage of mobile fraction ± SEM; n values are as follows: GFP-JPH1 ( n = 20); GFP-JPH1ΔMORN I-VIII ( n = 12), GFP-JPH1ΔTMD ( n = 7), and GFP-TMD-JPH1 ( n = 10). Asterisks indicate statistical significance compared with the mobile fraction of GFP-JPH1, as evaluated by the Kruskal–Wallis multiple comparisons test (** P ≤ 0.05; *** P ≤ 0.01). ( C ) Immunoprecipitation experiments on HEK293T cells coexpressing myc-JPH1 and GFP-JPH1 or myc-JPH1 and GFP-JPH2, or myc-JPH2 and GFP-JPH2. Total lysates from HEK293T cells were immunoprecipitated with anti-myc conjugated agarose beads. Immunocomplexes were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected by mouse monoclonal anti-myc or anti-GFP antibodies. The vertical black line at Bottom indicates that an unrelated lane was eliminated from the figure. ( D ) GST pull-down experiments on the microsomal fraction of mouse skeletal muscles and of HEK293T cells. A total of 500 µg of the microsomal fraction from mouse skeletal muscles or HEK293T cells expressing either GFP-JPH1 or GFP-JPH2 was used in GST pull-down experiments using GST-joining JPH1 or GST-joining JPH2 fusion proteins. Proteins were separated by SDS/PAGE, transferred to nitrocellulose membranes, and detected by specific antibodies. A total of 30 µg of solubilized microsomes was loaded as control.

Techniques Used: Bimolecular Fluorescence Complementation Assay, Expressing, Immunoprecipitation, SDS Page

17) Product Images from "Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation"

Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2018.02.011

α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
Figure Legend Snippet: α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

Techniques Used: Selection, Incubation, Binding Assay, Amplification, Polymerase Chain Reaction, Purification, Magnetic Beads, Sequencing

18) Product Images from "Involvement of Alpha-PAK-Interacting Exchange Factor in the PAK1-c-Jun NH2-Terminal Kinase 1 Activation and Apoptosis Induced by Benzo[a]pyrene"

Article Title: Involvement of Alpha-PAK-Interacting Exchange Factor in the PAK1-c-Jun NH2-Terminal Kinase 1 Activation and Apoptosis Induced by Benzo[a]pyrene

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.20.6796-6807.2001

αPIX and the JNK pathway kinases facilitate the apoptotic cell death induced by B(a)P. (A) αPIX (ΔCH) and PAK1 (T423E) accelerate B(a)P-induced apoptotic cell death. HeLa cells were cotransfected with an eGFP expression vector and test plasmids as indicated. At 24 h posttransfection, the cells were placed in medium with 0.5% FBS and incubated for a further 12 h. They were then treated with 10 μ M B(a)P for 24 h or left untreated and were subjected to analysis. (Top) Quantitation of apoptotic cell death by using nuclear morphology. (Center) DNA integrity of transfected cells. Total DNA from the transfected samples was isolated, and an equal amount of DNA from each was separated on a 1.5% agarose gel. (Bottom) Expression levels of myc-tagged αPIX and PAK1, HA-tagged SEK1, and Flag-tagged Bcl-2 proteins as determined by immunoblot analysis. (B) αPIX (ΔSH3), PAK1 (K299R) and SEK1 (AL) inhibit apoptotic cell death induced by B(a)P. HeLa cells were cotransfected with an eGFP expression vector and test plasmids as indicated. At 36 h posttransfection, the cells were treated with 10μ M B(a)P for 36 h and subjected to analysis. (C) SEK1 (AL) blocks apoptosis accelerated by αPIX (ΔCH) in cells treated with B(a)P. HeLa cells were cotransfected with an eGFP expression vector and plasmids encoding myc-tagged αPIX (ΔCH) and HA-tagged SEK1 (AL) or Flag-tagged Bcl-2 as indicated. The total amount of transfected DNA was made constant by adding vector pCS2+ DNA. At 36 h posttransfection, the cells were treated with 10 μM B(a)P for 24 h and subjected to analysis. (D) The caspase inhibitor Z-Asp-CH2-DCB inhibits apoptosis accelerated by αPIX (Δ CH) in cells treated with B(a)P. HeLa cells were cotransfected with an eGFP expression vector and plasmids encoding myc-tagged αPIX (Δ CH) or mock vector as indicated. At 36 h posttransfection, the cells were preincubated with Z-Asp-CH2-DCB (50 μM) or vehicle (DMSO) for 90 min and treated with 10 μ M B(a)P for 24 h. Then the cells were harvested and subjected to analysis. Similar results were obtained in three independent experiments. The data shown are the mean and standard deviation for three independent experiments. SEK1 (AL), SEK1 (K220A, K224L).
Figure Legend Snippet: αPIX and the JNK pathway kinases facilitate the apoptotic cell death induced by B(a)P. (A) αPIX (ΔCH) and PAK1 (T423E) accelerate B(a)P-induced apoptotic cell death. HeLa cells were cotransfected with an eGFP expression vector and test plasmids as indicated. At 24 h posttransfection, the cells were placed in medium with 0.5% FBS and incubated for a further 12 h. They were then treated with 10 μ M B(a)P for 24 h or left untreated and were subjected to analysis. (Top) Quantitation of apoptotic cell death by using nuclear morphology. (Center) DNA integrity of transfected cells. Total DNA from the transfected samples was isolated, and an equal amount of DNA from each was separated on a 1.5% agarose gel. (Bottom) Expression levels of myc-tagged αPIX and PAK1, HA-tagged SEK1, and Flag-tagged Bcl-2 proteins as determined by immunoblot analysis. (B) αPIX (ΔSH3), PAK1 (K299R) and SEK1 (AL) inhibit apoptotic cell death induced by B(a)P. HeLa cells were cotransfected with an eGFP expression vector and test plasmids as indicated. At 36 h posttransfection, the cells were treated with 10μ M B(a)P for 36 h and subjected to analysis. (C) SEK1 (AL) blocks apoptosis accelerated by αPIX (ΔCH) in cells treated with B(a)P. HeLa cells were cotransfected with an eGFP expression vector and plasmids encoding myc-tagged αPIX (ΔCH) and HA-tagged SEK1 (AL) or Flag-tagged Bcl-2 as indicated. The total amount of transfected DNA was made constant by adding vector pCS2+ DNA. At 36 h posttransfection, the cells were treated with 10 μM B(a)P for 24 h and subjected to analysis. (D) The caspase inhibitor Z-Asp-CH2-DCB inhibits apoptosis accelerated by αPIX (Δ CH) in cells treated with B(a)P. HeLa cells were cotransfected with an eGFP expression vector and plasmids encoding myc-tagged αPIX (Δ CH) or mock vector as indicated. At 36 h posttransfection, the cells were preincubated with Z-Asp-CH2-DCB (50 μM) or vehicle (DMSO) for 90 min and treated with 10 μ M B(a)P for 24 h. Then the cells were harvested and subjected to analysis. Similar results were obtained in three independent experiments. The data shown are the mean and standard deviation for three independent experiments. SEK1 (AL), SEK1 (K220A, K224L).

Techniques Used: Expressing, Plasmid Preparation, Incubation, Quantitation Assay, Transfection, Isolation, Agarose Gel Electrophoresis, Standard Deviation

19) Product Images from "The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases"

Article Title: The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201705068

TRAPPII complex activates the Rab GTPases Rab1 and Rab11, whereas TRAPPIII only shows GEF activity toward Rab1. (A) Coomassie blue–stained protein gels of recombinant Drosophila TRAPP complexes purified from Sf9 cells coexpressing the subunits of each complex. FLAG tags on C10 or C11 allowed isolation of TRAPPII or TRAPPIII, respectively. PreScission protease (GST-HRV-3C protease) was used to cleave the tags (asterisks) and was subsequently removed using glutathione Sepharose beads. C10 also copurified with C9 in the absence of the shared subunits (TRAPPII lane). The Hsc70 chaperone (CG4264) is a contaminant of the TRAPPII purification protocol. Molecular masses are given in kilodaltons. (B) Release of mant-GDP from 250 nM of Rab-His 6 by 50 nM of TRAPPII or TRAPPIII in the presence or absence of synthetic fly Golgi mix liposomes. Traces are the mean of at least three experiments. Error bars show SEM.
Figure Legend Snippet: TRAPPII complex activates the Rab GTPases Rab1 and Rab11, whereas TRAPPIII only shows GEF activity toward Rab1. (A) Coomassie blue–stained protein gels of recombinant Drosophila TRAPP complexes purified from Sf9 cells coexpressing the subunits of each complex. FLAG tags on C10 or C11 allowed isolation of TRAPPII or TRAPPIII, respectively. PreScission protease (GST-HRV-3C protease) was used to cleave the tags (asterisks) and was subsequently removed using glutathione Sepharose beads. C10 also copurified with C9 in the absence of the shared subunits (TRAPPII lane). The Hsc70 chaperone (CG4264) is a contaminant of the TRAPPII purification protocol. Molecular masses are given in kilodaltons. (B) Release of mant-GDP from 250 nM of Rab-His 6 by 50 nM of TRAPPII or TRAPPIII in the presence or absence of synthetic fly Golgi mix liposomes. Traces are the mean of at least three experiments. Error bars show SEM.

Techniques Used: Activity Assay, Staining, Recombinant, Purification, Isolation

20) Product Images from "Cupidin, an Isoform of Homer/Vesl, Interacts with the Actin Cytoskeleton and Activated Rho Family Small GTPases and Is Expressed in Developing Mouse Cerebellar Granule Cells"

Article Title: Cupidin, an Isoform of Homer/Vesl, Interacts with the Actin Cytoskeleton and Activated Rho Family Small GTPases and Is Expressed in Developing Mouse Cerebellar Granule Cells

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.19-19-08389.1999

Cupidin interacts with F-actin and mGluR1α. A , F-actin binding to Cupidin in a cosedimentation assay. GST, GST-CPD/N, and GST-CPD/C were incubated with (+) or without (−) F-actin prepared from chicken skeletal muscles and then centrifuged. Equivalent protein amounts of the supernatant ( S ) and pellet ( P ) fractions were separated by SDS-PAGE and stained with Coomassie Brilliant Blue ( CBB ). A representative result of five independent experiments is shown here. The arrows indicate the position of actin, and the arrowheads represent each GST-fusion protein. B , Binding of Cupidin to mGluR1α in a pull down assay. The S1 fraction prepared from P7 mouse cerebellum or cerebrum was incubated with GST-CPD/N protein and then immobilized onto glutathione-Sepharose beads. After extensive washing, GST-CPD/N-bound proteins were extracted with SDS-PAGE sample buffer and were analyzed by Western blotting using an anti-mGluR1α polyclonal antibody. Lane 1 , Input (the same amounts of lysates used for the assay were loaded); lane 2 , eluate from a GST-bound column; lane 3 , eluate from a GST-CPD/N bound column. C , Coimmunoprecipitation of both mGluR1 and actin from P7 mouse cerebellar lysates using the anti-CPD antibody. The immunoprecipitates obtained with either the preimmune serum ( lane 2 ) or the affinity-purified anti-CPD polyclonal antibody were examined by Western blotting with the indicated antibodies (anti-CPD, anti-actin, and anti-mGluR1α). Lane 1 is the detergent extract of P7 mouse cerebellum. The arrow indicates Cupidin signal, and the asterisk indicates the heavy chain of IgG. D , Primary-cultured mouse cerebellar granule neurons at 7 DIV were triple-stained with an anti-CPD polyclonal antibody (FITC; a ), Texas Red-phalloidin ( b ), and an anti-synaptophysin monoclonal antibody (Cy5; c ) and observed by confocal microscopy. d is a superimposed composite images of a–c using three pseudocolors ( green for CPD, red for phalloidin, and blue for synaptophysin). Arrows indicate representative positions at which the three pseudocolors overlapped. Scale bar, 10 μm.
Figure Legend Snippet: Cupidin interacts with F-actin and mGluR1α. A , F-actin binding to Cupidin in a cosedimentation assay. GST, GST-CPD/N, and GST-CPD/C were incubated with (+) or without (−) F-actin prepared from chicken skeletal muscles and then centrifuged. Equivalent protein amounts of the supernatant ( S ) and pellet ( P ) fractions were separated by SDS-PAGE and stained with Coomassie Brilliant Blue ( CBB ). A representative result of five independent experiments is shown here. The arrows indicate the position of actin, and the arrowheads represent each GST-fusion protein. B , Binding of Cupidin to mGluR1α in a pull down assay. The S1 fraction prepared from P7 mouse cerebellum or cerebrum was incubated with GST-CPD/N protein and then immobilized onto glutathione-Sepharose beads. After extensive washing, GST-CPD/N-bound proteins were extracted with SDS-PAGE sample buffer and were analyzed by Western blotting using an anti-mGluR1α polyclonal antibody. Lane 1 , Input (the same amounts of lysates used for the assay were loaded); lane 2 , eluate from a GST-bound column; lane 3 , eluate from a GST-CPD/N bound column. C , Coimmunoprecipitation of both mGluR1 and actin from P7 mouse cerebellar lysates using the anti-CPD antibody. The immunoprecipitates obtained with either the preimmune serum ( lane 2 ) or the affinity-purified anti-CPD polyclonal antibody were examined by Western blotting with the indicated antibodies (anti-CPD, anti-actin, and anti-mGluR1α). Lane 1 is the detergent extract of P7 mouse cerebellum. The arrow indicates Cupidin signal, and the asterisk indicates the heavy chain of IgG. D , Primary-cultured mouse cerebellar granule neurons at 7 DIV were triple-stained with an anti-CPD polyclonal antibody (FITC; a ), Texas Red-phalloidin ( b ), and an anti-synaptophysin monoclonal antibody (Cy5; c ) and observed by confocal microscopy. d is a superimposed composite images of a–c using three pseudocolors ( green for CPD, red for phalloidin, and blue for synaptophysin). Arrows indicate representative positions at which the three pseudocolors overlapped. Scale bar, 10 μm.

Techniques Used: Binding Assay, Incubation, SDS Page, Staining, Pull Down Assay, Western Blot, Affinity Purification, Cell Culture, Confocal Microscopy

21) Product Images from "Determinants at the N- and C-termini of G?12 required for activation of Rho-mediated signaling"

Article Title: Determinants at the N- and C-termini of G?12 required for activation of Rho-mediated signaling

Journal: Journal of Molecular Signaling

doi: 10.1186/1750-2187-8-3

In vitro interaction of Gα 12 mutants with LARG. Immunoblot results for all LARG binding-impaired Gα 12 cassette mutants and selected other mutants are shown. HEK293 cells were transfected with the indicated plasmids (7.0 μg per 10-cm plate) and lysates were prepared for co-precipitation assays as described in Methods. Prior to this step, 5% of each lysate was set aside as starting material ( load ). Pulldown experiments were performed on 7–9 mutants per experiment, plus myc-Gα 12 QL as a positive control, using equal amounts of GST-LARG-RH ( LARG ) immobilized on glutathione-sepharose. Immobilized GST was utilized in parallel as a negative control. For all experimental samples, 20% of the volume was analyzed by SDS-PAGE and Coomassie blue staining to verify equal amounts of GST-LARG-RH and GST proteins in the precipitates (data not shown). Immunoblots displayed in this figure are representative of at least three trials per cassette mutant, except for mutants A - D , F - H , V , and KKK that showed minimal impairment in LARG binding after two trials. ( Inset) Coomassie blue analysis of GST-fusion constructs expressed in bacteria and immobilized on glutathione-sepharose: GST-LARG-RH ( LARG ), GST-p115-RH ( p115 ), and GST alone. Molecular weight standards (in kDa) are indicated at right.
Figure Legend Snippet: In vitro interaction of Gα 12 mutants with LARG. Immunoblot results for all LARG binding-impaired Gα 12 cassette mutants and selected other mutants are shown. HEK293 cells were transfected with the indicated plasmids (7.0 μg per 10-cm plate) and lysates were prepared for co-precipitation assays as described in Methods. Prior to this step, 5% of each lysate was set aside as starting material ( load ). Pulldown experiments were performed on 7–9 mutants per experiment, plus myc-Gα 12 QL as a positive control, using equal amounts of GST-LARG-RH ( LARG ) immobilized on glutathione-sepharose. Immobilized GST was utilized in parallel as a negative control. For all experimental samples, 20% of the volume was analyzed by SDS-PAGE and Coomassie blue staining to verify equal amounts of GST-LARG-RH and GST proteins in the precipitates (data not shown). Immunoblots displayed in this figure are representative of at least three trials per cassette mutant, except for mutants A - D , F - H , V , and KKK that showed minimal impairment in LARG binding after two trials. ( Inset) Coomassie blue analysis of GST-fusion constructs expressed in bacteria and immobilized on glutathione-sepharose: GST-LARG-RH ( LARG ), GST-p115-RH ( p115 ), and GST alone. Molecular weight standards (in kDa) are indicated at right.

Techniques Used: In Vitro, Binding Assay, Transfection, Positive Control, Negative Control, SDS Page, Staining, Western Blot, Mutagenesis, Construct, Molecular Weight

22) Product Images from "Multivalent interaction of ESCO2 with the replication machinery is required for cohesion"

Article Title: Multivalent interaction of ESCO2 with the replication machinery is required for cohesion

Journal: bioRxiv

doi: 10.1101/666115

PCNA interacts with ESCO2 motifs in vivo and in vitro . A. ESCO2N-GFP fusions . A cartoon depicting constructs in which the N terminal 375 a.a. of ESCO2 are fused directly to eGFP. Motifs A, B, and C were deleted independently, as shown; numbers indicate the number of amino acids deleted (not drawn to scale). B. Localization to replication foci . Confocal images of U2OS cells co-transfected with Ruby-PCNA and the GFP-fusion constructs. Colocalization is indicated in yellow in the merge image, as in Figure 2 . C. Box C is critical for PCNA recruitment by tethered ESCO2 . mCherry-lacI-ESCO2 (full-length) fusions with the deletions indicated in panel A were co-expressed with GFP-tagged PCNA as in Figure 2 , and the colocalization at nuclear foci was scored by fluorescence intensity profile analysis as in Figure 2 . D. MCM4 recruitment to tethered ESCO2 is dependent upon Box A . The experiment in Panel C was repeated, only in this case the ESCO2 constructs were co-expressed with mEmerald-MCM4. Recruitment to tethered ESCO2 was scored as in Figure 2 . E. Pull down assay using GST fusion proteins . Short peptide sequences including Box A, Box B, Box C, or the ESCO2 PIP box motifs were expressed as GST-fusion proteins. A parallel set was made in which alanine substitutions were made at the invariant amino acids (shown in red). The PIP-box from p21 was used as a positive control. F. Co-precipitation from cell free extracts . GST fusion proteins shown in panel A were mixed with Xenopus egg extract and incubated with glutathione sepharose beads. The beads were washed and bound proteins were eluted and probed for PCNA by immunoblot. A duplicate gel was stained with Coomassie dye to detect the GST fusion proteins. G. Co-precipitation of purified proteins . The indicated GST fusion proteins (panel E) were mixed with purified recombinant PCNA, pulled down with glutathione agarose beads and analyzed as in F for PCNA.
Figure Legend Snippet: PCNA interacts with ESCO2 motifs in vivo and in vitro . A. ESCO2N-GFP fusions . A cartoon depicting constructs in which the N terminal 375 a.a. of ESCO2 are fused directly to eGFP. Motifs A, B, and C were deleted independently, as shown; numbers indicate the number of amino acids deleted (not drawn to scale). B. Localization to replication foci . Confocal images of U2OS cells co-transfected with Ruby-PCNA and the GFP-fusion constructs. Colocalization is indicated in yellow in the merge image, as in Figure 2 . C. Box C is critical for PCNA recruitment by tethered ESCO2 . mCherry-lacI-ESCO2 (full-length) fusions with the deletions indicated in panel A were co-expressed with GFP-tagged PCNA as in Figure 2 , and the colocalization at nuclear foci was scored by fluorescence intensity profile analysis as in Figure 2 . D. MCM4 recruitment to tethered ESCO2 is dependent upon Box A . The experiment in Panel C was repeated, only in this case the ESCO2 constructs were co-expressed with mEmerald-MCM4. Recruitment to tethered ESCO2 was scored as in Figure 2 . E. Pull down assay using GST fusion proteins . Short peptide sequences including Box A, Box B, Box C, or the ESCO2 PIP box motifs were expressed as GST-fusion proteins. A parallel set was made in which alanine substitutions were made at the invariant amino acids (shown in red). The PIP-box from p21 was used as a positive control. F. Co-precipitation from cell free extracts . GST fusion proteins shown in panel A were mixed with Xenopus egg extract and incubated with glutathione sepharose beads. The beads were washed and bound proteins were eluted and probed for PCNA by immunoblot. A duplicate gel was stained with Coomassie dye to detect the GST fusion proteins. G. Co-precipitation of purified proteins . The indicated GST fusion proteins (panel E) were mixed with purified recombinant PCNA, pulled down with glutathione agarose beads and analyzed as in F for PCNA.

Techniques Used: In Vivo, In Vitro, Construct, Transfection, Fluorescence, Pull Down Assay, Positive Control, Incubation, Staining, Purification, Recombinant

23) Product Images from "The DUSP26 phosphatase activator adenylate kinase 2 regulates FADD phosphorylation and cell growth"

Article Title: The DUSP26 phosphatase activator adenylate kinase 2 regulates FADD phosphorylation and cell growth

Journal: Nature Communications

doi: 10.1038/ncomms4351

Isolation of DUSP26 as a FADD phosphatase and an AK2-binding partner. ( a ) HEK293T cells were co-transfected with p-FADD-HA and each phosphatase cDNA for 24 h, cell extracts were subjected to western blotting using anti-p-FADD and anti-FADD antibodies and to phosphatase assays with pNPP. Bars represent mean±s.d. ( n =3). ( b ) HEK293T cells were co-transfected with FADD-HA and pcDNA3, Flag-DUSP26 or Flag-DUSP26 C152S for 24 h, and cell extracts were then analysed by western blotting. ( c ) HeLa cells were transfected with pSuper, pDUSP26 or pDUSP26 shRNA for 36 h, after which cell extracts were subjected to western blotting using anti-p-FADD, anti-FADD and anti-α-tubulin antibodies. Total RNAs were purified and analysed with RT–PCR using DUSP26- or GAPDH-specific synthetic oligonucleotides as primers. ( d ) HEK293T cells were transfected with p3 × Flag or p3 × Flag-DUSP26 for 36 h, and cell extracts were prepared and subjected to the pull-down assay using anti-FLAG agarose-beads. The bound proteins were resolved by SDS–PAGE, stained with Coomassie-blue, and analysed by LC-MS/MS or analysed by western blotting using the indicated antibodies. NS indicates non-specific signal. ( e ) HEK293T cells were co-transfected with pAK2-HA and pFlag-DUSP26 for 36 h and cell extracts were subjected to immunoprecipitation (IP) assay using anti-HA or anti-Flag antibody. Whole-cell lysates and the immunoprecipitates were probed by western blotting with anti-FLAG or anti-HA antibody. ( f ) HeLa cell extracts were subjected to IP analysis using anti-AK2 or anti-DUSP26 antibodies and then the immunoprecipitates were analysed by western blotting with the indicated antibodies. ( g ) AK2 over-expression enhances the binding of DUSP26 to FADD. HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either GFP or AK2-GFP for 36 h, after which cells extracts were subjected to IP analysis using anti-HA antibody. Expression levels of Flag-DUSP26, FADD-HA, GFP and AK2-GFP in whole-cell lysates were examined by western blotting with the indicated antibodies. ( h ) HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either pSuper or AK2 shRNA for 36 h. Cell lysates were subjected to IP analysis with anti-HA antibody, and expression levels of Flag-DUSP26, FADD-HA and AK2 in whole-cell lysates were examined by western blotting.
Figure Legend Snippet: Isolation of DUSP26 as a FADD phosphatase and an AK2-binding partner. ( a ) HEK293T cells were co-transfected with p-FADD-HA and each phosphatase cDNA for 24 h, cell extracts were subjected to western blotting using anti-p-FADD and anti-FADD antibodies and to phosphatase assays with pNPP. Bars represent mean±s.d. ( n =3). ( b ) HEK293T cells were co-transfected with FADD-HA and pcDNA3, Flag-DUSP26 or Flag-DUSP26 C152S for 24 h, and cell extracts were then analysed by western blotting. ( c ) HeLa cells were transfected with pSuper, pDUSP26 or pDUSP26 shRNA for 36 h, after which cell extracts were subjected to western blotting using anti-p-FADD, anti-FADD and anti-α-tubulin antibodies. Total RNAs were purified and analysed with RT–PCR using DUSP26- or GAPDH-specific synthetic oligonucleotides as primers. ( d ) HEK293T cells were transfected with p3 × Flag or p3 × Flag-DUSP26 for 36 h, and cell extracts were prepared and subjected to the pull-down assay using anti-FLAG agarose-beads. The bound proteins were resolved by SDS–PAGE, stained with Coomassie-blue, and analysed by LC-MS/MS or analysed by western blotting using the indicated antibodies. NS indicates non-specific signal. ( e ) HEK293T cells were co-transfected with pAK2-HA and pFlag-DUSP26 for 36 h and cell extracts were subjected to immunoprecipitation (IP) assay using anti-HA or anti-Flag antibody. Whole-cell lysates and the immunoprecipitates were probed by western blotting with anti-FLAG or anti-HA antibody. ( f ) HeLa cell extracts were subjected to IP analysis using anti-AK2 or anti-DUSP26 antibodies and then the immunoprecipitates were analysed by western blotting with the indicated antibodies. ( g ) AK2 over-expression enhances the binding of DUSP26 to FADD. HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either GFP or AK2-GFP for 36 h, after which cells extracts were subjected to IP analysis using anti-HA antibody. Expression levels of Flag-DUSP26, FADD-HA, GFP and AK2-GFP in whole-cell lysates were examined by western blotting with the indicated antibodies. ( h ) HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either pSuper or AK2 shRNA for 36 h. Cell lysates were subjected to IP analysis with anti-HA antibody, and expression levels of Flag-DUSP26, FADD-HA and AK2 in whole-cell lysates were examined by western blotting.

Techniques Used: Isolation, Binding Assay, Transfection, Western Blot, shRNA, Purification, Reverse Transcription Polymerase Chain Reaction, Pull Down Assay, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunoprecipitation, Over Expression, Expressing

24) Product Images from "Trs20 is Required for TRAPP II Assembly"

Article Title: Trs20 is Required for TRAPP II Assembly

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/tra.12065

TRAPP purified from trs20ts mutant cells does not contain TRAPP II and does not act as a Ypt32 GEF A. The protein level of Trs120-myc is significantly lower in purified TRAPP complexes, but not in lysates, from trs20ts when compared to wild type cells. GST-Bet5 and GST, as a negative control, were over-expressed in wild type (NSY1471) and trs20ts (NSY1472) mutant cells also expressing endogenously tagged Trs120-myc. Cells grown to mid-log phase were either left at 26° or shifted to 37° for 70 minutes and then harvested. Cell lysates were prepared and GST-Bet5 complexes were purified on glutathione sepharose resin. The level of Trs120-myc was determined in lysates (top) and pull-downs (bottom) using immuno-blot analysis; G6PDH level was used as a loading control for lysates; GST-Bet5 and GST levels are used for the pull down yield. B. The protein level of Trs130-HA is significantly lower in lysates and TRAPP complexes purified from trs20ts when compared to wild type cells. Same as in panel A, except that cells were expressing endogenously tagged Trs130-HA, and the pull-down of the TRAPP I/II subunit Bet3 was verified using anti-Bet3 antibodies. The partial degradation of over-expressed GST-Bet5 in trs20ts mutant cells in likely due to the instability of TRAPP complexes in these cells. For panels A and B, the level of Trs120-myc or Trs130-HA was quantified and shown under the immuno-blots as percent of wild type cells. Protein level in lysates was corrected for the loading control, while in pull downs it was corrected for the full-length GST-Bet5; +/− represents SEM; P values are shown on the right (values
Figure Legend Snippet: TRAPP purified from trs20ts mutant cells does not contain TRAPP II and does not act as a Ypt32 GEF A. The protein level of Trs120-myc is significantly lower in purified TRAPP complexes, but not in lysates, from trs20ts when compared to wild type cells. GST-Bet5 and GST, as a negative control, were over-expressed in wild type (NSY1471) and trs20ts (NSY1472) mutant cells also expressing endogenously tagged Trs120-myc. Cells grown to mid-log phase were either left at 26° or shifted to 37° for 70 minutes and then harvested. Cell lysates were prepared and GST-Bet5 complexes were purified on glutathione sepharose resin. The level of Trs120-myc was determined in lysates (top) and pull-downs (bottom) using immuno-blot analysis; G6PDH level was used as a loading control for lysates; GST-Bet5 and GST levels are used for the pull down yield. B. The protein level of Trs130-HA is significantly lower in lysates and TRAPP complexes purified from trs20ts when compared to wild type cells. Same as in panel A, except that cells were expressing endogenously tagged Trs130-HA, and the pull-down of the TRAPP I/II subunit Bet3 was verified using anti-Bet3 antibodies. The partial degradation of over-expressed GST-Bet5 in trs20ts mutant cells in likely due to the instability of TRAPP complexes in these cells. For panels A and B, the level of Trs120-myc or Trs130-HA was quantified and shown under the immuno-blots as percent of wild type cells. Protein level in lysates was corrected for the loading control, while in pull downs it was corrected for the full-length GST-Bet5; +/− represents SEM; P values are shown on the right (values

Techniques Used: Purification, Mutagenesis, Activated Clotting Time Assay, Negative Control, Expressing, Western Blot

Trs20 is required for interaction of Trs120 with recombinant TRAPP I A. Pull down of His 6 -Trs120 with GST-Bet5 purified complexes. GST was pulled down, using glutathione sepharose resin, from lysates of bacteria expressing core TRAPP I (GST-Bet5, Trs23-S, Trs31-myc, and Bet3-MBP), core TRAPP I plus Trs33 (His 6 -Trs33), core TRAPP I plus Trs20 (Trs20-HA), or core TRAPP I plus Trs20 and Trs33. Cleared lysate (S100) from different bacterial cells expressing His 6 -Trs120 was then incubated with the resin and the level of proteins associated with the resin after precipitation was determined using immuno-blot analysis and antibodies against the tags (Trs120, Bet5, Trs33, Trs20) or the protein (Bet3). Trs120 co-purifies with GST-Bet5 complex ( > 5%), but not with GST, and only in the presence of Trs20. More Trs120 co-purifies with TRAPP when Trs33 is present ( > 10%). The expression levels of the different proteins in lysates are shown on the left (10% input for the Trs120 lysate). The full anti-His 6 . B. Pull down of His 6 -Trs120 with Bet3-MBP purified complexes. Binding of His 6 -Trs120 TRAPP I was determined as in part A (using the same lysates), except that amylose resin was used to pull down Bet3-MBP within the core TRAPP I complex. Trs120 co-purifies with TRAPP I only in the presence of Trs20 ( > 5%), and this level is higher in the presence of Trs33 ( > 10%). C. Pull down of core TRAPP I with His 6 -Trs120. Lysates from bacteria expressing His 6 -Trs120, or empty plasmid (θ) as a negative control, were purified on Ni 2+ resin. The resin was then incubated with lysates from cells expressing either core TRAPP I, or core TRAPP I plus Trs20-HA. The level of proteins associated with the resin after precipitation was determined using immuno-blot analysis and antibodies against the tags. TRAPP I, scored by the level of Trs31-myc, co-purified with Trs120, but not with the empty plasmid control, and only in the presence of Trs20. In A–C, asterisks indicate the tagged protein being bound directly to the resin. Results in this figure are representative of at least two independent experiments.
Figure Legend Snippet: Trs20 is required for interaction of Trs120 with recombinant TRAPP I A. Pull down of His 6 -Trs120 with GST-Bet5 purified complexes. GST was pulled down, using glutathione sepharose resin, from lysates of bacteria expressing core TRAPP I (GST-Bet5, Trs23-S, Trs31-myc, and Bet3-MBP), core TRAPP I plus Trs33 (His 6 -Trs33), core TRAPP I plus Trs20 (Trs20-HA), or core TRAPP I plus Trs20 and Trs33. Cleared lysate (S100) from different bacterial cells expressing His 6 -Trs120 was then incubated with the resin and the level of proteins associated with the resin after precipitation was determined using immuno-blot analysis and antibodies against the tags (Trs120, Bet5, Trs33, Trs20) or the protein (Bet3). Trs120 co-purifies with GST-Bet5 complex ( > 5%), but not with GST, and only in the presence of Trs20. More Trs120 co-purifies with TRAPP when Trs33 is present ( > 10%). The expression levels of the different proteins in lysates are shown on the left (10% input for the Trs120 lysate). The full anti-His 6 . B. Pull down of His 6 -Trs120 with Bet3-MBP purified complexes. Binding of His 6 -Trs120 TRAPP I was determined as in part A (using the same lysates), except that amylose resin was used to pull down Bet3-MBP within the core TRAPP I complex. Trs120 co-purifies with TRAPP I only in the presence of Trs20 ( > 5%), and this level is higher in the presence of Trs33 ( > 10%). C. Pull down of core TRAPP I with His 6 -Trs120. Lysates from bacteria expressing His 6 -Trs120, or empty plasmid (θ) as a negative control, were purified on Ni 2+ resin. The resin was then incubated with lysates from cells expressing either core TRAPP I, or core TRAPP I plus Trs20-HA. The level of proteins associated with the resin after precipitation was determined using immuno-blot analysis and antibodies against the tags. TRAPP I, scored by the level of Trs31-myc, co-purified with Trs120, but not with the empty plasmid control, and only in the presence of Trs20. In A–C, asterisks indicate the tagged protein being bound directly to the resin. Results in this figure are representative of at least two independent experiments.

Techniques Used: Recombinant, Purification, Expressing, Incubation, Binding Assay, Plasmid Preparation, Negative Control

25) Product Images from "Three SAUR proteins SAUR76, SAUR77 and SAUR78 promote plant growth in Arabidopsis"

Article Title: Three SAUR proteins SAUR76, SAUR77 and SAUR78 promote plant growth in Arabidopsis

Journal: Scientific Reports

doi: 10.1038/srep12477

Interaction of SAUR76-78 with ETR2 and their co-localization analysis. ( a ) Expressions of GST-SAUR fusion proteins. Arrows indicate positions of the corresponding GST-SAURs. GST was also noted as a degradation product. Numbers on the left indicate protein size markers. kD: kilodalton. ( b ) SAUR76-78 physically interact with ETR2 and EIN4 by GST pulldown. Upper panel: Each of the GST-SAURs can pulldown [ 35 S]-labeled ETR2 and EIN4. GST was used as a negative control. Lower panel: loading of the proteins by western analysis using anti-GST antibody. ( c ) Interaction of SAUR78 and SAUR76 with ETR2 by co-immunoprecipitation (Co-IP). Co-IP was performed with agarose beads conjugated with anti-Myc monoclonal antibody. The presence of the Flag-SAUR78, Flag-SAUR76 or Myc-ETR2 in the immunocomplex was detected with the anti-Flag or anti-Myc antibody by Western blotting. ( d ) Bimolecular fluorescence complementation (BiFC) assay. The Agrobacteria GV3101 haboring each of the two plasmids were co-infiltrated into tobacco leaves ( Nicotiana Benthamiana ). The samples were observed 48 h later under a confocal microscope. YFP fluorescence was excited at a wavelength of 488 nm. Bars indicate 25 μm. ( e ) Co-localization analysis of SAUR78 with ETR2. pGWB405-ETR2-GFP and pGWB454-SAUR78-RFP were transfected into Agrobacteria EHA105 and co-infiltrated into tobacco leaves. After infection for 3 d, fluorescence was observed under a confocal microscope. Bars indicate 25 μm.
Figure Legend Snippet: Interaction of SAUR76-78 with ETR2 and their co-localization analysis. ( a ) Expressions of GST-SAUR fusion proteins. Arrows indicate positions of the corresponding GST-SAURs. GST was also noted as a degradation product. Numbers on the left indicate protein size markers. kD: kilodalton. ( b ) SAUR76-78 physically interact with ETR2 and EIN4 by GST pulldown. Upper panel: Each of the GST-SAURs can pulldown [ 35 S]-labeled ETR2 and EIN4. GST was used as a negative control. Lower panel: loading of the proteins by western analysis using anti-GST antibody. ( c ) Interaction of SAUR78 and SAUR76 with ETR2 by co-immunoprecipitation (Co-IP). Co-IP was performed with agarose beads conjugated with anti-Myc monoclonal antibody. The presence of the Flag-SAUR78, Flag-SAUR76 or Myc-ETR2 in the immunocomplex was detected with the anti-Flag or anti-Myc antibody by Western blotting. ( d ) Bimolecular fluorescence complementation (BiFC) assay. The Agrobacteria GV3101 haboring each of the two plasmids were co-infiltrated into tobacco leaves ( Nicotiana Benthamiana ). The samples were observed 48 h later under a confocal microscope. YFP fluorescence was excited at a wavelength of 488 nm. Bars indicate 25 μm. ( e ) Co-localization analysis of SAUR78 with ETR2. pGWB405-ETR2-GFP and pGWB454-SAUR78-RFP were transfected into Agrobacteria EHA105 and co-infiltrated into tobacco leaves. After infection for 3 d, fluorescence was observed under a confocal microscope. Bars indicate 25 μm.

Techniques Used: Labeling, Negative Control, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Bimolecular Fluorescence Complementation Assay, Microscopy, Fluorescence, Transfection, Infection

26) Product Images from "TCS1, a Microtubule-Binding Protein, Interacts with KCBP/ZWICHEL to Regulate Trichome Cell Shape in Arabidopsis thaliana"

Article Title: TCS1, a Microtubule-Binding Protein, Interacts with KCBP/ZWICHEL to Regulate Trichome Cell Shape in Arabidopsis thaliana

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1006266

TCS1 physically and genetically interacts with KCBP to control the number of trichome branches. (A) TCS1 interacts with KCBP in yeast cells. (B) TCS1 physically interacts with KCBP in vitro . MBP-TCS1 was pulled down (PD) by GST-KCBP immobilized on Glutathione Sepharose 4B and analyzed by immunoblotting (IB) using an anti-MBP antibody. MBP was used as a negative control. (C) TCS1 interacts with KCBP in vivo . Total proteins from pTCS1 : TCS1-GFP;35SMyc-KCBP and 35S : GFP; 35SMyc-KCBP plants were immunoprecipitated with GFP-Trap-A (IP), and the immunoblots (IB) were probed with anti-GFP and anti-Myc antibodies, respectively. Myc-KCBP was detected in the immunoprecipitated TCS1-GFP complex. (D) Trichome branch (br) distribution of Col-0, tcs1-2 , zwi-101 and zwi-101 tcs1-2 first pair of leaves at 15 days after germination (DAG). Values are given as mean ± SE. (E) The average number of Col-0, tcs1-2 , zwi-101 , zwi-101 tcs1-2 trichome branches treated with (T) or without (UT) 20 μM oryzalin for 2 hours. The branch number of Col-0, tcs1-2 , zwi-101 , zwi-101 tcs1-2 trichomes was examined after a 10-day recovery on ½ MS medium.
Figure Legend Snippet: TCS1 physically and genetically interacts with KCBP to control the number of trichome branches. (A) TCS1 interacts with KCBP in yeast cells. (B) TCS1 physically interacts with KCBP in vitro . MBP-TCS1 was pulled down (PD) by GST-KCBP immobilized on Glutathione Sepharose 4B and analyzed by immunoblotting (IB) using an anti-MBP antibody. MBP was used as a negative control. (C) TCS1 interacts with KCBP in vivo . Total proteins from pTCS1 : TCS1-GFP;35SMyc-KCBP and 35S : GFP; 35SMyc-KCBP plants were immunoprecipitated with GFP-Trap-A (IP), and the immunoblots (IB) were probed with anti-GFP and anti-Myc antibodies, respectively. Myc-KCBP was detected in the immunoprecipitated TCS1-GFP complex. (D) Trichome branch (br) distribution of Col-0, tcs1-2 , zwi-101 and zwi-101 tcs1-2 first pair of leaves at 15 days after germination (DAG). Values are given as mean ± SE. (E) The average number of Col-0, tcs1-2 , zwi-101 , zwi-101 tcs1-2 trichome branches treated with (T) or without (UT) 20 μM oryzalin for 2 hours. The branch number of Col-0, tcs1-2 , zwi-101 , zwi-101 tcs1-2 trichomes was examined after a 10-day recovery on ½ MS medium.

Techniques Used: In Vitro, Negative Control, In Vivo, Immunoprecipitation, Western Blot, Mass Spectrometry

27) Product Images from "Hrs recruits clathrin to early endosomes"

Article Title: Hrs recruits clathrin to early endosomes

Journal: The EMBO Journal

doi: 10.1093/emboj/20.17.5008

Fig. 3. The C-terminus of Hrs binds clathrin TD. ( A ). This illustrates the existence of a potential clathrin-binding motif within residues 770–775 of Hrs. Hrs is the only protein that has the clathrin box motif at the very C-terminus. (B–E) Interaction of Hrs with clathrin. ( B and C ) L40 reporter yeast cells were transformed with bait constructs in pLexA and prey constructs in pGAD. Reporter β-galactosidase activities (in arbitrary units) indicate binding and are represented as mean values of two independent experiments performed in duplicate. Error bars denote ± SEM. In (B), a clathrin triskelion (consisting of three heavy chains) is illustrated, with the terminal domain (TD), distal domain (DD) and hub domain (HD) indicated. ( D ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3) were immobilized on glutathione–Sepharose beads and incubated with pig brain cytosol. The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin with anti-clathrin heavy chain antibodies (upper panel). ( E ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3), were immobilized on glutathione–Sepharose beads and incubated with purified recombinant clathrin terminal domain (TD 1–579 ). The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin-TD 1–579 with anti-clathrin heavy chain (upper panel). Lane 4 represents the total amount of recombinant clathrin-TD 1–579 added to the beads.
Figure Legend Snippet: Fig. 3. The C-terminus of Hrs binds clathrin TD. ( A ). This illustrates the existence of a potential clathrin-binding motif within residues 770–775 of Hrs. Hrs is the only protein that has the clathrin box motif at the very C-terminus. (B–E) Interaction of Hrs with clathrin. ( B and C ) L40 reporter yeast cells were transformed with bait constructs in pLexA and prey constructs in pGAD. Reporter β-galactosidase activities (in arbitrary units) indicate binding and are represented as mean values of two independent experiments performed in duplicate. Error bars denote ± SEM. In (B), a clathrin triskelion (consisting of three heavy chains) is illustrated, with the terminal domain (TD), distal domain (DD) and hub domain (HD) indicated. ( D ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3) were immobilized on glutathione–Sepharose beads and incubated with pig brain cytosol. The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin with anti-clathrin heavy chain antibodies (upper panel). ( E ) Recombinant GST (lane 1), GST–Hrs 707–775 (lane 2) or GST–Hrs 707–770 (lane 3), were immobilized on glutathione–Sepharose beads and incubated with purified recombinant clathrin terminal domain (TD 1–579 ). The beads were recovered by centrifugation and washed. Pellet fractions were resolved by SDS–PAGE and transferred to nitrocellulose. The blot was stained with Ponceau S (lower panel) prior to detection of clathrin-TD 1–579 with anti-clathrin heavy chain (upper panel). Lane 4 represents the total amount of recombinant clathrin-TD 1–579 added to the beads.

Techniques Used: Binding Assay, Transformation Assay, Construct, Recombinant, Incubation, Centrifugation, SDS Page, Staining, Purification

28) Product Images from "NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2"

Article Title: NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2

Journal: EMBO Reports

doi: 10.15252/embr.201540505

NAT 10 interacts with p53 and Mdm2 U2 OS cells were transfected with Flag‐ NAT 10 or control vectors. Forty‐eight hours later, cells were harvested and whole‐cell extracts were immunoprecipitated with Flag antibody affinity resin. The NAT 10‐binding proteins were resolved by SDS – PAGE and detected by silver staining. U2 OS , HCT 116 p53 +/+ , or HCT 116 p53 −/− cell lysates were immunoprecipitated with control IgG, anti‐ NAT 10 (B and F), anti‐p53 (C), and anti‐Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies. Purified NAT 10 was incubated with GST , GST ‐p53, or GST ‐Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by Western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel. Full‐length GST ‐p53 fusion protein, its deletion mutants, or GST protein was used in pull‐down experiments with purified NAT 10 protein. The levels of the GST fusion proteins are shown in the left panel. GST pull‐down assay was performed using purified GST ‐ NAT 10 deletion mutants or GST protein and overexpressed Flag‐p53 or Mdm2 protein in HEK 293T cells. Schematic diagram represents the constructs of GST ‐ NAT 10 deletion mutants (right panel).
Figure Legend Snippet: NAT 10 interacts with p53 and Mdm2 U2 OS cells were transfected with Flag‐ NAT 10 or control vectors. Forty‐eight hours later, cells were harvested and whole‐cell extracts were immunoprecipitated with Flag antibody affinity resin. The NAT 10‐binding proteins were resolved by SDS – PAGE and detected by silver staining. U2 OS , HCT 116 p53 +/+ , or HCT 116 p53 −/− cell lysates were immunoprecipitated with control IgG, anti‐ NAT 10 (B and F), anti‐p53 (C), and anti‐Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies. Purified NAT 10 was incubated with GST , GST ‐p53, or GST ‐Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by Western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel. Full‐length GST ‐p53 fusion protein, its deletion mutants, or GST protein was used in pull‐down experiments with purified NAT 10 protein. The levels of the GST fusion proteins are shown in the left panel. GST pull‐down assay was performed using purified GST ‐ NAT 10 deletion mutants or GST protein and overexpressed Flag‐p53 or Mdm2 protein in HEK 293T cells. Schematic diagram represents the constructs of GST ‐ NAT 10 deletion mutants (right panel).

Techniques Used: Transfection, Immunoprecipitation, Binding Assay, SDS Page, Silver Staining, Purification, Incubation, Western Blot, Pull Down Assay, Construct

29) Product Images from "Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis"

Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis

Journal: Cell Death and Differentiation

doi: 10.1038/s41418-017-0037-8

Direct physical interaction between TRF1 and Speedy A a Coomassie blue-stained gels showing the purification of relevant proteins. b Direct physical interaction between TRF1 and Speedy A were determined by GST pull-down assays. GST-TRF1 or GST conjugated sepharose beads were used to pull down His-FLAG-Speedy A. Bound proteins were detected by immunoblotting analysis with anti-FLAG and anti-GST antibodies. c No direct physical interaction between TRF1 and Cdk2, which was determined by GST pull-down assays. GST-TRF1 or GST conjugated sepharose beads were used to pull down His-MYC-Cdk2. Bound proteins were detected by immunoblotting analysis with anti-MYC and anti-GST antibodies. d Direct physical interaction between CDK2 and Speedy A was determined by GST pull-down assays. GST-CDK2 or GST conjugated sepharose beads were used to pull down purified His-FLAG-Speedy A. Bound proteins were detected by immunoblotting analysis with anti-FLAG and anti-GST antibodies. e Speedy A-mediated interaction between TRF1 and Cdk2. GST-TRF1 or GST conjugated sepharose beads were used to pull down His-FLAG-Speedy A and (or) His-MYC-Cdk2. Bound proteins were detected by immunoblotting analysis with anti-FLAG or anti-MYC antibody. Asterisks indicate the bands of Speedy A. Input for His-FLAG-Speedy A and His-MYC-Cdk2 were 10% and 1%, respectively. f TRF1 and Cdk2 do not compete for interaction with Speedy A. GST-TRF1 or GST conjugated sepharose beads were used to pull down His-FLAG-Speedy A in the presence of increasing amounts of His-MYC-Cdk2. Bound proteins were detected by immunoblotting analysis with anti-FLAG and anti-MYC antibodies. Asterisks indicate signals of Speedy A.
Figure Legend Snippet: Direct physical interaction between TRF1 and Speedy A a Coomassie blue-stained gels showing the purification of relevant proteins. b Direct physical interaction between TRF1 and Speedy A were determined by GST pull-down assays. GST-TRF1 or GST conjugated sepharose beads were used to pull down His-FLAG-Speedy A. Bound proteins were detected by immunoblotting analysis with anti-FLAG and anti-GST antibodies. c No direct physical interaction between TRF1 and Cdk2, which was determined by GST pull-down assays. GST-TRF1 or GST conjugated sepharose beads were used to pull down His-MYC-Cdk2. Bound proteins were detected by immunoblotting analysis with anti-MYC and anti-GST antibodies. d Direct physical interaction between CDK2 and Speedy A was determined by GST pull-down assays. GST-CDK2 or GST conjugated sepharose beads were used to pull down purified His-FLAG-Speedy A. Bound proteins were detected by immunoblotting analysis with anti-FLAG and anti-GST antibodies. e Speedy A-mediated interaction between TRF1 and Cdk2. GST-TRF1 or GST conjugated sepharose beads were used to pull down His-FLAG-Speedy A and (or) His-MYC-Cdk2. Bound proteins were detected by immunoblotting analysis with anti-FLAG or anti-MYC antibody. Asterisks indicate the bands of Speedy A. Input for His-FLAG-Speedy A and His-MYC-Cdk2 were 10% and 1%, respectively. f TRF1 and Cdk2 do not compete for interaction with Speedy A. GST-TRF1 or GST conjugated sepharose beads were used to pull down His-FLAG-Speedy A in the presence of increasing amounts of His-MYC-Cdk2. Bound proteins were detected by immunoblotting analysis with anti-FLAG and anti-MYC antibodies. Asterisks indicate signals of Speedy A.

Techniques Used: Staining, Purification

30) Product Images from "Subnuclear domain proteins in cancer cells support the functions of RUNX2 in the DNA damage response"

Article Title: Subnuclear domain proteins in cancer cells support the functions of RUNX2 in the DNA damage response

Journal: Journal of Cell Science

doi: 10.1242/jcs.160051

Interaction of RUNX2 with RUVBL2, INTS3 and BAZ1B. (A) The functional domains of RUNX2, RUVBL2, INTS3 and BAZ1B, and the location of peptide sequences identified by mass spectrometry. The peptide fragment identified by mass spectrometry is indicated as a closed bar. Functional domains in each peptide are indicated. RHD, Runt homolog domain; NMTS, nuclear matrix targeting sequences; AAA, ATPase associated with a variety of cellular activities; KD, kinase domain; DDT, DNA binding homeobox and different transcription factors; PHD, plant homeodomain; BRD, bromodomain. (B) Co-immunoprecipitation of RUNX2 with interacting proteins was analyzed by western blotting. To detect RUNX2–RUVBL2, RUNX2–INTS3 or RUNX2–BAZ1B endogenous interactions, 5 mg of whole-cell lysates from Saos2 or U2OS cells were immunoprecipitated (IP) with 5 µg of anti-RUNX2 antibodies or 5 µg of normal rabbit IgG as a negative control. Immunoprecipitation products were then analyzed by western blotting, using anti-RUVBL2, anti-INTS3 or anti-BAZ1B antibodies. Note that no clear immunoprecipitation products were seen using anti-INTS3 antibodies and the results are not shown. (C) Co-immunoprecipitation of FLAG–RUVBL2 protein with full-length RUNX2 [wildtype (WT), amino acids 1–528] or C-terminally deleted mutant (ΔC, amino acids 1–376). U2OS cells were transiently co-transfected with a FLAG–RUVBL2 expression construct and either full-length or C-terminally deleted RUNX2 construct. Whole-cell lysates were incubated with anti-FLAG M2 agarose beads (Sigma). Washed beads were subjected to SDS-PAGE and analyzed by western blotting (WB) using specific antibodies against the indicated proteins. Asterisks (*) mark bands caused by nonspecific interactions. (D) Bacterially expressed GST (‘G’), GST fused to the Runt homolog domain of RUNX2 (amino acids 107–241; GST-R) or GST fused to the C-terminus of RUNX2 (amino acids 240–528; GST-C) proteins were immobilized on glutathione beads and incubated with whole-cell lysates from Saos2 cells. After extensive washing, proteins bound to the beads were eluted in protein sample buffer and analyzed by western blotting with antibodies against the indicated proteins.
Figure Legend Snippet: Interaction of RUNX2 with RUVBL2, INTS3 and BAZ1B. (A) The functional domains of RUNX2, RUVBL2, INTS3 and BAZ1B, and the location of peptide sequences identified by mass spectrometry. The peptide fragment identified by mass spectrometry is indicated as a closed bar. Functional domains in each peptide are indicated. RHD, Runt homolog domain; NMTS, nuclear matrix targeting sequences; AAA, ATPase associated with a variety of cellular activities; KD, kinase domain; DDT, DNA binding homeobox and different transcription factors; PHD, plant homeodomain; BRD, bromodomain. (B) Co-immunoprecipitation of RUNX2 with interacting proteins was analyzed by western blotting. To detect RUNX2–RUVBL2, RUNX2–INTS3 or RUNX2–BAZ1B endogenous interactions, 5 mg of whole-cell lysates from Saos2 or U2OS cells were immunoprecipitated (IP) with 5 µg of anti-RUNX2 antibodies or 5 µg of normal rabbit IgG as a negative control. Immunoprecipitation products were then analyzed by western blotting, using anti-RUVBL2, anti-INTS3 or anti-BAZ1B antibodies. Note that no clear immunoprecipitation products were seen using anti-INTS3 antibodies and the results are not shown. (C) Co-immunoprecipitation of FLAG–RUVBL2 protein with full-length RUNX2 [wildtype (WT), amino acids 1–528] or C-terminally deleted mutant (ΔC, amino acids 1–376). U2OS cells were transiently co-transfected with a FLAG–RUVBL2 expression construct and either full-length or C-terminally deleted RUNX2 construct. Whole-cell lysates were incubated with anti-FLAG M2 agarose beads (Sigma). Washed beads were subjected to SDS-PAGE and analyzed by western blotting (WB) using specific antibodies against the indicated proteins. Asterisks (*) mark bands caused by nonspecific interactions. (D) Bacterially expressed GST (‘G’), GST fused to the Runt homolog domain of RUNX2 (amino acids 107–241; GST-R) or GST fused to the C-terminus of RUNX2 (amino acids 240–528; GST-C) proteins were immobilized on glutathione beads and incubated with whole-cell lysates from Saos2 cells. After extensive washing, proteins bound to the beads were eluted in protein sample buffer and analyzed by western blotting with antibodies against the indicated proteins.

Techniques Used: Functional Assay, Mass Spectrometry, Binding Assay, Immunoprecipitation, Western Blot, Negative Control, Mutagenesis, Transfection, Expressing, Construct, Incubation, SDS Page

31) Product Images from "Post-transcriptional regulation of thioredoxin by the stress inducible heterogenous ribonucleoprotein A18"

Article Title: Post-transcriptional regulation of thioredoxin by the stress inducible heterogenous ribonucleoprotein A18

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkj519

hnRNP A18 is associated with ribosomes. Sucrose gradient analysis was performed as described in the text on RKO cells exposed or not (Control) to 14 J m −2 UV radiation. The fractions were collected from the bottom of the tubes and their proteins and RNA contents were analyzed. Western blot analysis was used to measure the levels of hnRNP A18 (A18), ribosomal protein S6 (rpS6) and GAPDH. The levels of TRX and GAPDH transcripts were evaluated by RT–PCR. Total amounts of RNA were evaluated by agarose gels. The positions of the tRNA, 18 and 28S ribosomal RNA are indicated. The absence of ribosomal RNA in the first fraction corresponds to the non-ribosomal fraction (Unb:unbound). The fractions 2 and 3 contain the Monoribosomes (Mono.) and the low and high molecular weight polysomes are in the fractions 4 to 10.
Figure Legend Snippet: hnRNP A18 is associated with ribosomes. Sucrose gradient analysis was performed as described in the text on RKO cells exposed or not (Control) to 14 J m −2 UV radiation. The fractions were collected from the bottom of the tubes and their proteins and RNA contents were analyzed. Western blot analysis was used to measure the levels of hnRNP A18 (A18), ribosomal protein S6 (rpS6) and GAPDH. The levels of TRX and GAPDH transcripts were evaluated by RT–PCR. Total amounts of RNA were evaluated by agarose gels. The positions of the tRNA, 18 and 28S ribosomal RNA are indicated. The absence of ribosomal RNA in the first fraction corresponds to the non-ribosomal fraction (Unb:unbound). The fractions 2 and 3 contain the Monoribosomes (Mono.) and the low and high molecular weight polysomes are in the fractions 4 to 10.

Techniques Used: Western Blot, Reverse Transcription Polymerase Chain Reaction, Molecular Weight

hnRNP A18 binds to TRX 3′-UTR in solution. ( A ) Schematic representation of the different probes used. FL; full-length, nt; nucleotides. ( B ) RNA band shift was performed as described in the text. Increasing amounts (0.12 to 4 µg) of GST–hnRNP A18 was incubated with the indicated labeled TRX probe and run on a 1% agarose gel. ( C ) hnRNP A18 RBD and RGG domain are required for maximal RNA binding. Northwestern analysis was performed with increasing amounts (0.5 to 2 µg) of either full-length (FL) GST–hnRNP A18 (lanes 8–10), GST–hnRNP A18 RGG domain (RGG; lanes 2–4), GST–hnRNP A18 RBD (lanes 5–7) or GST alone (lane 1) and labeled TRX 3′-UTR as described in the text.
Figure Legend Snippet: hnRNP A18 binds to TRX 3′-UTR in solution. ( A ) Schematic representation of the different probes used. FL; full-length, nt; nucleotides. ( B ) RNA band shift was performed as described in the text. Increasing amounts (0.12 to 4 µg) of GST–hnRNP A18 was incubated with the indicated labeled TRX probe and run on a 1% agarose gel. ( C ) hnRNP A18 RBD and RGG domain are required for maximal RNA binding. Northwestern analysis was performed with increasing amounts (0.5 to 2 µg) of either full-length (FL) GST–hnRNP A18 (lanes 8–10), GST–hnRNP A18 RGG domain (RGG; lanes 2–4), GST–hnRNP A18 RBD (lanes 5–7) or GST alone (lane 1) and labeled TRX 3′-UTR as described in the text.

Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Labeling, Agarose Gel Electrophoresis, RNA Binding Assay

32) Product Images from "H2A.Z Is Required for Global Chromatin Integrity and for Recruitment of RNA Polymerase II under Specific Conditions"

Article Title: H2A.Z Is Required for Global Chromatin Integrity and for Recruitment of RNA Polymerase II under Specific Conditions

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.18.6270-6279.2001

The C-terminal region of H2A.Z interacts with components of the transcriptional machinery. (A) Aligned amino acid sequences of yeast H2A.Z and H2A using BLAST (National Center for Biotechnology Information). Boxed areas represent the C-terminal regions that were fused to GST for the experiments illustrated in panels B and C (red and white) and the M6 region in Drosophila ). (B) GST, GST-H2A (aa 96 to 132), and GST-Z (GST-H2A.Z [aa 103 to 134]) proteins bound to glutathione-Sepharose beads were incubated with a chromatin-enriched yeast extract. L, 2% input of the mixture; S, 2% of the supernatant after pelleting the Sepharose beads; P, washed Sepharose pellet. Samples were analyzed by SDS-PAGE followed by immunoblotting with either an anti-RNA polII antibody or an anti-TBP antibody. (C) The H2A.Z-RNA polII interaction is not mediated by the indirect bridging effect of nucleic acids. The chromatin-enriched extract was treated with or without DNase and RNase and then loaded in a 500-μl glutathione-Sepharose column coupled to GST-H2A.Z (aa 103 to 134). The column was washed and eluted with potassium acetate. L, input of the total reaction; E1 and E2, elutions. Sup-40K is an extract not enriched in chromatin; Pel-40K −DNase is a chromatin-enriched extract not treated with nucleases; Pel-40K +DNase represents the chromatin-enriched extract treated with nucleases. Samples were analyzed as for panel B with an anti-RNA polII antibody.
Figure Legend Snippet: The C-terminal region of H2A.Z interacts with components of the transcriptional machinery. (A) Aligned amino acid sequences of yeast H2A.Z and H2A using BLAST (National Center for Biotechnology Information). Boxed areas represent the C-terminal regions that were fused to GST for the experiments illustrated in panels B and C (red and white) and the M6 region in Drosophila ). (B) GST, GST-H2A (aa 96 to 132), and GST-Z (GST-H2A.Z [aa 103 to 134]) proteins bound to glutathione-Sepharose beads were incubated with a chromatin-enriched yeast extract. L, 2% input of the mixture; S, 2% of the supernatant after pelleting the Sepharose beads; P, washed Sepharose pellet. Samples were analyzed by SDS-PAGE followed by immunoblotting with either an anti-RNA polII antibody or an anti-TBP antibody. (C) The H2A.Z-RNA polII interaction is not mediated by the indirect bridging effect of nucleic acids. The chromatin-enriched extract was treated with or without DNase and RNase and then loaded in a 500-μl glutathione-Sepharose column coupled to GST-H2A.Z (aa 103 to 134). The column was washed and eluted with potassium acetate. L, input of the total reaction; E1 and E2, elutions. Sup-40K is an extract not enriched in chromatin; Pel-40K −DNase is a chromatin-enriched extract not treated with nucleases; Pel-40K +DNase represents the chromatin-enriched extract treated with nucleases. Samples were analyzed as for panel B with an anti-RNA polII antibody.

Techniques Used: Incubation, SDS Page

33) Product Images from "Autophosphorylation of a Bacterial Serine/Threonine Kinase, AfsK, Is Inhibited by KbpA, an AfsK-Binding Protein"

Article Title: Autophosphorylation of a Bacterial Serine/Threonine Kinase, AfsK, Is Inhibited by KbpA, an AfsK-Binding Protein

Journal: Journal of Bacteriology

doi: 10.1128/JB.183.19.5506-5512.2001

Interaction of KbpA and AfsK. (A) The pull-down of GST-AfsK with glutathione-Sepharose coprecipitated TRX-KbpA, which was detected with S protein by Western blotting (immunoblotting [IB]) (lane 4). TRX-KbpA was not recovered when GST or GST-AfsR was used. The pull-down of GST-AfsR (132 kDa), GST-AfsK (110 kDa), and GST (28 kDa) with glutathione-Sepharose was apparent by Western blotting with the antibody for GST (α-GST). The small protein found in lanes 5 and 6 is a degradation product derived from GST-AfsR. (B) Autophosphorylation of TRX-KΔC wt (51 kDa) and phosphorylation of GST-AfsR by TRX-KΔC wt . TRX-KΔC wt (5 μg) was incubated at 30°C for 10 min in the presence of [γ- 32 P]ATP, subjected to SDS-PAGE, and analyzed by autoradiography. GST-AfsR (3 μg) in the reaction mixture was also phosphorylated. Neither autophosphorylation nor phosphorylation of GST-AfsR occurred for TRX-KΔC K44A . (C) SDS-PAGE of the TRX-KΔC proteins labeled in vivo. Coomassie brilliant blue (CBB) staining revealed smeared bands for TRX-KΔC wt , as indicated by an asterisk, which represent phosphorylated forms of TRX-KΔC wt , as found by autoradiography. (D) The pull-down of GST-KbpA (54 kDa) with glutathione-Sepharose coprecipitated TRX-KΔC wt (lane 3) and TRX-KΔC K44A (lane 4) without recovering smeared, phosphorylated forms of TRX-KΔC wt . The small protein recovered by anti-GST antibody in lanes 3 and 4 is a degradation product. GST itself did not pull down the TRX-KΔC proteins (lanes 1 and 2). TRX-KΔC wt gave smeared bands (lane 5), but TRX-KΔC K44A did not (lane 6).
Figure Legend Snippet: Interaction of KbpA and AfsK. (A) The pull-down of GST-AfsK with glutathione-Sepharose coprecipitated TRX-KbpA, which was detected with S protein by Western blotting (immunoblotting [IB]) (lane 4). TRX-KbpA was not recovered when GST or GST-AfsR was used. The pull-down of GST-AfsR (132 kDa), GST-AfsK (110 kDa), and GST (28 kDa) with glutathione-Sepharose was apparent by Western blotting with the antibody for GST (α-GST). The small protein found in lanes 5 and 6 is a degradation product derived from GST-AfsR. (B) Autophosphorylation of TRX-KΔC wt (51 kDa) and phosphorylation of GST-AfsR by TRX-KΔC wt . TRX-KΔC wt (5 μg) was incubated at 30°C for 10 min in the presence of [γ- 32 P]ATP, subjected to SDS-PAGE, and analyzed by autoradiography. GST-AfsR (3 μg) in the reaction mixture was also phosphorylated. Neither autophosphorylation nor phosphorylation of GST-AfsR occurred for TRX-KΔC K44A . (C) SDS-PAGE of the TRX-KΔC proteins labeled in vivo. Coomassie brilliant blue (CBB) staining revealed smeared bands for TRX-KΔC wt , as indicated by an asterisk, which represent phosphorylated forms of TRX-KΔC wt , as found by autoradiography. (D) The pull-down of GST-KbpA (54 kDa) with glutathione-Sepharose coprecipitated TRX-KΔC wt (lane 3) and TRX-KΔC K44A (lane 4) without recovering smeared, phosphorylated forms of TRX-KΔC wt . The small protein recovered by anti-GST antibody in lanes 3 and 4 is a degradation product. GST itself did not pull down the TRX-KΔC proteins (lanes 1 and 2). TRX-KΔC wt gave smeared bands (lane 5), but TRX-KΔC K44A did not (lane 6).

Techniques Used: Western Blot, Derivative Assay, Incubation, SDS Page, Autoradiography, Labeling, In Vivo, Staining

34) Product Images from "IKKα controls ATG16L1 degradation to prevent ER stress during inflammation"

Article Title: IKKα controls ATG16L1 degradation to prevent ER stress during inflammation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20161867

IKKα phosphorylates ATG16L1. (A) Coimmunoprecipitation (Co-IP) and immunoblot (IB) analysis showing interaction of FLAG-IKKα and mCherry-ATG16L1. HEK293T cells were transfected as indicated, and lysates were subjected to coimmunoprecipitation assays. Immunoblots from 40-µg input were used to examine protein expression levels. (B) Coimmunoprecipitation of endogenous IKKα and ATG16L1 in WT colonic organoids. 600 µg of protein lysates from colon organoids were immunoprecipitated with ATG16L1 or control IgG. The immunoprecipitates and 40 µg of input lysates were analyzed by immunoblot analysis using the indicated antibodies. Data are representative of two experiments. (C) HEK293T cells were transfected with FLAG-IKKα, and whole-cell extracts were subjected to a pull down assay using various forms of GST-ATG16L1 and GST-Sepharose beads. The bead-bound proteins were analyzed by immunoblot analysis using anti-FLAG antibody. Data are representative of two experiments. (D) Schematic representation of the full-length ATG16L1 and different construct domains used for the expression of GST-fusion proteins. (E) HEK293T cells were transfected with FLAG-IKKα Wt or FLAG-IKKα AA . After 48 h, the cells were collected and lysed, and whole-cell extracts were subjected to immunoprecipitation using protein A/G plus agarose beads and anti-FLAG antibody. Then, immunoprecipitates were subjected to in vitro kinase assay with GST-ATG16L1(231–352). The FLAG immunoblot shows equal amounts of immunopurified FLAG-IKKα Wt or FLAG-IKKα AA . Phosphorylation of GST-ATG16L1(231–352) was confirmed by mass spectrometry. Data are representative of three experiments. (F) Mass spectrometric fragment ion scan of the peptide corresponding to phosphorylated serine 278 in ATG16L1. Data are representative of two experiments. (G) In vitro kinase assay of FLAG-IKKα Wt overexpressing HEK293T cells and different point-mutated GST-ATG16L1(231–352) substrates. Phosphorylated GST-ATG16L1(231–352) was visualized by autoradiography. Ponceau S staining shows the equal amounts of GST-ATG16L1 substrates. Data are representative of two experiments. EV, empty vector. (H) Endogenous IKK complex was immunoprecipitated using anti-IKKγ from untreated or 20 ng/ml TNF–treated (15 min) colonic organoids isolated from Ikkα Wt/Wt and Ikkα AA/AA mice and then subjected to in vitro kinase assay using GST-ATG16L1(231–352) as a substrate. Phosphorylated GST-ATG16L1(231–352) was visualized by autoradiography. Ponceau S staining and immunoblotting against IKKα and IKKβ show the equal amount of GST-ATG16L1 and immunoprecipitation efficiency, respectively. Data are representative of two experiments. (I) ATG16L1 proteolytic cleavage was assessed by immunoblot analysis in HeLa cells expressing mCherry-ATG16L1 Wt and mutants. HeLa cells were pretreated for 1 h with DMSO or 10 µM pan-caspase inhibitor (zVADfmk), followed by TNF stimulation (20 ng/ml) in the presence of 10 µg/ml cycloheximide (CHX) for 3 h. (J) Caspase 3–mediated in vitro ATG16L1 cleavage was assessed by immunoblot analysis. mCherry-ATG16L1 Wt and mutants were immunoprecipitated from HEK293T cells. Then, immunoprecipitates were subjected to in vitro cleavage assay using recombinant active caspase 3. Data are representative of two experiments. (I and J) CL, cleaved; FL, full length. (K) ATG16L1 proteolytic cleavage was assessed by immunoblot analysis in colonic organoids. Organoids were pretreated with DMSO or pan-caspase inhibitor (zVADfmk), followed by stimulation with NOD ligands, 20 µg/ml L-18MDP, and 20 µg/ml C12-iE-DAP, in the presence of 10 µg/ml cycloheximide for 3.5 h. Data are representative of two experiments. cl., cleaved. (L) Endogenous IKK complex was immunoprecipitated from untreated or L18-MDP–treated (20 µg/ml for 15 min) colonic organoids and then subjected to in vitro kinase assay as described in H. Data are representative of two experiments. (G, H, and L) Single asterisks indicate nonspecific signal determined by mass spectrometry.
Figure Legend Snippet: IKKα phosphorylates ATG16L1. (A) Coimmunoprecipitation (Co-IP) and immunoblot (IB) analysis showing interaction of FLAG-IKKα and mCherry-ATG16L1. HEK293T cells were transfected as indicated, and lysates were subjected to coimmunoprecipitation assays. Immunoblots from 40-µg input were used to examine protein expression levels. (B) Coimmunoprecipitation of endogenous IKKα and ATG16L1 in WT colonic organoids. 600 µg of protein lysates from colon organoids were immunoprecipitated with ATG16L1 or control IgG. The immunoprecipitates and 40 µg of input lysates were analyzed by immunoblot analysis using the indicated antibodies. Data are representative of two experiments. (C) HEK293T cells were transfected with FLAG-IKKα, and whole-cell extracts were subjected to a pull down assay using various forms of GST-ATG16L1 and GST-Sepharose beads. The bead-bound proteins were analyzed by immunoblot analysis using anti-FLAG antibody. Data are representative of two experiments. (D) Schematic representation of the full-length ATG16L1 and different construct domains used for the expression of GST-fusion proteins. (E) HEK293T cells were transfected with FLAG-IKKα Wt or FLAG-IKKα AA . After 48 h, the cells were collected and lysed, and whole-cell extracts were subjected to immunoprecipitation using protein A/G plus agarose beads and anti-FLAG antibody. Then, immunoprecipitates were subjected to in vitro kinase assay with GST-ATG16L1(231–352). The FLAG immunoblot shows equal amounts of immunopurified FLAG-IKKα Wt or FLAG-IKKα AA . Phosphorylation of GST-ATG16L1(231–352) was confirmed by mass spectrometry. Data are representative of three experiments. (F) Mass spectrometric fragment ion scan of the peptide corresponding to phosphorylated serine 278 in ATG16L1. Data are representative of two experiments. (G) In vitro kinase assay of FLAG-IKKα Wt overexpressing HEK293T cells and different point-mutated GST-ATG16L1(231–352) substrates. Phosphorylated GST-ATG16L1(231–352) was visualized by autoradiography. Ponceau S staining shows the equal amounts of GST-ATG16L1 substrates. Data are representative of two experiments. EV, empty vector. (H) Endogenous IKK complex was immunoprecipitated using anti-IKKγ from untreated or 20 ng/ml TNF–treated (15 min) colonic organoids isolated from Ikkα Wt/Wt and Ikkα AA/AA mice and then subjected to in vitro kinase assay using GST-ATG16L1(231–352) as a substrate. Phosphorylated GST-ATG16L1(231–352) was visualized by autoradiography. Ponceau S staining and immunoblotting against IKKα and IKKβ show the equal amount of GST-ATG16L1 and immunoprecipitation efficiency, respectively. Data are representative of two experiments. (I) ATG16L1 proteolytic cleavage was assessed by immunoblot analysis in HeLa cells expressing mCherry-ATG16L1 Wt and mutants. HeLa cells were pretreated for 1 h with DMSO or 10 µM pan-caspase inhibitor (zVADfmk), followed by TNF stimulation (20 ng/ml) in the presence of 10 µg/ml cycloheximide (CHX) for 3 h. (J) Caspase 3–mediated in vitro ATG16L1 cleavage was assessed by immunoblot analysis. mCherry-ATG16L1 Wt and mutants were immunoprecipitated from HEK293T cells. Then, immunoprecipitates were subjected to in vitro cleavage assay using recombinant active caspase 3. Data are representative of two experiments. (I and J) CL, cleaved; FL, full length. (K) ATG16L1 proteolytic cleavage was assessed by immunoblot analysis in colonic organoids. Organoids were pretreated with DMSO or pan-caspase inhibitor (zVADfmk), followed by stimulation with NOD ligands, 20 µg/ml L-18MDP, and 20 µg/ml C12-iE-DAP, in the presence of 10 µg/ml cycloheximide for 3.5 h. Data are representative of two experiments. cl., cleaved. (L) Endogenous IKK complex was immunoprecipitated from untreated or L18-MDP–treated (20 µg/ml for 15 min) colonic organoids and then subjected to in vitro kinase assay as described in H. Data are representative of two experiments. (G, H, and L) Single asterisks indicate nonspecific signal determined by mass spectrometry.

Techniques Used: Co-Immunoprecipitation Assay, Transfection, Western Blot, Expressing, Immunoprecipitation, Pull Down Assay, Construct, In Vitro, Kinase Assay, Mass Spectrometry, Autoradiography, Staining, Plasmid Preparation, Isolation, Mouse Assay, Cleavage Assay, Recombinant

35) Product Images from "Direct interaction between centralspindlin and PRC1 reinforces mechanical resilience of the central spindle"

Article Title: Direct interaction between centralspindlin and PRC1 reinforces mechanical resilience of the central spindle

Journal: Nature Communications

doi: 10.1038/ncomms8290

Physical interaction between SPD-1 and CYK-4 sensitive to SPD-1 R83W mutation. ( a ) In vitro translated full-length CYK-4 was pulled down by full-length SPD-1 immobilized on chitin beads via a chitin-binding domain (CBD) tag. ( b ) In vitro translated full-length SPD-1 was pulled down by full-length CYK-4 or the centralspindlin holocomplex (CYK-4/ZEN-4) immobilized on glutathione-Sepharose beads via a glutathione- S -transferase (GST) tag. ( c ) Schematic drawings of SPD-1 and CYK-4. R83W indicates the mutation found in the spd-1(oj5) mutant exhibiting central spindle defects. ( d ) Yeast 2-hybrid assay of the indicated combinations of bait and prey. Growth on histidine-deficient medium containing 3-amino-1,2,4-triazole (–His+3AT) indicates a positive interaction between the bait and prey. ( e ) SPD-1 1-228 fragment with or without the R83W mutation (WT: wild type) was pulled down by CYK-4 constructs expressed as fusion proteins with maltose-binding protein (MBP) and detected with an anti-SPD-1 antibody. The CYK-4 tail region is necessary and, if dimerized, sufficient for efficient binding. ( f ) The R83W mutation does not affect the mobility of the SPD-1 full-length protein in Superdex 200 size-exclusion chromatography (blue: wild type; red: R83W). The elution profile of a mixture of standard proteins (Thy, thyroglobulin; IgG, gamma globulin; Ova, ovalbumin; MyG, myoglobin; VB12, vitamin B12) is shown in grey. ( g , h ) The R83W mutation does not affect the interaction of SPD-1 with microtubules. ( g ) Wild-type and R83W SPD-1 were incubated with microtubules or control buffer and sedimented by ultracentrifugation. P, pellet; S, supernatant; T, total. Increased recovery in the pellet in the presence of microtubules indicates the co-precipitation of SPD-1 with the microtubules. ( h ) Microtubules were incubated with SPD-1 with or without the R83W mutation and visualized by immunofluorescence following fixation. Scale bar, 20 μm.
Figure Legend Snippet: Physical interaction between SPD-1 and CYK-4 sensitive to SPD-1 R83W mutation. ( a ) In vitro translated full-length CYK-4 was pulled down by full-length SPD-1 immobilized on chitin beads via a chitin-binding domain (CBD) tag. ( b ) In vitro translated full-length SPD-1 was pulled down by full-length CYK-4 or the centralspindlin holocomplex (CYK-4/ZEN-4) immobilized on glutathione-Sepharose beads via a glutathione- S -transferase (GST) tag. ( c ) Schematic drawings of SPD-1 and CYK-4. R83W indicates the mutation found in the spd-1(oj5) mutant exhibiting central spindle defects. ( d ) Yeast 2-hybrid assay of the indicated combinations of bait and prey. Growth on histidine-deficient medium containing 3-amino-1,2,4-triazole (–His+3AT) indicates a positive interaction between the bait and prey. ( e ) SPD-1 1-228 fragment with or without the R83W mutation (WT: wild type) was pulled down by CYK-4 constructs expressed as fusion proteins with maltose-binding protein (MBP) and detected with an anti-SPD-1 antibody. The CYK-4 tail region is necessary and, if dimerized, sufficient for efficient binding. ( f ) The R83W mutation does not affect the mobility of the SPD-1 full-length protein in Superdex 200 size-exclusion chromatography (blue: wild type; red: R83W). The elution profile of a mixture of standard proteins (Thy, thyroglobulin; IgG, gamma globulin; Ova, ovalbumin; MyG, myoglobin; VB12, vitamin B12) is shown in grey. ( g , h ) The R83W mutation does not affect the interaction of SPD-1 with microtubules. ( g ) Wild-type and R83W SPD-1 were incubated with microtubules or control buffer and sedimented by ultracentrifugation. P, pellet; S, supernatant; T, total. Increased recovery in the pellet in the presence of microtubules indicates the co-precipitation of SPD-1 with the microtubules. ( h ) Microtubules were incubated with SPD-1 with or without the R83W mutation and visualized by immunofluorescence following fixation. Scale bar, 20 μm.

Techniques Used: Mutagenesis, In Vitro, Binding Assay, Y2H Assay, Construct, Size-exclusion Chromatography, Incubation, Immunofluorescence

36) Product Images from "The ON:OFF switch, σ1R-HINT1 protein, controls GPCR-NMDA receptor cross-regulation: Implications in neurological disorders"

Article Title: The ON:OFF switch, σ1R-HINT1 protein, controls GPCR-NMDA receptor cross-regulation: Implications in neurological disorders

Journal: Oncotarget

doi:

HINT1 and σ1R binding to the MOR and NMDAR NR1 subunit C-terminal sequence C0-C1-C2 a. In vitro HINT1 binding to σ1R and NR1 subunits . Because HINT1 forms homodimers, the protomer was used at 200 nM, whereas the GST-σ1R and GST-NR1 C0-C1-C2 peptides were used at 100 nM (GST alone did not bind to the HINT1 protein: lane 1, negative control). Glutathione Sepharose 4B captured the GST fusion protein, and the pellets were then washed, solubilized in 2x Laemmli buffer and resolved by SDS-PAGE. The presence of HINT1 and GST was analyzed sequentially in Western blots (WB). b. Effect of calcium on the association of σ1R with NR1 subunits and MORs . The recombinant proteins were used at 100 nM. The assay was performed in the presence of increasing amounts of calcium chloride (0, 0.25, 0.75, or 2.5 mM). Bait proteins (GST-NR1 C0-C1-C2 and GST-MOR) were immobilized by covalent attachment to NHS-activated Sepharose. Prey protein (σ1R) alone did not bind to either the NHS-Sepharose or the recombinant GST (negative controls). The pellets obtained were processed as described. *Significantly different from the immuno-signals of the 0 mM CaCl 2 group assigned an arbitrary value of 1; ANOVA, total DF = 15, followed by Dunnett multiple comparisons vs control group, p
Figure Legend Snippet: HINT1 and σ1R binding to the MOR and NMDAR NR1 subunit C-terminal sequence C0-C1-C2 a. In vitro HINT1 binding to σ1R and NR1 subunits . Because HINT1 forms homodimers, the protomer was used at 200 nM, whereas the GST-σ1R and GST-NR1 C0-C1-C2 peptides were used at 100 nM (GST alone did not bind to the HINT1 protein: lane 1, negative control). Glutathione Sepharose 4B captured the GST fusion protein, and the pellets were then washed, solubilized in 2x Laemmli buffer and resolved by SDS-PAGE. The presence of HINT1 and GST was analyzed sequentially in Western blots (WB). b. Effect of calcium on the association of σ1R with NR1 subunits and MORs . The recombinant proteins were used at 100 nM. The assay was performed in the presence of increasing amounts of calcium chloride (0, 0.25, 0.75, or 2.5 mM). Bait proteins (GST-NR1 C0-C1-C2 and GST-MOR) were immobilized by covalent attachment to NHS-activated Sepharose. Prey protein (σ1R) alone did not bind to either the NHS-Sepharose or the recombinant GST (negative controls). The pellets obtained were processed as described. *Significantly different from the immuno-signals of the 0 mM CaCl 2 group assigned an arbitrary value of 1; ANOVA, total DF = 15, followed by Dunnett multiple comparisons vs control group, p

Techniques Used: Binding Assay, Sequencing, In Vitro, Negative Control, SDS Page, Western Blot, Recombinant

Calcium-dependent binding of σ1Rs to MORs and NR1 subunits: Influence of σ1R regulation a. The σ1R agonist pregnenolone sulfate stabilizes the σ1R-NR1 interaction while diminishing σ1R binding to MORs . The recombinant MOR, NR1 C0-C1-C2 and σ1R were used at 100 nM. The assay was performed in the presence of increasing amounts of calcium chloride (0, 0.25, 0.75, 2.5 mM). Bait proteins (GST-NR1 C0-C1-C2 and GST-MOR) were immobilized by covalent attachment to NHS-activated Sepharose. Prey proteins alone did not bind either to the NHS-Sepharose or to the recombinant GST (negative controls). The pellets obtained were processed as described to determine σ1Rs in Western blots (see the Methods section). The bars are the mean ± S.E.M of three independent assays. Effect of calcium. For each interaction of σ1R, MOR-σ1R and NR1-σ1R, the effects of increasing calcium availability are shown relative to the data obtained in the absence of calcium control group (C): arbitrary value of 1): *Significant differences, ANOVA (DF = 11), Dunnett multiple comparisons vs control group, p
Figure Legend Snippet: Calcium-dependent binding of σ1Rs to MORs and NR1 subunits: Influence of σ1R regulation a. The σ1R agonist pregnenolone sulfate stabilizes the σ1R-NR1 interaction while diminishing σ1R binding to MORs . The recombinant MOR, NR1 C0-C1-C2 and σ1R were used at 100 nM. The assay was performed in the presence of increasing amounts of calcium chloride (0, 0.25, 0.75, 2.5 mM). Bait proteins (GST-NR1 C0-C1-C2 and GST-MOR) were immobilized by covalent attachment to NHS-activated Sepharose. Prey proteins alone did not bind either to the NHS-Sepharose or to the recombinant GST (negative controls). The pellets obtained were processed as described to determine σ1Rs in Western blots (see the Methods section). The bars are the mean ± S.E.M of three independent assays. Effect of calcium. For each interaction of σ1R, MOR-σ1R and NR1-σ1R, the effects of increasing calcium availability are shown relative to the data obtained in the absence of calcium control group (C): arbitrary value of 1): *Significant differences, ANOVA (DF = 11), Dunnett multiple comparisons vs control group, p

Techniques Used: Binding Assay, Recombinant, Western Blot

37) Product Images from "Involvement of 14-3-3 Proteins in the Second Epidermal Growth Factor-induced Wave of Rac1 Activation in the Process of Cell Migration *"

Article Title: Involvement of 14-3-3 Proteins in the Second Epidermal Growth Factor-induced Wave of Rac1 Activation in the Process of Cell Migration *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.255489

Identification of Tiam1 as a RacGEF which is responsible for the second EGF-induced wave of Rac1 activation. A , GST pulldown assay. Lysates of A431 cells stimulated with EGF for 12 h were incubated with GST or GST-14-3-3ζ and glutathione-Sepharose
Figure Legend Snippet: Identification of Tiam1 as a RacGEF which is responsible for the second EGF-induced wave of Rac1 activation. A , GST pulldown assay. Lysates of A431 cells stimulated with EGF for 12 h were incubated with GST or GST-14-3-3ζ and glutathione-Sepharose

Techniques Used: Activation Assay, GST Pulldown Assay, Incubation

38) Product Images from "Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis"

Article Title: Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1001482

Binding of RNA by dihydrolipoamide acetyltransferases might be a global phenomenon. (A) Hexahistidine-tagged E2 fusion proteins from C. reinhardtii (Cr), S. cerevisiae (Sc), Synechocystis sp. 6803 (Syn), and H. sapiens (Hs) along with two control proteins (PratA and RBP40) were purified on Ni-NTA Sepharose, separated by SDS-PAGE, and Coomassie-stained. To exclude an unspecific RNA binding of contaminating E. coli proteins in (B), we used the same volumes as used for the C. reinhardtii E2 protein of an elution fraction obtained from the bacterial host strain transformed with the empty expression vector served as control (eV). Recombinant proteins are indicated by arrows. Proteins in the preparation of the human E2 subunit (Hs) that are specifically recognized by an anti-histidine antibody are marked by an asterisk. (B) RNA binding assay. One of the 20 (∼100 ng) recombinant proteins shown in (A) was used for UV cross-linking to psbA mRNA. Lanes “ psbA* ” and “ psbA ” show the radiolabeled psbA RNA without and with RNase treatment, respectively. Due to the high intensity of the psbA * signal, a lower exposure of this lane is shown. Specific radioactive signals are indicated by arrows.
Figure Legend Snippet: Binding of RNA by dihydrolipoamide acetyltransferases might be a global phenomenon. (A) Hexahistidine-tagged E2 fusion proteins from C. reinhardtii (Cr), S. cerevisiae (Sc), Synechocystis sp. 6803 (Syn), and H. sapiens (Hs) along with two control proteins (PratA and RBP40) were purified on Ni-NTA Sepharose, separated by SDS-PAGE, and Coomassie-stained. To exclude an unspecific RNA binding of contaminating E. coli proteins in (B), we used the same volumes as used for the C. reinhardtii E2 protein of an elution fraction obtained from the bacterial host strain transformed with the empty expression vector served as control (eV). Recombinant proteins are indicated by arrows. Proteins in the preparation of the human E2 subunit (Hs) that are specifically recognized by an anti-histidine antibody are marked by an asterisk. (B) RNA binding assay. One of the 20 (∼100 ng) recombinant proteins shown in (A) was used for UV cross-linking to psbA mRNA. Lanes “ psbA* ” and “ psbA ” show the radiolabeled psbA RNA without and with RNase treatment, respectively. Due to the high intensity of the psbA * signal, a lower exposure of this lane is shown. Specific radioactive signals are indicated by arrows.

Techniques Used: Binding Assay, Purification, SDS Page, Staining, RNA Binding Assay, Transformation Assay, Expressing, Plasmid Preparation, Recombinant

39) Product Images from "TMEM74 promotes tumor cell survival by inducing autophagy via interactions with ATG16L1 and ATG9A"

Article Title: TMEM74 promotes tumor cell survival by inducing autophagy via interactions with ATG16L1 and ATG9A

Journal: Cell Death & Disease

doi: 10.1038/cddis.2017.370

TMEM74 associates with ATG16L1 via its C-terminal and influences the interaction between ATG5 and ATG16L1. ( a ) Schematic representations of WT ATG16L1 and its mutants: ATG16L1(1–320), and ATG16L1 △(1–320) . ( b ) HeLa cells were co-transfected with GFP-ATG16L1 and mCherry-TMEM74 for 24 h, Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( c ) HeLa cells were co-transfected with GFP-TMEM74 and mCherry-ATG16L1 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, ATG16L1 was detected in the washed beads using an anti-ATG16L1 IgG by western blotting. ( d ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG16L1, GFP-ATG16L1 was detected in the washed beads by western blotting. ( e , f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG16L1(1–320), or GFP-ATG16L1 △(1–320) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 were detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG16L1 for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, ATG5-ATG12 complex pulled down was detected in the immunoprecipitates using anti-ATG5 by western blotting. Quantification of ATG5-ATG12 pulled down relative to GFP-ATG16L1 was shown as column. Data are means±S.D. of three experiments. * P
Figure Legend Snippet: TMEM74 associates with ATG16L1 via its C-terminal and influences the interaction between ATG5 and ATG16L1. ( a ) Schematic representations of WT ATG16L1 and its mutants: ATG16L1(1–320), and ATG16L1 △(1–320) . ( b ) HeLa cells were co-transfected with GFP-ATG16L1 and mCherry-TMEM74 for 24 h, Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( c ) HeLa cells were co-transfected with GFP-TMEM74 and mCherry-ATG16L1 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, ATG16L1 was detected in the washed beads using an anti-ATG16L1 IgG by western blotting. ( d ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG16L1, GFP-ATG16L1 was detected in the washed beads by western blotting. ( e , f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG16L1(1–320), or GFP-ATG16L1 △(1–320) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 were detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG16L1 for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, ATG5-ATG12 complex pulled down was detected in the immunoprecipitates using anti-ATG5 by western blotting. Quantification of ATG5-ATG12 pulled down relative to GFP-ATG16L1 was shown as column. Data are means±S.D. of three experiments. * P

Techniques Used: Transfection, Western Blot, Incubation

TMEM74 associates with ATG9A via its N-terminal and influences the interaction between ATG9 and WIPI1. ( a ) HeLa cells were co-transfected with GFP-ATG9A and mCherry-TMEM74 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( b ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG9A, GFP-ATG9A was detected in the washed beads using an anti-GFP IgG by western blotting. ( c ) Schematic representations of WT-ATG9A and its mutants: ATG9A(1–495), and ATG9A △(1–495) , and ATG9A △(153–289) . ( d – f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG9A(1–495), GFP-ATG9A △(1–495) , or GFP-ATG9A △(153–289) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 was detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG9A for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, WIPI1 pulled down was detected in the immunoprecipitates using the anti-WIPI1 antibody by western blotting. Quantification of WIPI1 pulled down relative to GFP-ATG9A was shown as column. Data are means±S.D. of three experiments. * P
Figure Legend Snippet: TMEM74 associates with ATG9A via its N-terminal and influences the interaction between ATG9 and WIPI1. ( a ) HeLa cells were co-transfected with GFP-ATG9A and mCherry-TMEM74 for 24 h. Total cell extracts were subjected to IP using either an anti-GFP or an isotype control IgG, TMEM74 was detected in the washed beads using anti-TMEM74 IgG by western blotting. ( b ) GST and GST-TMEM74 fusion protein immobilized on glutainione-sepharose beads were incubated with HeLa cell lysates containing GFP-ATG9A, GFP-ATG9A was detected in the washed beads using an anti-GFP IgG by western blotting. ( c ) Schematic representations of WT-ATG9A and its mutants: ATG9A(1–495), and ATG9A △(1–495) , and ATG9A △(153–289) . ( d – f ) HeLa cells were co-transfected with mCherry-TMEM74 and GFP-ATG9A(1–495), GFP-ATG9A △(1–495) , or GFP-ATG9A △(153–289) respectively for 24 h. Total cell extracts were subjected to IP using an anti-GFP or an isotype control IgG, as indicated. TMEM74 was detected in the washed beads by western blotting. ( g , h ) HeLa cells were firstly treated by siTMEM74-1 , siTMEM74-2 or siControl for 24 h, then transfected with GFP-ATG9A for 24 h, meanwhile treated with EBSS for at least 8 h. Total cell extracts were subjected to IP using an anti-GFP or a non-specific control IgG, WIPI1 pulled down was detected in the immunoprecipitates using the anti-WIPI1 antibody by western blotting. Quantification of WIPI1 pulled down relative to GFP-ATG9A was shown as column. Data are means±S.D. of three experiments. * P

Techniques Used: Transfection, Western Blot, Incubation

40) Product Images from "Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of β-catenin mRNA"

Article Title: Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of β-catenin mRNA

Journal: Journal of Cell Science

doi: 10.1242/jcs.056176

Dvl3-KSRP interaction is RNA dependent and Dvl3 complex harbors Ctnnb1 mRNA. ( A ) F9 cells were co-transfected with HA-Dvl3-GFP2 and FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-HA affinity matrix. Interaction of KSRP with exogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( B ) F9 cells were transfected with FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-Dvl3 antibodies. Interaction of KSRP with endogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( C ) To test whether the in vitro interaction of KSRP and Dvl3 is also RNA dependent, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST or GST-KSRP-Sepharose beads in the absence or presence (5 μg) of either 3′-UTR of Ctnnb1 or Gapdh . The interaction was visualized by SDS-PAGE and autoradiography. ( D ) RNA immunoprecipitation assay was performed on F9 cell lysates with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by RT-PCR with primers specific for Ctnnb1 , Myc or Fzd7 . Representative gel of two independent experiments that proved highly reproducible is displayed. ( E ) F9 cells were treated with Wnt3a (10 ng/ml) for indicated periods of time and RNA immunoprecipitation assay was performed with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by quantitative real-time PCR with β-catenin specific primers. Representative blots of two independent experiments that proved highly reproducible were displayed. The data represent mean values ± s.e.m. from two independent experiments whose results were in high agreement. In the lower panel, a representative gel is displayed. ## P
Figure Legend Snippet: Dvl3-KSRP interaction is RNA dependent and Dvl3 complex harbors Ctnnb1 mRNA. ( A ) F9 cells were co-transfected with HA-Dvl3-GFP2 and FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-HA affinity matrix. Interaction of KSRP with exogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( B ) F9 cells were transfected with FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-Dvl3 antibodies. Interaction of KSRP with endogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( C ) To test whether the in vitro interaction of KSRP and Dvl3 is also RNA dependent, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST or GST-KSRP-Sepharose beads in the absence or presence (5 μg) of either 3′-UTR of Ctnnb1 or Gapdh . The interaction was visualized by SDS-PAGE and autoradiography. ( D ) RNA immunoprecipitation assay was performed on F9 cell lysates with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by RT-PCR with primers specific for Ctnnb1 , Myc or Fzd7 . Representative gel of two independent experiments that proved highly reproducible is displayed. ( E ) F9 cells were treated with Wnt3a (10 ng/ml) for indicated periods of time and RNA immunoprecipitation assay was performed with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by quantitative real-time PCR with β-catenin specific primers. Representative blots of two independent experiments that proved highly reproducible were displayed. The data represent mean values ± s.e.m. from two independent experiments whose results were in high agreement. In the lower panel, a representative gel is displayed. ## P

Techniques Used: Transfection, Lysis, Incubation, In Vitro, Synthesized, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

KSRP interacts with Dvl3. ( A ) F9 cells were transiently transfected with FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. Asterisks indicate the bands of immunoglobulin heavy and light chains. ( B ) F9 cells were treated with 100 nM of Dvl3 siRNA for 24 hours followed by transient expression of FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. ( C ) F9 cell lysates were immunoprecipitated with either rabbit control IgG or rabbit anti-KSRP polyclonal antibody and the interaction of KSRP with Dvl3 was visualized by probing the blots with anti-Dvl3 mouse monoclonal antibody. ( D ) F9 cells were transiently transfected with empty vector or FLAG-KSRP for 24 hours. The cells were then treated with Wnt3a (10 ng/ml) for indicated period of time followed by cell lysis and affinity pull-downs with anti-Dvl3 specific antibodies followed by immunoblotting with anti-FLAG antibodies. ( E ) To test the direct interaction of Dvl3 with KSRP, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST- or GST-KSRP-Sepharose beads in the presence of 0.8% BSA. The interaction was visualized by SDS-PAGE and autoradiography. Representative blots of three independent experiments that proved highly reproducible are shown. * P
Figure Legend Snippet: KSRP interacts with Dvl3. ( A ) F9 cells were transiently transfected with FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. Asterisks indicate the bands of immunoglobulin heavy and light chains. ( B ) F9 cells were treated with 100 nM of Dvl3 siRNA for 24 hours followed by transient expression of FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. ( C ) F9 cell lysates were immunoprecipitated with either rabbit control IgG or rabbit anti-KSRP polyclonal antibody and the interaction of KSRP with Dvl3 was visualized by probing the blots with anti-Dvl3 mouse monoclonal antibody. ( D ) F9 cells were transiently transfected with empty vector or FLAG-KSRP for 24 hours. The cells were then treated with Wnt3a (10 ng/ml) for indicated period of time followed by cell lysis and affinity pull-downs with anti-Dvl3 specific antibodies followed by immunoblotting with anti-FLAG antibodies. ( E ) To test the direct interaction of Dvl3 with KSRP, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST- or GST-KSRP-Sepharose beads in the presence of 0.8% BSA. The interaction was visualized by SDS-PAGE and autoradiography. Representative blots of three independent experiments that proved highly reproducible are shown. * P

Techniques Used: Transfection, Lysis, Expressing, Immunoprecipitation, Plasmid Preparation, In Vitro, Synthesized, Labeling, SDS Page, Autoradiography

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Centrifugation:

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Article Title: Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis
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Incubation:

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    GE Healthcare glutatione sepharose 4b
    VP35 IID associates with the NP (481-500) region. A: Localization of deletion mutants of VP35 and NP. Wild-type and deletion mutants of NP were co-transfected with VP35 wild-type and deletion mutants in HuH-7 cells. 48 hours after transfection, cells were stained with anti-FLAG (NP, red) and anti-myc (VP35, green). Merged green/red fields are shown. B: NP(1-500) and related mutants as indicated were co-transfected with VP35-myc wild-type and deletion mutants in 293T/17 cells and cells were lysed 48-50 hours after transfection. An anti-myc antibody was used for immunoprecipitation and co-immunoprecipitated proteins were analyzed by western blotting. GAPDH is the loading control of the lysate. Top panel: immunoprecipitates were separated by SDS-PAGE, blotted and probed with the indicated antibodies. The left and right sides of the data shown were sourced from the same image, with several lanes deleted between lanes 5 and 6 of the figure. Bottom panel: Crude lysates were separated by SDS-PAGE, blotted and probed with the indicated antibodies to determine protein expression levels. C: NP(1-500) and NP(1-641) were co-transfected with VP35-myc wild-type or indicated mutants in 293T/17 cells and cells were lysed 48-50 hours after transfection. An anti-myc antibody was used for immunoprecipitation and co-immunoprecipitated proteins were analyzed by western blotting. Top panel: immunoprecipitates were separated by SDS-PAGE, blotted and probed with the indicated antibodies. Bottom panel: Crude lysates were separated by SDS-PAGE, blotted and probed with the indicated antibodies to determine protein expression levels. D: Pull down assay of E.coli expressed proteins. GST-NPs with the indicated regions of NP or His-tagged VP35-IID were expressed in E.coli and purified using glutathione <t>Sepharose</t> or Ni-NTA <t>agarose.</t> Purified proteins were dialyzed, subjected to SDS-PAGE and stained with CBB (Input panel). Purified proteins were quantified and equal amounts of GST-NPs or GST were mixed with His-VP35-IID protein. After overnight incubation with the indicated beads followed by washing, protein bound to the beads was extracted with 2x sample buffer, and equal volumes of the eluents were subjected to SDS-PAGE and stained with CBB.
    Glutatione Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione sepharose 4b
    HDX-MS mapping of NDP52 interactions with the ULK1 complex. (A-D) Difference of Hydrogen Deuterium Exchange percentages of the ATG13 (A), ATG101 (B), ULK1 (C) and FIP200 (D) in ULK1 complex vs in ULK1 complex with NDP52 at the 60s time point. All values are mean ± SD. (E) Pull-down efficiency of GFP-tagged wild type ULK1 complex or GFP-FIP200 by glutathione <t>sepharose</t> beads coated with different concentrations of GST-NDP52 as baits. N=3 biological replicates. All values are Mean ± SD. (F) Pull-down assays of mutant FIP200 constructs (M1-M8) and wild type with NDP52. Both GSH and Amylose resin were used to pull down GST-FIP200(1274-C):MBP-NDP52 complex from lysate of overexpressing HEK cells. The pull-down results were visualized by SDS-PAGE and Coomassie blue staining.
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    GE Healthcare glutathione sepharose beads
    S273-paxillin phosphorylation by PAK regulates paxillin–GIT1 binding. (a) CHO-K1 lysates treated (right) and untreated (left) with 5 nM CalyculinA (CalA) were probed (top) using a phospho–S273-paxillin–specific antibody. Total paxillin levels were assayed with an anti-paxillin antibody (bottom). A single band corresponding to the molecular mass of paxillin (∼68 kD) was detected in treated lysates. (b) Kinase assay was performed with FLAG-WT-paxillin and either KD- or CA-myc-PAK synthesized in vitro, and S273-paxillin phosphorylation was assessed with a phospho–S273-paxillin antibody. Bottom blots show equal loading by probing with anti-FLAG and anti-myc antibodies, respectively. Phospho–S273-paxillin levels increased eightfold with CA-PAK compared with KD-PAK. (c) Paxillin was immunoprecipitated using a GFP antibody from CHO-K1 lysates expressing paxillin-GFP and either KD- or CA-myc-PAK, and S273-paxillin phosphorylation levels were assayed using a phospho–S273-paxillin antibody. The lower two panels show equal levels of paxillin–GFP and myc-PAK, and the GFP blot shows equal loading in the lysates. S273-paxillin phosphorylation increased eightfold with CA-PAK as compared with KD-PAK. (d) A GFP antibody was used to immunoprecipitate paxillin from CHO-K1 lysates expressing GFP control or WT-, S273A-, or S273D-paxillin-GFP and FLAG-GIT1. GIT1 binding was probed using an anti-FLAG antibody. The bottom two panels show equivalent expression of S273-paxillin mutants and FLAG-GIT1 in the lysates. GIT1 binding to S273D-paxillin increased threefold, whereas it was reduced twofold with S273A-paxillin, when compared with WT-paxillin. (e) Paxillin was immunoprecipitated from CHO-K1 lysates expressing GFP control or WT-, S273A-, or S273D-paxillin-GFP and myc-FAK using a GFP antibody, and FAK binding was assessed with an anti-myc antibody. The bottom two panels show equivalent expression of S273-paxillin mutants and myc-FAK in the lysates. S273-paxillin phosphorylation only marginally affected FAK binding. (f) GIT1 was immunoprecipitated from in vitro mixtures of FLAG-GIT1, untagged WT-paxillin, and either KD- or CA-PAK using anti-FLAG M2-conjugated <t>agarose,</t> and phospho–S273-paxillin binding was probed using a phospho–S273-paxillin antibody. The middle blot shows equal levels of FLAG-GIT1 using an anti-FLAG antibody. (bottom) Equal loading of the lysates using anti-myc and anti-paxillin antibodies, respectively. Phospho–S273-paxillin–GIT1 binding increased sevenfold with CA-PAK compared with KD-PAK. (g) Anti-FLAG M2-conjugated agarose was used to immunoprecipitate GIT1 from in vitro mixtures of FLAG-GIT1, untagged WT-paxillin, and CA-PAK preincubated with 500-fold molar excess of phospho– or nonphospho–S273-paxillin peptide, and phospho–S273-paxillin binding was assessed with a phospho–S273-paxillin antibody. Very low levels of phospho–S273-paxillin–GIT1 binding was detected with the competitive phosphopeptide (left), whereas a robust signal was observed with the noncompetitive peptide (right), confirming that the PAK-mediated increase in phospho–S273-paxillin–GIT1 binding is specific to S273-paxillin phosphorylation.
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    GE Healthcare glutathione sepharose binding buffer
    Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM <t>sepharose.</t> Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P
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    VP35 IID associates with the NP (481-500) region. A: Localization of deletion mutants of VP35 and NP. Wild-type and deletion mutants of NP were co-transfected with VP35 wild-type and deletion mutants in HuH-7 cells. 48 hours after transfection, cells were stained with anti-FLAG (NP, red) and anti-myc (VP35, green). Merged green/red fields are shown. B: NP(1-500) and related mutants as indicated were co-transfected with VP35-myc wild-type and deletion mutants in 293T/17 cells and cells were lysed 48-50 hours after transfection. An anti-myc antibody was used for immunoprecipitation and co-immunoprecipitated proteins were analyzed by western blotting. GAPDH is the loading control of the lysate. Top panel: immunoprecipitates were separated by SDS-PAGE, blotted and probed with the indicated antibodies. The left and right sides of the data shown were sourced from the same image, with several lanes deleted between lanes 5 and 6 of the figure. Bottom panel: Crude lysates were separated by SDS-PAGE, blotted and probed with the indicated antibodies to determine protein expression levels. C: NP(1-500) and NP(1-641) were co-transfected with VP35-myc wild-type or indicated mutants in 293T/17 cells and cells were lysed 48-50 hours after transfection. An anti-myc antibody was used for immunoprecipitation and co-immunoprecipitated proteins were analyzed by western blotting. Top panel: immunoprecipitates were separated by SDS-PAGE, blotted and probed with the indicated antibodies. Bottom panel: Crude lysates were separated by SDS-PAGE, blotted and probed with the indicated antibodies to determine protein expression levels. D: Pull down assay of E.coli expressed proteins. GST-NPs with the indicated regions of NP or His-tagged VP35-IID were expressed in E.coli and purified using glutathione Sepharose or Ni-NTA agarose. Purified proteins were dialyzed, subjected to SDS-PAGE and stained with CBB (Input panel). Purified proteins were quantified and equal amounts of GST-NPs or GST were mixed with His-VP35-IID protein. After overnight incubation with the indicated beads followed by washing, protein bound to the beads was extracted with 2x sample buffer, and equal volumes of the eluents were subjected to SDS-PAGE and stained with CBB.

    Journal: bioRxiv

    Article Title: Ebola virus inclusion body formation and RNA synthesis are controlled by a novel domain of NP interacting with VP35

    doi: 10.1101/2020.04.06.028423

    Figure Lengend Snippet: VP35 IID associates with the NP (481-500) region. A: Localization of deletion mutants of VP35 and NP. Wild-type and deletion mutants of NP were co-transfected with VP35 wild-type and deletion mutants in HuH-7 cells. 48 hours after transfection, cells were stained with anti-FLAG (NP, red) and anti-myc (VP35, green). Merged green/red fields are shown. B: NP(1-500) and related mutants as indicated were co-transfected with VP35-myc wild-type and deletion mutants in 293T/17 cells and cells were lysed 48-50 hours after transfection. An anti-myc antibody was used for immunoprecipitation and co-immunoprecipitated proteins were analyzed by western blotting. GAPDH is the loading control of the lysate. Top panel: immunoprecipitates were separated by SDS-PAGE, blotted and probed with the indicated antibodies. The left and right sides of the data shown were sourced from the same image, with several lanes deleted between lanes 5 and 6 of the figure. Bottom panel: Crude lysates were separated by SDS-PAGE, blotted and probed with the indicated antibodies to determine protein expression levels. C: NP(1-500) and NP(1-641) were co-transfected with VP35-myc wild-type or indicated mutants in 293T/17 cells and cells were lysed 48-50 hours after transfection. An anti-myc antibody was used for immunoprecipitation and co-immunoprecipitated proteins were analyzed by western blotting. Top panel: immunoprecipitates were separated by SDS-PAGE, blotted and probed with the indicated antibodies. Bottom panel: Crude lysates were separated by SDS-PAGE, blotted and probed with the indicated antibodies to determine protein expression levels. D: Pull down assay of E.coli expressed proteins. GST-NPs with the indicated regions of NP or His-tagged VP35-IID were expressed in E.coli and purified using glutathione Sepharose or Ni-NTA agarose. Purified proteins were dialyzed, subjected to SDS-PAGE and stained with CBB (Input panel). Purified proteins were quantified and equal amounts of GST-NPs or GST were mixed with His-VP35-IID protein. After overnight incubation with the indicated beads followed by washing, protein bound to the beads was extracted with 2x sample buffer, and equal volumes of the eluents were subjected to SDS-PAGE and stained with CBB.

    Article Snippet: Protein concentration was set to 1μM of His tagged proteins and to 2 μM of GST-tagged proteins for HIS-Select HF Nickel Affinity Gel pull down, and was set to 1 μM of GST tagged proteins and to 2 μM of His-fusion proteins for Glutatione Sepharose 4B pull down.

    Techniques: Transfection, Staining, Immunoprecipitation, Western Blot, SDS Page, Expressing, Pull Down Assay, Purification, Incubation

    HDX-MS mapping of NDP52 interactions with the ULK1 complex. (A-D) Difference of Hydrogen Deuterium Exchange percentages of the ATG13 (A), ATG101 (B), ULK1 (C) and FIP200 (D) in ULK1 complex vs in ULK1 complex with NDP52 at the 60s time point. All values are mean ± SD. (E) Pull-down efficiency of GFP-tagged wild type ULK1 complex or GFP-FIP200 by glutathione sepharose beads coated with different concentrations of GST-NDP52 as baits. N=3 biological replicates. All values are Mean ± SD. (F) Pull-down assays of mutant FIP200 constructs (M1-M8) and wild type with NDP52. Both GSH and Amylose resin were used to pull down GST-FIP200(1274-C):MBP-NDP52 complex from lysate of overexpressing HEK cells. The pull-down results were visualized by SDS-PAGE and Coomassie blue staining.

    Journal: bioRxiv

    Article Title: The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment

    doi: 10.1101/2020.05.19.104315

    Figure Lengend Snippet: HDX-MS mapping of NDP52 interactions with the ULK1 complex. (A-D) Difference of Hydrogen Deuterium Exchange percentages of the ATG13 (A), ATG101 (B), ULK1 (C) and FIP200 (D) in ULK1 complex vs in ULK1 complex with NDP52 at the 60s time point. All values are mean ± SD. (E) Pull-down efficiency of GFP-tagged wild type ULK1 complex or GFP-FIP200 by glutathione sepharose beads coated with different concentrations of GST-NDP52 as baits. N=3 biological replicates. All values are Mean ± SD. (F) Pull-down assays of mutant FIP200 constructs (M1-M8) and wild type with NDP52. Both GSH and Amylose resin were used to pull down GST-FIP200(1274-C):MBP-NDP52 complex from lysate of overexpressing HEK cells. The pull-down results were visualized by SDS-PAGE and Coomassie blue staining.

    Article Snippet: The supernatant was incubated with Glutathione Sepharose 4B (GE Healthcare) or Ni-NTA Resins (Qiagen) as appropriate, with gentle shaking for 2 hours at 4 °C.

    Techniques: Mutagenesis, Construct, SDS Page, Staining

    NDP52 allosterically activates membrane association of ULK1 complex. (A) Microscopy-based bead protein interaction assay with glutathione sepharose beads coated with GST-NDP52 as baits and incubated with GFP-tagged wild type ULK1 complex or mutant as prey. Representative confocal micrographs are shown. Scale bars, 50 µm. (B) Quantification of the GFP-ULK1 complex signal intensity measured on glutathione sepharose beads coated with GST-NDP52 (means ± SDs; N = 20). (C) Representative confocal micrographs showing the membrane recruitment of GFP-ULK1 complex. GFP-tagged wild type ULK1 complex or mutant was mixed with GUVs in the presence of GST-NDP52 and GST-4xUb at room temperature. Images taken at indicated time points were shown. Scale bars, 10 µm. (D) Quantitation of the kinetics of ULK1 complex recruitment to the membrane from individual GUV tracing in A (means ± SDs; N= 22 (WT); 25 (ΔMR); 22 (ΔNDP52)).

    Journal: bioRxiv

    Article Title: The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment

    doi: 10.1101/2020.05.19.104315

    Figure Lengend Snippet: NDP52 allosterically activates membrane association of ULK1 complex. (A) Microscopy-based bead protein interaction assay with glutathione sepharose beads coated with GST-NDP52 as baits and incubated with GFP-tagged wild type ULK1 complex or mutant as prey. Representative confocal micrographs are shown. Scale bars, 50 µm. (B) Quantification of the GFP-ULK1 complex signal intensity measured on glutathione sepharose beads coated with GST-NDP52 (means ± SDs; N = 20). (C) Representative confocal micrographs showing the membrane recruitment of GFP-ULK1 complex. GFP-tagged wild type ULK1 complex or mutant was mixed with GUVs in the presence of GST-NDP52 and GST-4xUb at room temperature. Images taken at indicated time points were shown. Scale bars, 10 µm. (D) Quantitation of the kinetics of ULK1 complex recruitment to the membrane from individual GUV tracing in A (means ± SDs; N= 22 (WT); 25 (ΔMR); 22 (ΔNDP52)).

    Article Snippet: The supernatant was incubated with Glutathione Sepharose 4B (GE Healthcare) or Ni-NTA Resins (Qiagen) as appropriate, with gentle shaking for 2 hours at 4 °C.

    Techniques: Microscopy, Protein Interaction Assay, Incubation, Mutagenesis, Quantitation Assay

    Purified ULK1 complex is functional. (A) ADP-Glo Kinase assay of ULK1 complex with ULKtide as substrate. N=5 biological replicates. All values are Mean ± SD. (B) Microscopy-based bead protein interaction assay with glutathione sepharose beads coated with GST-NDP52 as baits and incubated with GFP-tagged wild type ULK1 complex. Representative confocal micrographs are shown. Scale bars, 50 µm. (C) Pull-down efficiency of GFP-tagged wild type ULK1 complex by glutathione sepharose beads coated with GST-NDP52 or GST-4xUb as baits. N=3 biological replicates. All values are Mean ± SD.

    Journal: bioRxiv

    Article Title: The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment

    doi: 10.1101/2020.05.19.104315

    Figure Lengend Snippet: Purified ULK1 complex is functional. (A) ADP-Glo Kinase assay of ULK1 complex with ULKtide as substrate. N=5 biological replicates. All values are Mean ± SD. (B) Microscopy-based bead protein interaction assay with glutathione sepharose beads coated with GST-NDP52 as baits and incubated with GFP-tagged wild type ULK1 complex. Representative confocal micrographs are shown. Scale bars, 50 µm. (C) Pull-down efficiency of GFP-tagged wild type ULK1 complex by glutathione sepharose beads coated with GST-NDP52 or GST-4xUb as baits. N=3 biological replicates. All values are Mean ± SD.

    Article Snippet: The supernatant was incubated with Glutathione Sepharose 4B (GE Healthcare) or Ni-NTA Resins (Qiagen) as appropriate, with gentle shaking for 2 hours at 4 °C.

    Techniques: Purification, Functional Assay, Kinase Assay, Microscopy, Protein Interaction Assay, Incubation

    S273-paxillin phosphorylation by PAK regulates paxillin–GIT1 binding. (a) CHO-K1 lysates treated (right) and untreated (left) with 5 nM CalyculinA (CalA) were probed (top) using a phospho–S273-paxillin–specific antibody. Total paxillin levels were assayed with an anti-paxillin antibody (bottom). A single band corresponding to the molecular mass of paxillin (∼68 kD) was detected in treated lysates. (b) Kinase assay was performed with FLAG-WT-paxillin and either KD- or CA-myc-PAK synthesized in vitro, and S273-paxillin phosphorylation was assessed with a phospho–S273-paxillin antibody. Bottom blots show equal loading by probing with anti-FLAG and anti-myc antibodies, respectively. Phospho–S273-paxillin levels increased eightfold with CA-PAK compared with KD-PAK. (c) Paxillin was immunoprecipitated using a GFP antibody from CHO-K1 lysates expressing paxillin-GFP and either KD- or CA-myc-PAK, and S273-paxillin phosphorylation levels were assayed using a phospho–S273-paxillin antibody. The lower two panels show equal levels of paxillin–GFP and myc-PAK, and the GFP blot shows equal loading in the lysates. S273-paxillin phosphorylation increased eightfold with CA-PAK as compared with KD-PAK. (d) A GFP antibody was used to immunoprecipitate paxillin from CHO-K1 lysates expressing GFP control or WT-, S273A-, or S273D-paxillin-GFP and FLAG-GIT1. GIT1 binding was probed using an anti-FLAG antibody. The bottom two panels show equivalent expression of S273-paxillin mutants and FLAG-GIT1 in the lysates. GIT1 binding to S273D-paxillin increased threefold, whereas it was reduced twofold with S273A-paxillin, when compared with WT-paxillin. (e) Paxillin was immunoprecipitated from CHO-K1 lysates expressing GFP control or WT-, S273A-, or S273D-paxillin-GFP and myc-FAK using a GFP antibody, and FAK binding was assessed with an anti-myc antibody. The bottom two panels show equivalent expression of S273-paxillin mutants and myc-FAK in the lysates. S273-paxillin phosphorylation only marginally affected FAK binding. (f) GIT1 was immunoprecipitated from in vitro mixtures of FLAG-GIT1, untagged WT-paxillin, and either KD- or CA-PAK using anti-FLAG M2-conjugated agarose, and phospho–S273-paxillin binding was probed using a phospho–S273-paxillin antibody. The middle blot shows equal levels of FLAG-GIT1 using an anti-FLAG antibody. (bottom) Equal loading of the lysates using anti-myc and anti-paxillin antibodies, respectively. Phospho–S273-paxillin–GIT1 binding increased sevenfold with CA-PAK compared with KD-PAK. (g) Anti-FLAG M2-conjugated agarose was used to immunoprecipitate GIT1 from in vitro mixtures of FLAG-GIT1, untagged WT-paxillin, and CA-PAK preincubated with 500-fold molar excess of phospho– or nonphospho–S273-paxillin peptide, and phospho–S273-paxillin binding was assessed with a phospho–S273-paxillin antibody. Very low levels of phospho–S273-paxillin–GIT1 binding was detected with the competitive phosphopeptide (left), whereas a robust signal was observed with the noncompetitive peptide (right), confirming that the PAK-mediated increase in phospho–S273-paxillin–GIT1 binding is specific to S273-paxillin phosphorylation.

    Journal: The Journal of Cell Biology

    Article Title: Paxillin phosphorylation at Ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics

    doi: 10.1083/jcb.200509075

    Figure Lengend Snippet: S273-paxillin phosphorylation by PAK regulates paxillin–GIT1 binding. (a) CHO-K1 lysates treated (right) and untreated (left) with 5 nM CalyculinA (CalA) were probed (top) using a phospho–S273-paxillin–specific antibody. Total paxillin levels were assayed with an anti-paxillin antibody (bottom). A single band corresponding to the molecular mass of paxillin (∼68 kD) was detected in treated lysates. (b) Kinase assay was performed with FLAG-WT-paxillin and either KD- or CA-myc-PAK synthesized in vitro, and S273-paxillin phosphorylation was assessed with a phospho–S273-paxillin antibody. Bottom blots show equal loading by probing with anti-FLAG and anti-myc antibodies, respectively. Phospho–S273-paxillin levels increased eightfold with CA-PAK compared with KD-PAK. (c) Paxillin was immunoprecipitated using a GFP antibody from CHO-K1 lysates expressing paxillin-GFP and either KD- or CA-myc-PAK, and S273-paxillin phosphorylation levels were assayed using a phospho–S273-paxillin antibody. The lower two panels show equal levels of paxillin–GFP and myc-PAK, and the GFP blot shows equal loading in the lysates. S273-paxillin phosphorylation increased eightfold with CA-PAK as compared with KD-PAK. (d) A GFP antibody was used to immunoprecipitate paxillin from CHO-K1 lysates expressing GFP control or WT-, S273A-, or S273D-paxillin-GFP and FLAG-GIT1. GIT1 binding was probed using an anti-FLAG antibody. The bottom two panels show equivalent expression of S273-paxillin mutants and FLAG-GIT1 in the lysates. GIT1 binding to S273D-paxillin increased threefold, whereas it was reduced twofold with S273A-paxillin, when compared with WT-paxillin. (e) Paxillin was immunoprecipitated from CHO-K1 lysates expressing GFP control or WT-, S273A-, or S273D-paxillin-GFP and myc-FAK using a GFP antibody, and FAK binding was assessed with an anti-myc antibody. The bottom two panels show equivalent expression of S273-paxillin mutants and myc-FAK in the lysates. S273-paxillin phosphorylation only marginally affected FAK binding. (f) GIT1 was immunoprecipitated from in vitro mixtures of FLAG-GIT1, untagged WT-paxillin, and either KD- or CA-PAK using anti-FLAG M2-conjugated agarose, and phospho–S273-paxillin binding was probed using a phospho–S273-paxillin antibody. The middle blot shows equal levels of FLAG-GIT1 using an anti-FLAG antibody. (bottom) Equal loading of the lysates using anti-myc and anti-paxillin antibodies, respectively. Phospho–S273-paxillin–GIT1 binding increased sevenfold with CA-PAK compared with KD-PAK. (g) Anti-FLAG M2-conjugated agarose was used to immunoprecipitate GIT1 from in vitro mixtures of FLAG-GIT1, untagged WT-paxillin, and CA-PAK preincubated with 500-fold molar excess of phospho– or nonphospho–S273-paxillin peptide, and phospho–S273-paxillin binding was assessed with a phospho–S273-paxillin antibody. Very low levels of phospho–S273-paxillin–GIT1 binding was detected with the competitive phosphopeptide (left), whereas a robust signal was observed with the noncompetitive peptide (right), confirming that the PAK-mediated increase in phospho–S273-paxillin–GIT1 binding is specific to S273-paxillin phosphorylation.

    Article Snippet: Antibodies and reagents Blebbistatin and CalyculinA were obtained from Calbiochem; DME from GIBCO BRL; fibronectin, protease inhibitor cocktail, Protein A–agarose beads, mouse-IgG beads, and anti-FLAG M2-conjugated agarose from Sigma-Aldrich; glutathione–Sepharose beads and ECL detection system from GE Healthcare, CCM1 from Hyclone, Nucleofection kit from Amaxa Biosytsems, and TnT T7-coupled reticulocyte lysate system from Promega.

    Techniques: Binding Assay, Kinase Assay, Synthesized, In Vitro, Immunoprecipitation, Expressing

    Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM sepharose. Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P

    Journal: Nature Communications

    Article Title: Molecular basis of AKAP79 regulation by calmodulin

    doi: 10.1038/s41467-017-01715-w

    Figure Lengend Snippet: Crystal structure of CaM in complex with its AKAP79 binding site. a Cartoon representation showing one of the two copies (chains B and D) of AKAP79 peptide (orange) bound to CaM ( blue ) in the asymmetric unit. The C-lobe (lighter blue ) is in the open conformation with each of its two EF hands coordinating Ca 2+ (yellow). b Rotation of the complex through 90° highlighting the position of the four hydrophobic amino acids comprising the 1-4-7-8 motif. c Reduction in alphascreen signal between biotin-CaM and GST-AKAP79 (1–153) upon addition of 20-mer peptides derived from AKAP79 77–96 ( n = 4). The effects of point mutations within the disruptor peptide were compared. d Binding of purified full-length WT, Δ79–86 or W79A AKAP79 to CaM sepharose. Each AKAP79 variant was purified in complex with the D/D of RIIα. AKAP79 was released from the beads by incubation with EGTA, and detected by anti-AKAP79 IB. The experiment was performed in triplicate with each replicate leading to the same pattern of bands. e Limited Ramachandran plot showing dihedral angles for both copies of AKAP79 positions 80–85 in the asymmetric unit. Black triangles represent amino acids with angles characteristic of 3 10 helices; white diamonds are amino acids with α-helical geometry. f Representation of backbone H-bonds within the AKAP79 helix with distances shown in Å. The two α-helix-type bonds are shown by dotted lines; 3 10 -helical H-bonds as striped lines. The carbonyl group of S81 that does not H-bond to a backbone group is asterisked. g Rotation of the helix through 90°. The triangular backbone geometry of positions 83–86 is such that the side-chains of W79, L83 and T86 extend in the same direction. ** P

    Article Snippet: The eluted protein was buffer exchanged into glutathione sepharose binding buffer (25 mM Tris pH 7.4, 500 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mM Benzamidine) using a Sephadex G-25 column, before 3 h incubation with glutathione sepharose 4B (GE Life Sciences).

    Techniques: Chick Chorioallantoic Membrane Assay, Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Derivative Assay, Purification, Variant Assay, Incubation

    Delineation of key residues in AKAP79 required for CaM binding. a Pull-down of either WT, Δ33–48, Δ79–86, or Δ391–400 FLAG-tagged-AKAP79 (inputs shown in bottom panel) with either CaM sepharose (top panel) or cAMP agarose (middle panel). AKAP79 was detected by anti-FLAG immunoblotting. The experiment was performed in triplicate with each replicate producing the same pattern of bands. b Sequence LOGO for AKAP5 gene products aligned with predicted helical region. The cross-linking cluster between AKAP79 positions 90–99 and K94 in CaM is indicated along with the boundaries of peptides used in the following panels. c – e Determination of inhibitory constants for the peptides outlined in ( b ) in disrupting interaction between biotin-CaM and GST-AKAP79 (1–153) detected using the alphascreen assay ( n = 4 for all data points). K i constants were determined for the 9-mer and 11-mer peptides c , 16-mer and 20-mer peptides d , and for either WT or L101A 26-mer peptides e .***P

    Journal: Nature Communications

    Article Title: Molecular basis of AKAP79 regulation by calmodulin

    doi: 10.1038/s41467-017-01715-w

    Figure Lengend Snippet: Delineation of key residues in AKAP79 required for CaM binding. a Pull-down of either WT, Δ33–48, Δ79–86, or Δ391–400 FLAG-tagged-AKAP79 (inputs shown in bottom panel) with either CaM sepharose (top panel) or cAMP agarose (middle panel). AKAP79 was detected by anti-FLAG immunoblotting. The experiment was performed in triplicate with each replicate producing the same pattern of bands. b Sequence LOGO for AKAP5 gene products aligned with predicted helical region. The cross-linking cluster between AKAP79 positions 90–99 and K94 in CaM is indicated along with the boundaries of peptides used in the following panels. c – e Determination of inhibitory constants for the peptides outlined in ( b ) in disrupting interaction between biotin-CaM and GST-AKAP79 (1–153) detected using the alphascreen assay ( n = 4 for all data points). K i constants were determined for the 9-mer and 11-mer peptides c , 16-mer and 20-mer peptides d , and for either WT or L101A 26-mer peptides e .***P

    Article Snippet: The eluted protein was buffer exchanged into glutathione sepharose binding buffer (25 mM Tris pH 7.4, 500 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mM Benzamidine) using a Sephadex G-25 column, before 3 h incubation with glutathione sepharose 4B (GE Life Sciences).

    Techniques: Chick Chorioallantoic Membrane Assay, Binding Assay, Sequencing, Amplified Luminescent Proximity Homogenous Assay