Structured Review

GE Healthcare glutathione sepharose beads
PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After <t>glutathione-agarose</t> pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.
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Images

1) Product Images from "Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation *"

Article Title: Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.729699

PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After glutathione-agarose pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.
Figure Legend Snippet: PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After glutathione-agarose pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.

Techniques Used: In Vitro, Incubation, Recombinant, Autoradiography, Staining, SDS Page, Marker, Mutagenesis, Sequencing, Binding Assay, Western Blot

2) Product Images from "HCMV Encoded Glycoprotein M (UL100) Interacts with Rab11 Effector Protein FIP4"

Article Title: HCMV Encoded Glycoprotein M (UL100) Interacts with Rab11 Effector Protein FIP4

Journal: Traffic (Copenhagen, Denmark)

doi: 10.1111/j.1600-0854.2009.00967.x

FIP4 interact with the gM-CT (A) Pull down assay in which glutathione sepharose beads containing gM-CT constructs (GST-gM-CT, GST-gM-ac1, GST-gM-ac2) or purified GST alone were incubated with the HK293 cell lysate expressing FIP4- myc . After extensive washing protein samples were boiled in sample buffer, resolved by SDS-PAGE and assayed by western analysis. Western blots were probed with anti-myc (9E10) mab and detected with the HRP (top panel). The lower panel shows the Coomassie blue stained gels of the GST purified input of proteins used in pull down assay. (B) Pull down assay of FIP3 or FIP4 by cytoplasmic tail of gM. Glutathione sepharose beads containing gM-CT constructs or GST alone were incubated with HK293 cell lysates expressing FIP3- myc or FIP4- myc. After extensive washing, beads were boiled and eluted proteins resolved by SDS-PAGE followed by western blot analysis. Western blots were probed with anti-myc mab and detected with the HRP. (C) Fluorescence Resonance Energy Transfer (FRET) indicating strong FIP4 and gM interaction in HCMV infected cells. For FRET assays, HFF cells were electroporated with FIP4 or FIP3 myc- tagged constructs and plated on the coverslips. 24 hours later, the cells were infected with HCMV. The cells were fixed in 4% PFA and stained with IMP anti-gM monoclonal antibodies (specific for the C-terminal tail of gM) or anti- myc antibody followed by labeling with secondary antibody conjugated with FITC or TxRed. A non-bleaching region was selected as an internal control of FRET analysis detail description of the assay is provided in Materials and Methods.
Figure Legend Snippet: FIP4 interact with the gM-CT (A) Pull down assay in which glutathione sepharose beads containing gM-CT constructs (GST-gM-CT, GST-gM-ac1, GST-gM-ac2) or purified GST alone were incubated with the HK293 cell lysate expressing FIP4- myc . After extensive washing protein samples were boiled in sample buffer, resolved by SDS-PAGE and assayed by western analysis. Western blots were probed with anti-myc (9E10) mab and detected with the HRP (top panel). The lower panel shows the Coomassie blue stained gels of the GST purified input of proteins used in pull down assay. (B) Pull down assay of FIP3 or FIP4 by cytoplasmic tail of gM. Glutathione sepharose beads containing gM-CT constructs or GST alone were incubated with HK293 cell lysates expressing FIP3- myc or FIP4- myc. After extensive washing, beads were boiled and eluted proteins resolved by SDS-PAGE followed by western blot analysis. Western blots were probed with anti-myc mab and detected with the HRP. (C) Fluorescence Resonance Energy Transfer (FRET) indicating strong FIP4 and gM interaction in HCMV infected cells. For FRET assays, HFF cells were electroporated with FIP4 or FIP3 myc- tagged constructs and plated on the coverslips. 24 hours later, the cells were infected with HCMV. The cells were fixed in 4% PFA and stained with IMP anti-gM monoclonal antibodies (specific for the C-terminal tail of gM) or anti- myc antibody followed by labeling with secondary antibody conjugated with FITC or TxRed. A non-bleaching region was selected as an internal control of FRET analysis detail description of the assay is provided in Materials and Methods.

Techniques Used: Pull Down Assay, Construct, Purification, Incubation, Expressing, SDS Page, Western Blot, Staining, Fluorescence, Förster Resonance Energy Transfer, Infection, Labeling

FIP4 bound to the gM-CT recruits Rab11 but fails to bind Arf5 or Arf6. HK293 cells were transfected with FIP4- myc alone or co-transfected with FIP4-myc and either with HA-Arf5, HA-Arf6, GFP-Rab11, and Arf1-HA as a control. Two days post transfection cells were lysed and analyzed by immunoprecipitation with myc antibody-tagged magnetic beads as described in the Materials and Methods or incubated with sepharose beads containing purified GST-gM-CT or GST alone. After immunoprecipitation and extensive washing, precipitated protein were resolved by SDS-PAGE, transferred to membranes and probed with appropriate antibody. The asterisk indicates HA-Arf6 band which was visualized only after prolonged exposure of the membrane that had been probed with anti-HA and developed with HRP.
Figure Legend Snippet: FIP4 bound to the gM-CT recruits Rab11 but fails to bind Arf5 or Arf6. HK293 cells were transfected with FIP4- myc alone or co-transfected with FIP4-myc and either with HA-Arf5, HA-Arf6, GFP-Rab11, and Arf1-HA as a control. Two days post transfection cells were lysed and analyzed by immunoprecipitation with myc antibody-tagged magnetic beads as described in the Materials and Methods or incubated with sepharose beads containing purified GST-gM-CT or GST alone. After immunoprecipitation and extensive washing, precipitated protein were resolved by SDS-PAGE, transferred to membranes and probed with appropriate antibody. The asterisk indicates HA-Arf6 band which was visualized only after prolonged exposure of the membrane that had been probed with anti-HA and developed with HRP.

Techniques Used: Transfection, Immunoprecipitation, Magnetic Beads, Incubation, Purification, SDS Page

3) Product Images from "Sequential Protein Association with Nascent 60S Ribosomal Particles"

Article Title: Sequential Protein Association with Nascent 60S Ribosomal Particles

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.13.4449-4460.2003

Rlp24 and Nog1 genetically and physically interact. (A) The Rlp24-TAP strain was transformed with a high-copy-number LEU2 vector (pFL46S) carrying no insert or inserts corresponding to Nog1 or Rlp24. The rescue of the slow-growth phenotype was tested by spotting transformants on minimal-medium plates in 10 −1 -dilution steps. The plates were incubated at 25°C for 4 days. (B) Total protein extracts from bacteria expressing GST-Rlp24, GST-Rpl24B, and GST-Rpl5 were incubated with extracts containing Nog1 and then with glutathione-Sepharose beads (Pharmacia). After extensive washing, the bound proteins were eluted under denaturing conditions and separated by electrophoresis. The Coomassie blue-stained gels representing the input mixture of bacterial protein extracts (left) and the purified proteins (right) are shown side by side for comparison. A band of about 38 kDa that copurifies with GST-Rlp24 only in the presence of Nog1 (∗) is a C-terminal Nog1 fragment (matching peptides between amino acids 361 and 599 as determined by MALDI-TOF mass spectrometry and assignment of peptide masses to the Nog1 sequence).
Figure Legend Snippet: Rlp24 and Nog1 genetically and physically interact. (A) The Rlp24-TAP strain was transformed with a high-copy-number LEU2 vector (pFL46S) carrying no insert or inserts corresponding to Nog1 or Rlp24. The rescue of the slow-growth phenotype was tested by spotting transformants on minimal-medium plates in 10 −1 -dilution steps. The plates were incubated at 25°C for 4 days. (B) Total protein extracts from bacteria expressing GST-Rlp24, GST-Rpl24B, and GST-Rpl5 were incubated with extracts containing Nog1 and then with glutathione-Sepharose beads (Pharmacia). After extensive washing, the bound proteins were eluted under denaturing conditions and separated by electrophoresis. The Coomassie blue-stained gels representing the input mixture of bacterial protein extracts (left) and the purified proteins (right) are shown side by side for comparison. A band of about 38 kDa that copurifies with GST-Rlp24 only in the presence of Nog1 (∗) is a C-terminal Nog1 fragment (matching peptides between amino acids 361 and 599 as determined by MALDI-TOF mass spectrometry and assignment of peptide masses to the Nog1 sequence).

Techniques Used: Transformation Assay, Plasmid Preparation, Incubation, Expressing, Electrophoresis, Staining, Purification, Mass Spectrometry, Sequencing

4) Product Images from "She4p/Dim1p Interacts with the Motor Domain of Unconventional Myosins in the Budding Yeast, Saccharomyces cerevisiae"

Article Title: She4p/Dim1p Interacts with the Motor Domain of Unconventional Myosins in the Budding Yeast, Saccharomyces cerevisiae

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-09-0616

She4p interacts with Myo5p in a temperature-dependent manner. Myo5p-TAP from YKT323 lysate was adsorbed to IgG Sepharose. GST-She4p was purified from bacterial lysate. IgG Sepharose with or without Myo5p-TAP was incubated with GST-She4p at 4 or 30°C. The IgG Sepharose was collected, and bound proteins were analyzed by SDS-PAGE and SYPRO orange staining. The result shown is a representative of four experiments.
Figure Legend Snippet: She4p interacts with Myo5p in a temperature-dependent manner. Myo5p-TAP from YKT323 lysate was adsorbed to IgG Sepharose. GST-She4p was purified from bacterial lysate. IgG Sepharose with or without Myo5p-TAP was incubated with GST-She4p at 4 or 30°C. The IgG Sepharose was collected, and bound proteins were analyzed by SDS-PAGE and SYPRO orange staining. The result shown is a representative of four experiments.

Techniques Used: Purification, Incubation, SDS Page, Staining

5) Product Images from "PNUTS functions as a proto-oncogene by sequestering PTEN"

Article Title: PNUTS functions as a proto-oncogene by sequestering PTEN

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-12-1394

PNUTS is a novel PTEN associated protein (a) Co-immunoprecipitation of endogenous PTEN and PNUTS was carried out using extracts prepared from HEK 293T cells. The presence of PNUTS in PTEN complex and vice versa was evaluated by immunoblotting with the indicated antibodies. (b) Lysates from bacteria expressing MBP-PTEN were pulled down with GST alone or GST-PNUTS containing sepharose beads and immunoblotted with anti-MBP antibody. (c) Schematic representation of N-terminal SFB-tagged PTEN (FL), along with its various deletion mutants (D1-D5). (d) SFB-tagged PTEN Full Length and domain deletions were expressed in HEK 293T cells along with Full Length MYC-PNUTS, the cell lysates were pulled down with Streptavidin beads and the interaction of PNUTS was detected with anti-Myc antibody. The expression of PTEN and PNUTS was checked by immunoblotting with anti-Flag and anti-Myc antibodies respectively. (e) Schematic representation of N-terminal SFB-tagged PNUTS (FL), along with its deletion mutants (D1-D7). (f) SFB-tagged PNUTS constructs and Myc-PTEN were co-expressed in HEK293T cells, the cell lysates were pulled down with streptavidin beads and the interaction of PTEN was detected by immunoblotting with anti-Myc antibodies.
Figure Legend Snippet: PNUTS is a novel PTEN associated protein (a) Co-immunoprecipitation of endogenous PTEN and PNUTS was carried out using extracts prepared from HEK 293T cells. The presence of PNUTS in PTEN complex and vice versa was evaluated by immunoblotting with the indicated antibodies. (b) Lysates from bacteria expressing MBP-PTEN were pulled down with GST alone or GST-PNUTS containing sepharose beads and immunoblotted with anti-MBP antibody. (c) Schematic representation of N-terminal SFB-tagged PTEN (FL), along with its various deletion mutants (D1-D5). (d) SFB-tagged PTEN Full Length and domain deletions were expressed in HEK 293T cells along with Full Length MYC-PNUTS, the cell lysates were pulled down with Streptavidin beads and the interaction of PNUTS was detected with anti-Myc antibody. The expression of PTEN and PNUTS was checked by immunoblotting with anti-Flag and anti-Myc antibodies respectively. (e) Schematic representation of N-terminal SFB-tagged PNUTS (FL), along with its deletion mutants (D1-D7). (f) SFB-tagged PNUTS constructs and Myc-PTEN were co-expressed in HEK293T cells, the cell lysates were pulled down with streptavidin beads and the interaction of PTEN was detected by immunoblotting with anti-Myc antibodies.

Techniques Used: Immunoprecipitation, Expressing, Construct

6) Product Images from "Phosphorylation-induced Conformational Changes in Rap1b"

Article Title: Phosphorylation-induced Conformational Changes in Rap1b

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.011312

Effect of phosphorylation of Rap1b on its interaction with RalGDS. Purified proteins were loaded in vitro with GTP or GDP, and its interaction with RalGDS was analyzed by an RBD pulldown assay utilizing GST-RalGDS RBD precoupled to glutathione-Sepharose
Figure Legend Snippet: Effect of phosphorylation of Rap1b on its interaction with RalGDS. Purified proteins were loaded in vitro with GTP or GDP, and its interaction with RalGDS was analyzed by an RBD pulldown assay utilizing GST-RalGDS RBD precoupled to glutathione-Sepharose

Techniques Used: Purification, In Vitro

7) Product Images from "A proteomic approach to identify endosomal cargoes controlling cancer invasiveness"

Article Title: A proteomic approach to identify endosomal cargoes controlling cancer invasiveness

Journal: Journal of Cell Science

doi: 10.1242/jcs.190835

Rab17 is a key regulator and interacting protein of the endosomal v-SNARE Vamp8. (A) Kaplan–Meier plot showing the influence of Rab17 expression on overall survival from breast cancer. The red and black lines represent patients with Rab17 expression above and below the median, respectively. n =1778 patients (low Rab17); n =1776 patients (high Rab17). Log rank test, P =1.2×10 −11 . (B) MDA-MB-231 cells that had been SILAC-labelled with heavy- and light-isotope amino acids were transfected with non-targeting siRNA (si-Con) or an siRNA targeting Rab17 (si-Rab17), respectively [forward (Fw) experiment]. For the reverse (Rev) experiment, these labelling conditions were swapped. Scatter plot indicates the SILAC ratio between siRab17 and siCon cells (si-Rab17/si-Con; log 2 scale) for each protein obtained for the forward versus the reverse experiments for the whole-cell proteome. Blue dotted lines indicate the regions encompassing significantly affected proteins (significance B statistical test, FDR of 5%, Perseus software). (C–E) MDA-MB-231 cells were transfected with siRNAs targeting Rab17 (SMARTPool or individual oligonucleotide Rab17#1), Vamp8 [SMARTPool (SP)], ERK2 or non-targeting controls (si-Con#1 and si-Con#2). Vamp8, Vamp7, Vamp3 and ERK1 and ERK2 expression levels were then determined by western blotting. In E, Rab17 expression was determined by using quantitative PCR (qPCR) relative to that in control (con) (graph). Data are mean±s.e.m. (F) MDA-MB-231 cells were transfected with GFP or GFP–Rab17. GFP-tagged proteins were isolated using GFP-Trap_A agarose beads (Chromotek). Proteins eluted from the beads were resolved by SDS-PAGE, stained with Coomassie Blue (Expedeon) and in-gel digested for MS analysis. Statistical testing of differences between means (Welch’s t -test) was performed to show the most differentially expressed proteins across three independent experimental replicates. The red dotted line indicates the significance threshold. Blue dots denote the 95 significant interacting proteins identified. (G) MDA-MB-231 cells were transfected with GFP, GFP–Rab17 or GFP–Rab24. GFP-tagged proteins were isolated as described in A, and immunoprecipitates were analysed by western blotting with antibodies recognising GFP, Vamp8 or Vamp7. IP, immunoprecipitation. (H) Zoom of plot in Fig. 1 F showing the identity (gene name) of late-endosome- and lysosome-associated proteins (red dots) in the Rab17 interactome. 43% of the members of the Rab17 interactome belong to the lysosome and late-endosome category, and have been annotated according to Chapel et al. (2013) .
Figure Legend Snippet: Rab17 is a key regulator and interacting protein of the endosomal v-SNARE Vamp8. (A) Kaplan–Meier plot showing the influence of Rab17 expression on overall survival from breast cancer. The red and black lines represent patients with Rab17 expression above and below the median, respectively. n =1778 patients (low Rab17); n =1776 patients (high Rab17). Log rank test, P =1.2×10 −11 . (B) MDA-MB-231 cells that had been SILAC-labelled with heavy- and light-isotope amino acids were transfected with non-targeting siRNA (si-Con) or an siRNA targeting Rab17 (si-Rab17), respectively [forward (Fw) experiment]. For the reverse (Rev) experiment, these labelling conditions were swapped. Scatter plot indicates the SILAC ratio between siRab17 and siCon cells (si-Rab17/si-Con; log 2 scale) for each protein obtained for the forward versus the reverse experiments for the whole-cell proteome. Blue dotted lines indicate the regions encompassing significantly affected proteins (significance B statistical test, FDR of 5%, Perseus software). (C–E) MDA-MB-231 cells were transfected with siRNAs targeting Rab17 (SMARTPool or individual oligonucleotide Rab17#1), Vamp8 [SMARTPool (SP)], ERK2 or non-targeting controls (si-Con#1 and si-Con#2). Vamp8, Vamp7, Vamp3 and ERK1 and ERK2 expression levels were then determined by western blotting. In E, Rab17 expression was determined by using quantitative PCR (qPCR) relative to that in control (con) (graph). Data are mean±s.e.m. (F) MDA-MB-231 cells were transfected with GFP or GFP–Rab17. GFP-tagged proteins were isolated using GFP-Trap_A agarose beads (Chromotek). Proteins eluted from the beads were resolved by SDS-PAGE, stained with Coomassie Blue (Expedeon) and in-gel digested for MS analysis. Statistical testing of differences between means (Welch’s t -test) was performed to show the most differentially expressed proteins across three independent experimental replicates. The red dotted line indicates the significance threshold. Blue dots denote the 95 significant interacting proteins identified. (G) MDA-MB-231 cells were transfected with GFP, GFP–Rab17 or GFP–Rab24. GFP-tagged proteins were isolated as described in A, and immunoprecipitates were analysed by western blotting with antibodies recognising GFP, Vamp8 or Vamp7. IP, immunoprecipitation. (H) Zoom of plot in Fig. 1 F showing the identity (gene name) of late-endosome- and lysosome-associated proteins (red dots) in the Rab17 interactome. 43% of the members of the Rab17 interactome belong to the lysosome and late-endosome category, and have been annotated according to Chapel et al. (2013) .

Techniques Used: Expressing, Multiple Displacement Amplification, Transfection, Software, Western Blot, Real-time Polymerase Chain Reaction, Isolation, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation

8) Product Images from "Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap"

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.02119

Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).
Figure Legend Snippet: Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

Techniques Used: Staining, Purification, SDS Page, Pull Down Assay, Incubation, In Vitro, Labeling, Autoradiography, Immunoprecipitation

Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.
Figure Legend Snippet: Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.

Techniques Used: In Vitro, Luciferase, Synthesized, Centrifugation, Incubation, Activity Assay, In Vivo, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction

m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.
Figure Legend Snippet: m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.

Techniques Used: Binding Assay, Infection, Transfection, Plasmid Preparation, Incubation, Western Blot, Software, SDS Page

Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.
Figure Legend Snippet: Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

Techniques Used: Pull Down Assay, Purification, Incubation, In Vitro, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Transfection, Western Blot, Infection, CTL Assay, Confocal Microscopy, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining

9) Product Images from "Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap"

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.02119

Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).
Figure Legend Snippet: Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [ 35 S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

Techniques Used: Staining, Purification, SDS Page, Pull Down Assay, Incubation, In Vitro, Labeling, Autoradiography, Immunoprecipitation

Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.
Figure Legend Snippet: Effect of pVIII on capped mRNA translation. (A). In vitro . The TNT ® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo . 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ∗ statistically significant.

Techniques Used: In Vitro, Luciferase, Synthesized, Centrifugation, Incubation, Activity Assay, In Vivo, Transfection, Plasmid Preparation, Expressing, Reverse Transcription Polymerase Chain Reaction

m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.
Figure Legend Snippet: m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.

Techniques Used: Binding Assay, Infection, Transfection, Plasmid Preparation, Incubation, Western Blot, Software, SDS Page

Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.
Figure Legend Snippet: Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

Techniques Used: Pull Down Assay, Purification, Incubation, In Vitro, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Transfection, Western Blot, Infection, CTL Assay, Confocal Microscopy, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining

10) Product Images from "The Putative RNA Helicase HELZ Promotes Cell Proliferation, Translation Initiation and Ribosomal Protein S6 Phosphorylation"

Article Title: The Putative RNA Helicase HELZ Promotes Cell Proliferation, Translation Initiation and Ribosomal Protein S6 Phosphorylation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022107

HELZ interacts with the poly(A)-binding protein (PABP). GST pull-down using glutathione-sepharose beads was conducted by incubating crude HeLa cell lysates with recombinant GST, GST-Paip2 106–127 and GST-HELZ 1023–1199 ( A ), GST and the indicated GST-HELZ fragments ( D ), or crude HEK293 cell lysates with GST and GST-PABP 554–636 ( C ). Hypoxic HeLa cell lysates were incubated with GST or GST-HELZ 1023–1199 ( B ). Eluates were subjected to SDS-PAGE and immunoblotting using the indicated antibodies.
Figure Legend Snippet: HELZ interacts with the poly(A)-binding protein (PABP). GST pull-down using glutathione-sepharose beads was conducted by incubating crude HeLa cell lysates with recombinant GST, GST-Paip2 106–127 and GST-HELZ 1023–1199 ( A ), GST and the indicated GST-HELZ fragments ( D ), or crude HEK293 cell lysates with GST and GST-PABP 554–636 ( C ). Hypoxic HeLa cell lysates were incubated with GST or GST-HELZ 1023–1199 ( B ). Eluates were subjected to SDS-PAGE and immunoblotting using the indicated antibodies.

Techniques Used: Binding Assay, Recombinant, Incubation, SDS Page

11) Product Images from "Transgenic Evaluation of Activated Mutant Alleles of SOS2 Reveals a Critical Requirement for Its Kinase Activity and C-Terminal Regulatory Domain for Salt Tolerance in Arabidopsis thaliana"

Article Title: Transgenic Evaluation of Activated Mutant Alleles of SOS2 Reveals a Critical Requirement for Its Kinase Activity and C-Terminal Regulatory Domain for Salt Tolerance in Arabidopsis thaliana

Journal: The Plant Cell

doi: 10.1105/tpc.019174

Expression of T/DSOS2 in A. thaliana . T/DSOS2 transcript levels in the wild-type and WT/T/DSOS2 transgenic lines (A) . RNA gel blot analysis with total RNA extracted from the wild type and two WT/T/DSOS2 lines grown in the absence of NaCl. 25S rRNA (ethidium bromide stained) was used as a loading control. Total protein was extracted from the wild-type and transgenic plants and incubated with GST-SOS3 coupled to glutathione-Sepharose beads. The GST-SOS3-T/DSOS2/SOS2 protein complex was used for immunoblot analysis with anti-SOS2 antibody (B) and peptide phosphorylation assays (C) .
Figure Legend Snippet: Expression of T/DSOS2 in A. thaliana . T/DSOS2 transcript levels in the wild-type and WT/T/DSOS2 transgenic lines (A) . RNA gel blot analysis with total RNA extracted from the wild type and two WT/T/DSOS2 lines grown in the absence of NaCl. 25S rRNA (ethidium bromide stained) was used as a loading control. Total protein was extracted from the wild-type and transgenic plants and incubated with GST-SOS3 coupled to glutathione-Sepharose beads. The GST-SOS3-T/DSOS2/SOS2 protein complex was used for immunoblot analysis with anti-SOS2 antibody (B) and peptide phosphorylation assays (C) .

Techniques Used: Expressing, Transgenic Assay, Western Blot, Staining, Incubation

Expression of T/DSOS2 in sos2-2 and sos3-1 . RNA gel blot analysis of T/DSOS2 expression in sos3-1 , sos2-2 or sos3-1 and sos2-2 transgenic lines grown in the absence of NaCl (A) . 25S rRNA (ethidium bromide stained) was used as a loading control. Total protein was extracted from mutant and transgenic plants and incubated with GST-SOS3 coupled to glutathione-Sepharose beads. The GST-SOS3-T/DSOS2/SOS2 complex was used for immunoblot analysis (B) with protein from sos3-1 (1) and sos2-2 (3) mutants or sos3-1 (2) and sos2-2 (4) transgenic lines. Proteins were probed with anti-SOS2 antibody. The GST-SOS3-T/DSOS2/SOS2 complex was also used for peptide phosphorylation assays (C) with protein from sos3-1 (1), the sos3-1 transgenic line (2), sos2-2 (3), and the sos2-2 transgenic line (4).
Figure Legend Snippet: Expression of T/DSOS2 in sos2-2 and sos3-1 . RNA gel blot analysis of T/DSOS2 expression in sos3-1 , sos2-2 or sos3-1 and sos2-2 transgenic lines grown in the absence of NaCl (A) . 25S rRNA (ethidium bromide stained) was used as a loading control. Total protein was extracted from mutant and transgenic plants and incubated with GST-SOS3 coupled to glutathione-Sepharose beads. The GST-SOS3-T/DSOS2/SOS2 complex was used for immunoblot analysis (B) with protein from sos3-1 (1) and sos2-2 (3) mutants or sos3-1 (2) and sos2-2 (4) transgenic lines. Proteins were probed with anti-SOS2 antibody. The GST-SOS3-T/DSOS2/SOS2 complex was also used for peptide phosphorylation assays (C) with protein from sos3-1 (1), the sos3-1 transgenic line (2), sos2-2 (3), and the sos2-2 transgenic line (4).

Techniques Used: Expressing, Western Blot, Transgenic Assay, Staining, Mutagenesis, Incubation

12) Product Images from "Molecular and functional characterization of the only known hemiascomycete ortholog of the carboxyl terminus of Hsc70-interacting protein CHIP in the yeast Yarrowia lipolytica"

Article Title: Molecular and functional characterization of the only known hemiascomycete ortholog of the carboxyl terminus of Hsc70-interacting protein CHIP in the yeast Yarrowia lipolytica

Journal: Cell Stress & Chaperones

doi: 10.1007/s12192-011-0302-6

The interaction of Yl. Chn1p with S. cerevisiae Ssa1p is inhibited by Fes1p but not by Sse1p or Ydj1p. a GST or GST– Yl. Chn1p (10 μg) were bound to glutathione sepharose beads, and then S. cerevisiae Ssa1p (5 μg)
Figure Legend Snippet: The interaction of Yl. Chn1p with S. cerevisiae Ssa1p is inhibited by Fes1p but not by Sse1p or Ydj1p. a GST or GST– Yl. Chn1p (10 μg) were bound to glutathione sepharose beads, and then S. cerevisiae Ssa1p (5 μg)

Techniques Used:

13) Product Images from "SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity"

Article Title: SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

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

doi: 10.1073/pnas.1617981114

VAMP8 or VAMP2 bind to a recombinant STX11/GST–SNAP23 complex. ( A ) Equivalent amounts of recombinant GST (GST alone) or GST–SNAP23 were bound to glutathione-Sepharose beads, and increasing concentrations (0.5, 1.0, or 1.5 μg) of
Figure Legend Snippet: VAMP8 or VAMP2 bind to a recombinant STX11/GST–SNAP23 complex. ( A ) Equivalent amounts of recombinant GST (GST alone) or GST–SNAP23 were bound to glutathione-Sepharose beads, and increasing concentrations (0.5, 1.0, or 1.5 μg) of

Techniques Used: Recombinant

14) Product Images from "Kaposi Sarcoma-associated Herpesvirus vIRF-3 Protein Binds to F-box of Skp2 Protein and Acts as a Regulator of c-Myc Protein Function and Stability *"

Article Title: Kaposi Sarcoma-associated Herpesvirus vIRF-3 Protein Binds to F-box of Skp2 Protein and Acts as a Regulator of c-Myc Protein Function and Stability *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.335216

vIRF-3 neither inhibits c-Myc/Skp2 interaction nor blocks c-Myc ubiquitylation. A , increased binding of c-Myc to Skp2 in the presence of vIRF-3. Constant amounts of in vitro translated c-Myc-HA protein were incubated with Skp2-GST immobilized to glutathione-Sepharose
Figure Legend Snippet: vIRF-3 neither inhibits c-Myc/Skp2 interaction nor blocks c-Myc ubiquitylation. A , increased binding of c-Myc to Skp2 in the presence of vIRF-3. Constant amounts of in vitro translated c-Myc-HA protein were incubated with Skp2-GST immobilized to glutathione-Sepharose

Techniques Used: Binding Assay, In Vitro, Incubation

15) Product Images from "Defective in Mitotic Arrest 1 (Dma1) Ubiquitin Ligase Controls G1 Cyclin Degradation *"

Article Title: Defective in Mitotic Arrest 1 (Dma1) Ubiquitin Ligase Controls G1 Cyclin Degradation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.426593

Dma1 interacts and ubiquitinates Pcl1. A , binding in vitro assay. Recombinant GST-Pcl1 was purified from E. coli and incubated with yeast cell extracts of the strain YPC708 (with a genomic TAP-tagged Dma1) for 45 min, and purified with glutathione-Sepharose beads. Samples were analyzed by immunoblot analysis to detect Dma1-TAP proteins. WCE, whole cell extract; Input , quantity of Pcl1 used in the trapping. B , Pcl1-Pho85 complexes co-immunoprecipitate with Dma1 in vivo . Yeast extracts from strains containing untagged PHO85 or GST- PHO85 and TAP-tagged DMA1 (from the chromosomal locus) were precipitated with glutathione-Sepharose beads, and then probed using specific antibodies. C and D, Pcl1 is ubiquitinated in vitro by Dma1. Ubiquitination assays were done by in vitro reconstitution of E1-E2-Dma1 complexes (see “Experimental Procedures”). Panel C shows Dma1 activity associated to the Mms2-Ubc13 E2 complexes. Panel D shows Dma1 activity associated to the Ubc4 E2 enzyme. The reaction was started at time 0 by adding ATP. Samples were taken at 0 and 5 h and analyzed for ubiquitination levels by immunoblotting using α-Ub antibody. E , Pcl1 is ubiquitinated in vivo in a Dma1-dependent manner. The indicated strains carrying a centromeric plasmid with PCL1 -His 6x -TAP expressed under the GAL promoter was grown for 4 h in synthetic complete medium with galactose as a carbon source. The same amount of immunopurified Pcl1-His was separated by SDS-PAGE gels and visualized by immunoblot using an ubiquitin antibody ( top panels ). To validate the levels of Pcl1, the blot was also analyzed using an α-TAP antibody ( bottom panel ).
Figure Legend Snippet: Dma1 interacts and ubiquitinates Pcl1. A , binding in vitro assay. Recombinant GST-Pcl1 was purified from E. coli and incubated with yeast cell extracts of the strain YPC708 (with a genomic TAP-tagged Dma1) for 45 min, and purified with glutathione-Sepharose beads. Samples were analyzed by immunoblot analysis to detect Dma1-TAP proteins. WCE, whole cell extract; Input , quantity of Pcl1 used in the trapping. B , Pcl1-Pho85 complexes co-immunoprecipitate with Dma1 in vivo . Yeast extracts from strains containing untagged PHO85 or GST- PHO85 and TAP-tagged DMA1 (from the chromosomal locus) were precipitated with glutathione-Sepharose beads, and then probed using specific antibodies. C and D, Pcl1 is ubiquitinated in vitro by Dma1. Ubiquitination assays were done by in vitro reconstitution of E1-E2-Dma1 complexes (see “Experimental Procedures”). Panel C shows Dma1 activity associated to the Mms2-Ubc13 E2 complexes. Panel D shows Dma1 activity associated to the Ubc4 E2 enzyme. The reaction was started at time 0 by adding ATP. Samples were taken at 0 and 5 h and analyzed for ubiquitination levels by immunoblotting using α-Ub antibody. E , Pcl1 is ubiquitinated in vivo in a Dma1-dependent manner. The indicated strains carrying a centromeric plasmid with PCL1 -His 6x -TAP expressed under the GAL promoter was grown for 4 h in synthetic complete medium with galactose as a carbon source. The same amount of immunopurified Pcl1-His was separated by SDS-PAGE gels and visualized by immunoblot using an ubiquitin antibody ( top panels ). To validate the levels of Pcl1, the blot was also analyzed using an α-TAP antibody ( bottom panel ).

Techniques Used: Binding Assay, In Vitro, Recombinant, Purification, Incubation, In Vivo, Activity Assay, Plasmid Preparation, SDS Page

16) Product Images from "CD4 and Major Histocompatibility Complex Class I Downregulation by the Human Immunodeficiency Virus Type 1 Nef Protein in Pediatric AIDS Progression"

Article Title: CD4 and Major Histocompatibility Complex Class I Downregulation by the Human Immunodeficiency Virus Type 1 Nef Protein in Pediatric AIDS Progression

Journal: Journal of Virology

doi: 10.1128/JVI.77.21.11536-11545.2003

Binding of AP-1 to GST-Nef fusion proteins. GST alone, GST-Nef wild-type (NL4-3), or GST fused to the indicated Nef proteins derived from patients was immobilized on Sepharose beads and incubated with Jurkat cell lysates. Bound proteins were eluted, separated by SDS-PAGE, immunoblotted with anti-AP-1 γ subunit (top panels) or anti-GST antibody (lower panels), and quantified by densitometry. As a control, 20 μg of Jurkat cell lysate was loaded on the same gel. Relative AP-1 binding activity was calculated as the amount of AP-1 γ subunit normalized for the amount of GST fusion protein and expressed as a percentage of the value measured for GST-wild-type Nef. Data representative of one of three independent experiments are shown. CD4 and MHC-I downregulation activities relative to wild-type Nef are indicated.
Figure Legend Snippet: Binding of AP-1 to GST-Nef fusion proteins. GST alone, GST-Nef wild-type (NL4-3), or GST fused to the indicated Nef proteins derived from patients was immobilized on Sepharose beads and incubated with Jurkat cell lysates. Bound proteins were eluted, separated by SDS-PAGE, immunoblotted with anti-AP-1 γ subunit (top panels) or anti-GST antibody (lower panels), and quantified by densitometry. As a control, 20 μg of Jurkat cell lysate was loaded on the same gel. Relative AP-1 binding activity was calculated as the amount of AP-1 γ subunit normalized for the amount of GST fusion protein and expressed as a percentage of the value measured for GST-wild-type Nef. Data representative of one of three independent experiments are shown. CD4 and MHC-I downregulation activities relative to wild-type Nef are indicated.

Techniques Used: Binding Assay, Derivative Assay, Incubation, SDS Page, Activity Assay

17) Product Images from "LATS1 and LATS2 Phosphorylate CDC26 to Modulate Assembly of the Tetratricopeptide Repeat Subcomplex of APC/C"

Article Title: LATS1 and LATS2 Phosphorylate CDC26 to Modulate Assembly of the Tetratricopeptide Repeat Subcomplex of APC/C

Journal: PLoS ONE

doi: 10.1371/journal.pone.0118662

LATS1-mediated phosphorylation of CDC26 T7 inhibits the interaction between CDC26 and APC6. (A–C) Pull-down assays showing the interaction of wild-type and mutant CDC26 with APC6. (A) The interaction of T7-phosphorylated CDC26 with APC6. Recombinant CDC26 and or distilled water (Ctrl) was incubated with 0 or 2 μl (400 ng) of GST-LATS1 in a cold in vitro kinase assay and then incubated with GST-tagged APC6 bound to glutathione-Sepharose beads. The input fraction and the bead-bound proteins were subjected to immunoblot analyses with the indicated antibodies. (B) The interaction of the CDC26 T7A and T7D mutants with APC6. Lysates from HeLa cells expressing FLAG alone (FLAG-mock) or FLAG-tagged wild-type, T7A-mutated, or T7D-mutated CDC26 were incubated with GST-tagged APC6 bound to glutathione-Sepharose beads. The bead-bound proteins and 3% of the input fraction were subjected to immunoblot analyses using the indicated antibodies. (C) HeLa cells expressing FLAG alone (FLAG-mock) or FLAG-tagged wild-type, T7A-mutated, T7D-mutated, or T7E-mutated CDC26 were transfected with an expression vector harboring HA-tagged APC6. Lysates of the transfected cells were immunoprecipitated with control IgG and an anti-FLAG antibody. The immunoprecipitates and 3% of the input fractions were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies.
Figure Legend Snippet: LATS1-mediated phosphorylation of CDC26 T7 inhibits the interaction between CDC26 and APC6. (A–C) Pull-down assays showing the interaction of wild-type and mutant CDC26 with APC6. (A) The interaction of T7-phosphorylated CDC26 with APC6. Recombinant CDC26 and or distilled water (Ctrl) was incubated with 0 or 2 μl (400 ng) of GST-LATS1 in a cold in vitro kinase assay and then incubated with GST-tagged APC6 bound to glutathione-Sepharose beads. The input fraction and the bead-bound proteins were subjected to immunoblot analyses with the indicated antibodies. (B) The interaction of the CDC26 T7A and T7D mutants with APC6. Lysates from HeLa cells expressing FLAG alone (FLAG-mock) or FLAG-tagged wild-type, T7A-mutated, or T7D-mutated CDC26 were incubated with GST-tagged APC6 bound to glutathione-Sepharose beads. The bead-bound proteins and 3% of the input fraction were subjected to immunoblot analyses using the indicated antibodies. (C) HeLa cells expressing FLAG alone (FLAG-mock) or FLAG-tagged wild-type, T7A-mutated, T7D-mutated, or T7E-mutated CDC26 were transfected with an expression vector harboring HA-tagged APC6. Lysates of the transfected cells were immunoprecipitated with control IgG and an anti-FLAG antibody. The immunoprecipitates and 3% of the input fractions were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies.

Techniques Used: Mutagenesis, Recombinant, Incubation, In Vitro, Kinase Assay, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation

18) Product Images from "Phosphorylation-enabled binding of Sgo1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis"

Article Title: Phosphorylation-enabled binding of Sgo1-PP2A to cohesin protects sororin and centromeric cohesion during mitosis

Journal: Nature cell biology

doi: 10.1038/ncb2637

Sgo1 T346 phosphorylation promotes its binding to cohesin ( a ) Mitotic HeLa Tet-On cells stably expressing Myc-Sgo1 WT or T346A (TA) were lysed with or without Turbo Nuclease. The total cell lysates (Input) and IgG/α-Smc1 immunoprecipitates (IP) were blotted with the indicated antibodies. ( b ) HeLa Tet-On cells were either mock transfected or transfected with siSgo1, collected at 7 hr after a thymidine block (G2) or in mitosis (M), and lysed with the nuclease-containing buffer. The total cell lysates and α-Smc1 IP were blotted with the indicated antibodies. ( c ) Glutathione-Sepharose beads bound to GST, GST-Sgo1 WT, or T346A (TA) proteins treated with buffer or cyclin B–Cdk1 were incubated with mitotic HeLa Tet-On cell extracts. The proteins bound to beads were blotted with the indicated antibodies. ( d ) Lysates of mitotic HeLa Tet-On cells stably expressing StrepII-SA2 were incubated with Strep-Tactin beads. After washing, the beads were incubated with GST-Sgo1 WT or T346A (TA) that had been treated with buffer or cyclin B–Cdk1. The input Sgo1 proteins and proteins bound to beads were blotted with the indicated antibodies. ( e ) HeLa Tet-On cells stably expressing Myc-Sgo1 were mocked transfected or transfected with the indicated siRNAs, collected at mitosis, and lysed in the presence of nuclease. The total cell lysates (Input) and α-Smc1 IP were blotted with the indicated antibodies. IgG IP from mock transfected cells was used as a negative control. Note that the commercial Wapl antibody (Bethyl) failed to detect Wapl in α-Smc1 IPs. ( f ) GST or GST-Sgo1 pre-treated with buffer or cyclin B–Cdk1 were immobilized on glutathione-Sepharose beads. The beads were then incubated with lysates of Sf9 cells expressing recombinant human Scc1-His 6 and SA2. The Sf9 lysate (Input) and proteins bound to beads were blotted with the indicated antibodies.
Figure Legend Snippet: Sgo1 T346 phosphorylation promotes its binding to cohesin ( a ) Mitotic HeLa Tet-On cells stably expressing Myc-Sgo1 WT or T346A (TA) were lysed with or without Turbo Nuclease. The total cell lysates (Input) and IgG/α-Smc1 immunoprecipitates (IP) were blotted with the indicated antibodies. ( b ) HeLa Tet-On cells were either mock transfected or transfected with siSgo1, collected at 7 hr after a thymidine block (G2) or in mitosis (M), and lysed with the nuclease-containing buffer. The total cell lysates and α-Smc1 IP were blotted with the indicated antibodies. ( c ) Glutathione-Sepharose beads bound to GST, GST-Sgo1 WT, or T346A (TA) proteins treated with buffer or cyclin B–Cdk1 were incubated with mitotic HeLa Tet-On cell extracts. The proteins bound to beads were blotted with the indicated antibodies. ( d ) Lysates of mitotic HeLa Tet-On cells stably expressing StrepII-SA2 were incubated with Strep-Tactin beads. After washing, the beads were incubated with GST-Sgo1 WT or T346A (TA) that had been treated with buffer or cyclin B–Cdk1. The input Sgo1 proteins and proteins bound to beads were blotted with the indicated antibodies. ( e ) HeLa Tet-On cells stably expressing Myc-Sgo1 were mocked transfected or transfected with the indicated siRNAs, collected at mitosis, and lysed in the presence of nuclease. The total cell lysates (Input) and α-Smc1 IP were blotted with the indicated antibodies. IgG IP from mock transfected cells was used as a negative control. Note that the commercial Wapl antibody (Bethyl) failed to detect Wapl in α-Smc1 IPs. ( f ) GST or GST-Sgo1 pre-treated with buffer or cyclin B–Cdk1 were immobilized on glutathione-Sepharose beads. The beads were then incubated with lysates of Sf9 cells expressing recombinant human Scc1-His 6 and SA2. The Sf9 lysate (Input) and proteins bound to beads were blotted with the indicated antibodies.

Techniques Used: Binding Assay, Stable Transfection, Expressing, Transfection, Blocking Assay, Incubation, Negative Control, Recombinant

19) Product Images from "The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins"

Article Title: The Sec34/Sec35p complex, a Ypt1p effector required for retrograde intra-Golgi trafficking, interacts with Golgi SNAREs and COPI vesicle coat proteins

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200111081

GST-tagged TFI1, TFI2, and TFI3 interact with both Sed5p and COPI proteins. GST-tagged proteins were expressed in the cells of appropriate gene deletion strains, in which the GST-chimera was the only source of TFI1, TFI2, or TFI3 proteins. A membrane fraction was obtained after centrifugation at 150,000 g for 1 h at 4°C. Extracted membrane proteins (2 mg) were incubated with 50 μl prewashed glutathione–Sepharose beads. Each eluate was loaded on the 10% SDS-PAGE and analyzed by immunoblot with α-Sed5p and α-coatomer sera. The Coomassie blue staining of the samples presents on the left panel of the Fig. 1 B.
Figure Legend Snippet: GST-tagged TFI1, TFI2, and TFI3 interact with both Sed5p and COPI proteins. GST-tagged proteins were expressed in the cells of appropriate gene deletion strains, in which the GST-chimera was the only source of TFI1, TFI2, or TFI3 proteins. A membrane fraction was obtained after centrifugation at 150,000 g for 1 h at 4°C. Extracted membrane proteins (2 mg) were incubated with 50 μl prewashed glutathione–Sepharose beads. Each eluate was loaded on the 10% SDS-PAGE and analyzed by immunoblot with α-Sed5p and α-coatomer sera. The Coomassie blue staining of the samples presents on the left panel of the Fig. 1 B.

Techniques Used: Centrifugation, Incubation, SDS Page, Staining

Sec34p interacts with retrograde Golgi SNAREs and with COPI. GST-Sec34p was expressed in a Δsec34 yeast strain. Affinity chromatography on glutathione–Sepharose beads was employed to purify GST-Sec34p and associated proteins from a P100 fraction (Total) that was solubilized in CHN buffer (20 mM Hepes, pH 7.4, 1% CHAPS, 0.15 M NaCl). The beads were eluted with 10 mM glutathione (Eluate). As a control, the GST protein was expressed and purified from the membrane fraction of sec34/ Δ sec34 strain. The GST-Sec34p– and Sec34-associated proteins were identified by immunoblotting with antibodies to A, SNARE proteins and Sec35p; (B) anti-COPI, Sec21p, or Sec13p. (C) Coomassie blue staining of the GST and GST-Sec34p eluates.
Figure Legend Snippet: Sec34p interacts with retrograde Golgi SNAREs and with COPI. GST-Sec34p was expressed in a Δsec34 yeast strain. Affinity chromatography on glutathione–Sepharose beads was employed to purify GST-Sec34p and associated proteins from a P100 fraction (Total) that was solubilized in CHN buffer (20 mM Hepes, pH 7.4, 1% CHAPS, 0.15 M NaCl). The beads were eluted with 10 mM glutathione (Eluate). As a control, the GST protein was expressed and purified from the membrane fraction of sec34/ Δ sec34 strain. The GST-Sec34p– and Sec34-associated proteins were identified by immunoblotting with antibodies to A, SNARE proteins and Sec35p; (B) anti-COPI, Sec21p, or Sec13p. (C) Coomassie blue staining of the GST and GST-Sec34p eluates.

Techniques Used: Affinity Chromatography, Purification, Staining

The Sec34/35 complex is an effector of the Ypt1 protein . GST-Ypt1 or GST-Ypt6p (1 μg each), were preloaded with GDP or GTP as indicated, and bound to glutathione–Sepharose beads. Beads were incubated with 0.2 μg of purified Sec34/35 complex in 0.2 ml of binding buffer (40 mM Hepes pH 7.0, 150 mM KoAc, 2 mM MgOAc, 5% glycerol, 1 mM DTT) for 3 h at 4°C. Beads were washed with binding buffer and proteins were eluted with the sample buffer. Eluted proteins and 25% of the unbound material were loaded on 10% SDS-PAGE and immunoblotted with affinity-purified antibodies to GST, Sec34p, and Sec35p.
Figure Legend Snippet: The Sec34/35 complex is an effector of the Ypt1 protein . GST-Ypt1 or GST-Ypt6p (1 μg each), were preloaded with GDP or GTP as indicated, and bound to glutathione–Sepharose beads. Beads were incubated with 0.2 μg of purified Sec34/35 complex in 0.2 ml of binding buffer (40 mM Hepes pH 7.0, 150 mM KoAc, 2 mM MgOAc, 5% glycerol, 1 mM DTT) for 3 h at 4°C. Beads were washed with binding buffer and proteins were eluted with the sample buffer. Eluted proteins and 25% of the unbound material were loaded on 10% SDS-PAGE and immunoblotted with affinity-purified antibodies to GST, Sec34p, and Sec35p.

Techniques Used: Incubation, Purification, Binding Assay, SDS Page, Affinity Purification

Sec35p-TAP complex purified from yeast cytosol. (A) Sec35p-TAP is associated with four other proteins. Proteins eluted from calmodulin-agarose beads were separated by gel electrophoresis and stained with Coomassie blue. The indicated bands were identified by mass spectroscopy of tryptic fragments. The band labeled with double asterisks represents a proteolytic fragment of Tfi3p. No readable spectra were obtained from the minor bands labeled with asterisks. (B) GST-tagged TFI1, TFI2, and TFI3 interact with both Sec34p and Sec35p. The GST-tagged proteins were expressed in cells of appropriate gene deletion strains, in which the GST-chimera was the only source of TFI1, TFI2, or TFI3 protein. Membrane fractions were obtained after centrifugation at 150,000 g , 1 h, 4°C. Extracted membrane proteins (2 mg) were incubated with 50 μl prewashed glutathione–Sepharose beads. The eluates were loaded on the 10% SDS-PAGE and then analyzed by immunoblot with α Sec34p and α Sec35p (left), or stained with Coomassie blue (right).
Figure Legend Snippet: Sec35p-TAP complex purified from yeast cytosol. (A) Sec35p-TAP is associated with four other proteins. Proteins eluted from calmodulin-agarose beads were separated by gel electrophoresis and stained with Coomassie blue. The indicated bands were identified by mass spectroscopy of tryptic fragments. The band labeled with double asterisks represents a proteolytic fragment of Tfi3p. No readable spectra were obtained from the minor bands labeled with asterisks. (B) GST-tagged TFI1, TFI2, and TFI3 interact with both Sec34p and Sec35p. The GST-tagged proteins were expressed in cells of appropriate gene deletion strains, in which the GST-chimera was the only source of TFI1, TFI2, or TFI3 protein. Membrane fractions were obtained after centrifugation at 150,000 g , 1 h, 4°C. Extracted membrane proteins (2 mg) were incubated with 50 μl prewashed glutathione–Sepharose beads. The eluates were loaded on the 10% SDS-PAGE and then analyzed by immunoblot with α Sec34p and α Sec35p (left), or stained with Coomassie blue (right).

Techniques Used: Purification, Nucleic Acid Electrophoresis, Staining, Mass Spectrometry, Labeling, Centrifugation, Incubation, SDS Page

Sec34p and Sed5p interact genetically and physically. (A) Coimmunoprecipitations of putative partners of the Sec34/35 complex. A P100 membrane fraction (Total) from RSY1157 was solubilized in 20 mM Hepes-KOH, pH 7.4, with 0.1 M NaCl and 1% CHAPS. The extract was incubated with protein A Sepharose beads, to which different primary antibodies had been crosslinked. Rabbit IgGs were used as a control for unspecific binding. After washing of the beads, specifically bound proteins were eluted, run on 11%SDS-PAGE, and analyzed by immunoblotting. Two representative experiments performed under identical conditions are shown. Membranes (3% of total) were loaded on the first lane. Approximately 15% of Sec34p and 40% of Sec35p were recovered in the corresponding IP. Approximately 0.2% of Sed5p was specifically coprecipitated in both Sec34 and Sec35 IP's. (B) The Sec34/Sec35 complex interacts with Sed5p in vitro. Purified GST, GST-Sed5p, or GST-Sso1p (5 μg each) were mixed with 0.2 μg of purified Sec34/35 complex in 0.5 ml binding buffer, incubated for 3 h at 4°C, and centrifuged at 20,000 g for 10 min. The supernatant (0.45 ml) was incubated for 1 h at 4°C with 20 μl glutathione–Sepharose beads in the same buffer. Beads were washed and bound proteins were eluted with 10 mM glutathione, separated by 10% SDS-PAGE, and immunoblotted with affinity-purified antibodies to Sec34p and Sec35p. (C) Physical in vitro interaction of Sec34p with Sed5p. Purified proteins (as in B) were bound to glutathione–Sepharose beads, mixed with 5 μg of His 6 -Sec34p in 0.5 ml of binding buffer and incubated for 3h at 4°C with rotation. Beads were washed and bound proteins were eluted with 10 mM glutathione, separated by 11% SDS-PAGE and stained with Coomassie blue. Approximately 10% of His 6 -Sec34p were recovered with GST-Sed5p bound to glutathione–Sepharose beads under conditions used. (D) The sed5–1 and sec34–2 alleles display a synthetic lethal interaction. Diploid strains resulting from the mating of GWY234 ( sed5–1 ) with GWY95 ( sec34–2 ) (top) or GWY235 ( sed5–1 ) with GWY93 ( sec35–1 ) (bottom) were sporulated, tetrad dissected, and incubated on YPD plates at 30°C for 3 d. Eight representative tetrads for each dissection are shown.
Figure Legend Snippet: Sec34p and Sed5p interact genetically and physically. (A) Coimmunoprecipitations of putative partners of the Sec34/35 complex. A P100 membrane fraction (Total) from RSY1157 was solubilized in 20 mM Hepes-KOH, pH 7.4, with 0.1 M NaCl and 1% CHAPS. The extract was incubated with protein A Sepharose beads, to which different primary antibodies had been crosslinked. Rabbit IgGs were used as a control for unspecific binding. After washing of the beads, specifically bound proteins were eluted, run on 11%SDS-PAGE, and analyzed by immunoblotting. Two representative experiments performed under identical conditions are shown. Membranes (3% of total) were loaded on the first lane. Approximately 15% of Sec34p and 40% of Sec35p were recovered in the corresponding IP. Approximately 0.2% of Sed5p was specifically coprecipitated in both Sec34 and Sec35 IP's. (B) The Sec34/Sec35 complex interacts with Sed5p in vitro. Purified GST, GST-Sed5p, or GST-Sso1p (5 μg each) were mixed with 0.2 μg of purified Sec34/35 complex in 0.5 ml binding buffer, incubated for 3 h at 4°C, and centrifuged at 20,000 g for 10 min. The supernatant (0.45 ml) was incubated for 1 h at 4°C with 20 μl glutathione–Sepharose beads in the same buffer. Beads were washed and bound proteins were eluted with 10 mM glutathione, separated by 10% SDS-PAGE, and immunoblotted with affinity-purified antibodies to Sec34p and Sec35p. (C) Physical in vitro interaction of Sec34p with Sed5p. Purified proteins (as in B) were bound to glutathione–Sepharose beads, mixed with 5 μg of His 6 -Sec34p in 0.5 ml of binding buffer and incubated for 3h at 4°C with rotation. Beads were washed and bound proteins were eluted with 10 mM glutathione, separated by 11% SDS-PAGE and stained with Coomassie blue. Approximately 10% of His 6 -Sec34p were recovered with GST-Sed5p bound to glutathione–Sepharose beads under conditions used. (D) The sed5–1 and sec34–2 alleles display a synthetic lethal interaction. Diploid strains resulting from the mating of GWY234 ( sed5–1 ) with GWY95 ( sec34–2 ) (top) or GWY235 ( sed5–1 ) with GWY93 ( sec35–1 ) (bottom) were sporulated, tetrad dissected, and incubated on YPD plates at 30°C for 3 d. Eight representative tetrads for each dissection are shown.

Techniques Used: Incubation, Binding Assay, SDS Page, In Vitro, Purification, Affinity Purification, Staining, Dissection

20) Product Images from "Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2"

Article Title: Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029739

FRS2 directly interacts with Cbl-associated protein. (A) Yeast two-hybrid analysis of the interaction between FRS2 and CAP domains. (B) Structure of the CAP-GST constructs used. (C) and (D) Interaction of purified FRS2-His and CAP-GST proteins. CAP-GST fusion proteins were immobilized on sepharose and tested for the binding of purified FRS2-His. Upper blot shows the bound FRS2-His (anti-His antibody), lower blot the ponceau staining of the GST proteins. 1 µg of FRS2-His was used as a positive control. (E) Quantification of the binding of FRS2 to various CAP domains. A binding of FRS2 significantly higher than background was seen with the full-length CAP, delta-SoHo and the third SH3 domain. (F) Endogenous FRS2 was immunoprecipitated from Hep3B cells, and the binding of endogenous CAP was tested. Please note that several isoforms of CAP are present in Hep3B cells, of which only one appears to bind FRS2.
Figure Legend Snippet: FRS2 directly interacts with Cbl-associated protein. (A) Yeast two-hybrid analysis of the interaction between FRS2 and CAP domains. (B) Structure of the CAP-GST constructs used. (C) and (D) Interaction of purified FRS2-His and CAP-GST proteins. CAP-GST fusion proteins were immobilized on sepharose and tested for the binding of purified FRS2-His. Upper blot shows the bound FRS2-His (anti-His antibody), lower blot the ponceau staining of the GST proteins. 1 µg of FRS2-His was used as a positive control. (E) Quantification of the binding of FRS2 to various CAP domains. A binding of FRS2 significantly higher than background was seen with the full-length CAP, delta-SoHo and the third SH3 domain. (F) Endogenous FRS2 was immunoprecipitated from Hep3B cells, and the binding of endogenous CAP was tested. Please note that several isoforms of CAP are present in Hep3B cells, of which only one appears to bind FRS2.

Techniques Used: Construct, Purification, Binding Assay, Staining, Positive Control, Immunoprecipitation

Flot-1 and CAP compete for the binding to FRS2. CAP-GST was immobilized to sepharose and incubated with HeLa cell lysates in the presence of increasing amounts (1–5 µg) of purified FRS2-His. The binding of endogenous flot-1 from the lysates was analyzed by Western blot (upper blot). Middle panel shows the blot for FRS2-His and the lowermost one a ponceau staining of the GST proteins.
Figure Legend Snippet: Flot-1 and CAP compete for the binding to FRS2. CAP-GST was immobilized to sepharose and incubated with HeLa cell lysates in the presence of increasing amounts (1–5 µg) of purified FRS2-His. The binding of endogenous flot-1 from the lysates was analyzed by Western blot (upper blot). Middle panel shows the blot for FRS2-His and the lowermost one a ponceau staining of the GST proteins.

Techniques Used: Binding Assay, Incubation, Purification, Western Blot, Staining

Overexpression of FRS2 does not compensate for the signaling defects in flot-1 knockdown cells. FGF receptor and flot-1 compete for the binding to FRS2. (A) Flot-1 was knocked down in HeLa cells by means of siRNAs and the cells were transfected with FRS2-CFP. Starved cells were stimulated with FGF for 5 min, and the activation of Akt (uppermost blot) and ERK2 (3 rd blot) was measured with phospho-specific antibodies. The third blot from the bottom shows the analysis of the transfection efficiency of FRS2-CFP and of the 2 nd one the knockdown efficiency of flot-1. Lowermost blot (GAPDH) shows equal protein loading. (B) Purified FRS2-GST was immobilized on sepharose and incubated with lysates of HeLa cells transfected with increasing amounts of FGFR-myc (0.5 to 2 µg). The binding of endogenous flot-1 from these lysates was tested (upper blot). (C) Quantification of the flot-1 bound to FRS2. In the presence of increasing amounts of FGFR, the binding is significantly reduced. (D) Expression of FGFR was verified by Western blot.
Figure Legend Snippet: Overexpression of FRS2 does not compensate for the signaling defects in flot-1 knockdown cells. FGF receptor and flot-1 compete for the binding to FRS2. (A) Flot-1 was knocked down in HeLa cells by means of siRNAs and the cells were transfected with FRS2-CFP. Starved cells were stimulated with FGF for 5 min, and the activation of Akt (uppermost blot) and ERK2 (3 rd blot) was measured with phospho-specific antibodies. The third blot from the bottom shows the analysis of the transfection efficiency of FRS2-CFP and of the 2 nd one the knockdown efficiency of flot-1. Lowermost blot (GAPDH) shows equal protein loading. (B) Purified FRS2-GST was immobilized on sepharose and incubated with lysates of HeLa cells transfected with increasing amounts of FGFR-myc (0.5 to 2 µg). The binding of endogenous flot-1 from these lysates was tested (upper blot). (C) Quantification of the flot-1 bound to FRS2. In the presence of increasing amounts of FGFR, the binding is significantly reduced. (D) Expression of FGFR was verified by Western blot.

Techniques Used: Over Expression, Binding Assay, Transfection, Activation Assay, Purification, Incubation, Expressing, Western Blot

21) Product Images from "The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42"

Article Title: The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6606026

Interactions, regulation and stability of GEF16. ( A ) Yeast mating assay confirming that Tip-1 binds to short carboxyl terminal GEF16 fragments isolated from the primary Tip-1 yeast two-hybrid screen. ( B ) Glutathione transferase pull-down of Tip-1 with IVTT full-length wild-type and mutant GEF16 ( wt GEF16/ mut GEF16). The wt GEF16 contains the PDZ-binding domain ETDV, whereas mut GEF16 has the TDV residues deleted. Tip-1-GST fusion protein and GST control protein were bound to glutathione-sepharose beads. These were mixed with equal amounts of the IVTT wt GEF16 or mut GEF16 proteins, and then washed with binding buffer. Bound proteins were separated by SDS–PAGE and visualised by western immunoblotting with anti-GEF16. The GST-Tip-1 protein bound to the wt GEF16, but did not bind to the mutant form, whereas the GST control did not bind either product. ( C ) Competitive template RT–PCR and western blot analysis of GEF16 and Tip-1 expression in mRNAs and proteins extracted from C33A, C33AV, C33AT16 E6 and C33AT6 E6 cells. mRNA's were reverse transcribed and the resulting cDNAs PCR amplified using primers specific for GAPDH, HPV16 E6, GEF16 and Tip-1 by competitive template PCR. Total proteins were extracted from the same cells, separated by SDS–PAGE, electroblotted and immunoprobed with anti-GEF16 and anti-Tip-1. Anti-GAPDH was used as a loading control. ( D ) Immunoprecipitation with anti-GEF16 from lysates of C33AV, C33AT16 E6 and C33AT6 E6 cells treated with 10 μ of the selective proteasome inhibitor MG132 for 4 h. When immunoprobed with Tip-1 and Cdc42, it can be seen that Tip-1 is associated with GEF16 in both the presence and absence of T16 E6. Cdc42 was detected in association with the GEF16 complex in the presence of HPV type 16 E6, but not in type 6 or vector and parent control cells. (GEF16 has a putative Cdc42 binding site at amino acids 385–391 (QRTLQKL)).
Figure Legend Snippet: Interactions, regulation and stability of GEF16. ( A ) Yeast mating assay confirming that Tip-1 binds to short carboxyl terminal GEF16 fragments isolated from the primary Tip-1 yeast two-hybrid screen. ( B ) Glutathione transferase pull-down of Tip-1 with IVTT full-length wild-type and mutant GEF16 ( wt GEF16/ mut GEF16). The wt GEF16 contains the PDZ-binding domain ETDV, whereas mut GEF16 has the TDV residues deleted. Tip-1-GST fusion protein and GST control protein were bound to glutathione-sepharose beads. These were mixed with equal amounts of the IVTT wt GEF16 or mut GEF16 proteins, and then washed with binding buffer. Bound proteins were separated by SDS–PAGE and visualised by western immunoblotting with anti-GEF16. The GST-Tip-1 protein bound to the wt GEF16, but did not bind to the mutant form, whereas the GST control did not bind either product. ( C ) Competitive template RT–PCR and western blot analysis of GEF16 and Tip-1 expression in mRNAs and proteins extracted from C33A, C33AV, C33AT16 E6 and C33AT6 E6 cells. mRNA's were reverse transcribed and the resulting cDNAs PCR amplified using primers specific for GAPDH, HPV16 E6, GEF16 and Tip-1 by competitive template PCR. Total proteins were extracted from the same cells, separated by SDS–PAGE, electroblotted and immunoprobed with anti-GEF16 and anti-Tip-1. Anti-GAPDH was used as a loading control. ( D ) Immunoprecipitation with anti-GEF16 from lysates of C33AV, C33AT16 E6 and C33AT6 E6 cells treated with 10 μ of the selective proteasome inhibitor MG132 for 4 h. When immunoprobed with Tip-1 and Cdc42, it can be seen that Tip-1 is associated with GEF16 in both the presence and absence of T16 E6. Cdc42 was detected in association with the GEF16 complex in the presence of HPV type 16 E6, but not in type 6 or vector and parent control cells. (GEF16 has a putative Cdc42 binding site at amino acids 385–391 (QRTLQKL)).

Techniques Used: Isolation, Two Hybrid Screening, Mutagenesis, Binding Assay, SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Plasmid Preparation

22) Product Images from "Using Affinity Chromatography to Investigate Novel Protein-Protein Interactions in an Undergraduate Cell and Molecular Biology Lab Course"

Article Title: Using Affinity Chromatography to Investigate Novel Protein-Protein Interactions in an Undergraduate Cell and Molecular Biology Lab Course

Journal: CBE Life Sciences Education

doi: 10.1187/cbe.09-03-0019

Students did not identify a subdomain of GST-Nup1-C that associates with Mex67-GFP. (A) Students expressed GST-Nup1 fusion proteins in E. coli and purified the recombinant GST fusion proteins using glutathione-Sepharose beads. For each sample, a lane
Figure Legend Snippet: Students did not identify a subdomain of GST-Nup1-C that associates with Mex67-GFP. (A) Students expressed GST-Nup1 fusion proteins in E. coli and purified the recombinant GST fusion proteins using glutathione-Sepharose beads. For each sample, a lane

Techniques Used: Purification, Recombinant

23) Product Images from ""

Article Title:

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E06-09-0831

AAK1 isoforms share similar biochemical properties. (A) The N- and C-terminal regions (see Materials and Methods ) used for antibody production are indicated in the AAK1L schematic. (B) Immunoblot analysis of isolated clathrin-coated vesicles probed with N- and C-terminal AAK1 antisera (rabbits 6370 and 273, respectively) reveal a single immunoreactive band. (C) Full-length AAK1L or CBD2 (amino acids 823–960) fused to GST and GST alone were immobilized on glutathione-Sepharose beads and incubated with bovine brain clathrin triskelia or AP2. Clathrin and adaptor protein binding was detected by immunoblot analysis using the monoclonal antibodies TD.1 and AP.6, respectively. TD.1 recognizes the clathrin heavy chain (CHC) and AP.6 identifies the α-adaptin subunit of AP2. The percentage of clathrin or AP2 loaded onto the affinity matrix is indicated. (D) AAK1L- and AAKs-GST fusion proteins were incubated with [γ- 32 P]ATP in the absence or presence of mixed adaptor proteins. (E) Clathrin stimulation was then tested by supplementing kinase reactions with increasing concentrations of bovine clathrin triskelia, as indicated. (F) Clathrin (850 nM) stimulates AAK1L kinase activity toward μ1 (AP1) and μ2 (AP2). FSBA–inactivated AP1 or AP2 complexes were incubated with [γ- 32 P]ATP in the presence or absence of AAK1L and clathrin. μ phosphorylation is shown (E and F).
Figure Legend Snippet: AAK1 isoforms share similar biochemical properties. (A) The N- and C-terminal regions (see Materials and Methods ) used for antibody production are indicated in the AAK1L schematic. (B) Immunoblot analysis of isolated clathrin-coated vesicles probed with N- and C-terminal AAK1 antisera (rabbits 6370 and 273, respectively) reveal a single immunoreactive band. (C) Full-length AAK1L or CBD2 (amino acids 823–960) fused to GST and GST alone were immobilized on glutathione-Sepharose beads and incubated with bovine brain clathrin triskelia or AP2. Clathrin and adaptor protein binding was detected by immunoblot analysis using the monoclonal antibodies TD.1 and AP.6, respectively. TD.1 recognizes the clathrin heavy chain (CHC) and AP.6 identifies the α-adaptin subunit of AP2. The percentage of clathrin or AP2 loaded onto the affinity matrix is indicated. (D) AAK1L- and AAKs-GST fusion proteins were incubated with [γ- 32 P]ATP in the absence or presence of mixed adaptor proteins. (E) Clathrin stimulation was then tested by supplementing kinase reactions with increasing concentrations of bovine clathrin triskelia, as indicated. (F) Clathrin (850 nM) stimulates AAK1L kinase activity toward μ1 (AP1) and μ2 (AP2). FSBA–inactivated AP1 or AP2 complexes were incubated with [γ- 32 P]ATP in the presence or absence of AAK1L and clathrin. μ phosphorylation is shown (E and F).

Techniques Used: Isolation, Incubation, Protein Binding, Activity Assay

24) Product Images from "Nuclear import of cutaneous beta genus HPV8 E7 oncoprotein is mediated by hydrophobic interactions between its zinc-binding domain and FG nucleoporins"

Article Title: Nuclear import of cutaneous beta genus HPV8 E7 oncoprotein is mediated by hydrophobic interactions between its zinc-binding domain and FG nucleoporins

Journal: Virology

doi: 10.1016/j.virol.2013.11.020

A. HPV8 E7 interacts via its zinc-binding domain with the FG domain of Nup62 and mutations of hydrophobic residues disrupt its interaction. Hela cells were transfected with EGFP-8E7 (lane 1), EGFP-8E7 LRLFV65AAAAA (lane 2), EGFP-8E7 R66A (lane 3), EGFP-8cE7 (lane 4), EGFP-8cE7 LRLFV65AAAAA (lane 5), EGFP-8cE7 R66A (lane 6) and EGFP (lane 7) and cell lysates were prepared 24 h post-transfection and probed with a GFP antibody. HeLa cells lysate was also probed for Kap β2 (lane 8). GST-Nup62N (lanes 9-15) and GST (lanes 17 and 23) immobilized on glutathione-Sepharose were incubated with the cell lysates and the bound proteins were eluted and analyzed by immunobloting with a GFP antibody (lanes 9 and 17, EGFP-8E7; lanes 10 and 18, EGFP-8E7 LRLFV65AAAAA ; lanes 11 and 19, EGFP-8E7 R66A ; lanes 12 and 20, EGFP-8cE7; lanes 13 and 21, EGFP-8cE7 LRLFV65AAAAA ; lanes 14 and 22, EGFP-8cE7 R66A ; lanes 15 and 23, EGFP). Binding of Kap β2 to GST-Nup62N and GST was also analyzed (lanes 16 and 24). B. HPV8 E7 binds to Nup153 and mutations of hydrophobic residues within its zinc-binding domain inhibit this interaction. A. Hela cells were transfected with EGFP-8E7 (lane 1), EGFP-8E7 LRLFV65AAAAA (lane 2), EGFP-8E7 R66A (lane 3), EGFP-8cE7 (lane 4), EGFP-8cE7 LRLFV65AAAAA (lane 5), EGFP-8cE7 R66A (lane 6) and EGFP (lane 7) and cell lysates were prepared 24 h post transfection and probed with GFP antibody. HeLa cells lysate was also probed for Kap β2 (lane 8). GST-Nup153 (lanes 9 to 15) and GST (lanes 17 to 23) immobilized on glutathione-Sepharose were incubated with the cell lysates and the bound proteins were eluted and analyzed by immunobloting with a GFP antibody (lanes 9 and 17, EGFP-8E7; lanes 10 and 18, EGFP-8E7 LRLFV65AAAAA ; lanes 11 and 19, EGFP-8E7 R66A ; lanes 12 and 20, EGFP-8cE7; lanes 13 and 21, EGFP-8cE7 LRLFV65AAAAA ; lanes 14 and 22, EGFP-8cE7 R66A ; lanes 15 and 23, EGFP). Binding of Kap β2 to GST-Nup153 and GST was also analyzed (lanes 16 and 24).
Figure Legend Snippet: A. HPV8 E7 interacts via its zinc-binding domain with the FG domain of Nup62 and mutations of hydrophobic residues disrupt its interaction. Hela cells were transfected with EGFP-8E7 (lane 1), EGFP-8E7 LRLFV65AAAAA (lane 2), EGFP-8E7 R66A (lane 3), EGFP-8cE7 (lane 4), EGFP-8cE7 LRLFV65AAAAA (lane 5), EGFP-8cE7 R66A (lane 6) and EGFP (lane 7) and cell lysates were prepared 24 h post-transfection and probed with a GFP antibody. HeLa cells lysate was also probed for Kap β2 (lane 8). GST-Nup62N (lanes 9-15) and GST (lanes 17 and 23) immobilized on glutathione-Sepharose were incubated with the cell lysates and the bound proteins were eluted and analyzed by immunobloting with a GFP antibody (lanes 9 and 17, EGFP-8E7; lanes 10 and 18, EGFP-8E7 LRLFV65AAAAA ; lanes 11 and 19, EGFP-8E7 R66A ; lanes 12 and 20, EGFP-8cE7; lanes 13 and 21, EGFP-8cE7 LRLFV65AAAAA ; lanes 14 and 22, EGFP-8cE7 R66A ; lanes 15 and 23, EGFP). Binding of Kap β2 to GST-Nup62N and GST was also analyzed (lanes 16 and 24). B. HPV8 E7 binds to Nup153 and mutations of hydrophobic residues within its zinc-binding domain inhibit this interaction. A. Hela cells were transfected with EGFP-8E7 (lane 1), EGFP-8E7 LRLFV65AAAAA (lane 2), EGFP-8E7 R66A (lane 3), EGFP-8cE7 (lane 4), EGFP-8cE7 LRLFV65AAAAA (lane 5), EGFP-8cE7 R66A (lane 6) and EGFP (lane 7) and cell lysates were prepared 24 h post transfection and probed with GFP antibody. HeLa cells lysate was also probed for Kap β2 (lane 8). GST-Nup153 (lanes 9 to 15) and GST (lanes 17 to 23) immobilized on glutathione-Sepharose were incubated with the cell lysates and the bound proteins were eluted and analyzed by immunobloting with a GFP antibody (lanes 9 and 17, EGFP-8E7; lanes 10 and 18, EGFP-8E7 LRLFV65AAAAA ; lanes 11 and 19, EGFP-8E7 R66A ; lanes 12 and 20, EGFP-8cE7; lanes 13 and 21, EGFP-8cE7 LRLFV65AAAAA ; lanes 14 and 22, EGFP-8cE7 R66A ; lanes 15 and 23, EGFP). Binding of Kap β2 to GST-Nup153 and GST was also analyzed (lanes 16 and 24).

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

25) Product Images from "Regulation of CHMP4/ESCRT-III Function in Human Immunodeficiency Virus Type 1 Budding by CC2D1A"

Article Title: Regulation of CHMP4/ESCRT-III Function in Human Immunodeficiency Virus Type 1 Budding by CC2D1A

Journal: Journal of Virology

doi: 10.1128/JVI.06539-11

DM14 domain 1-dependent CHMP4B binding and inhibition of HIV-1 budding by an N-terminal CC2D1A fragment. (A) DM14 domain 1 is required for CHMP4B binding by an N-terminal CC2D1A fragment. The schematically illustrated GST fusion proteins were expressed in 293T cells together with CHMP4B-FLAG, and proteins precipitated from the cell lysates by glutathione-Sepharose beads were analyzed by Western blotting with anti-FLAG antibody or by Coomassie staining to detect the GST-DM14 fusion proteins. (B) Effects of the N-terminal CC2D1A fragments on the rescue of HIV-1 ΔPTAPP by ALIX.
Figure Legend Snippet: DM14 domain 1-dependent CHMP4B binding and inhibition of HIV-1 budding by an N-terminal CC2D1A fragment. (A) DM14 domain 1 is required for CHMP4B binding by an N-terminal CC2D1A fragment. The schematically illustrated GST fusion proteins were expressed in 293T cells together with CHMP4B-FLAG, and proteins precipitated from the cell lysates by glutathione-Sepharose beads were analyzed by Western blotting with anti-FLAG antibody or by Coomassie staining to detect the GST-DM14 fusion proteins. (B) Effects of the N-terminal CC2D1A fragments on the rescue of HIV-1 ΔPTAPP by ALIX.

Techniques Used: Binding Assay, Inhibition, Western Blot, Staining

CHMP4B binding by isolated DM14 domains and their effects on the function of ALIX in HIV-1 budding. (A) Ability of GST-DM14 domain fusion proteins to pull down CHMP4B. The schematically illustrated GST fusion proteins were expressed in 293T cells together with CHMP4B-HA, and proteins precipitated from the cell lysates by glutathione-Sepharose beads were analyzed by Western blotting with an anti-HA antibody or by Coomassie staining to detect the GST-DM14 fusion proteins. (B) Effects of the GST-DM14 domain fusion proteins on the rescue of HIV-1 ΔPTAPP by ALIX. 293T cells were cotransfected with ΔPTAPP HIV-1 proviral DNA, a vector expressing HA-ALIX, and vectors expressing GST or the indicated GST-DM14 domain fusion proteins. Virion pellets and the cell lysates were analyzed by Western blotting with anti-CA and anti-HA antibodies to detect Gag, Gag cleavage products, and ALIX.
Figure Legend Snippet: CHMP4B binding by isolated DM14 domains and their effects on the function of ALIX in HIV-1 budding. (A) Ability of GST-DM14 domain fusion proteins to pull down CHMP4B. The schematically illustrated GST fusion proteins were expressed in 293T cells together with CHMP4B-HA, and proteins precipitated from the cell lysates by glutathione-Sepharose beads were analyzed by Western blotting with an anti-HA antibody or by Coomassie staining to detect the GST-DM14 fusion proteins. (B) Effects of the GST-DM14 domain fusion proteins on the rescue of HIV-1 ΔPTAPP by ALIX. 293T cells were cotransfected with ΔPTAPP HIV-1 proviral DNA, a vector expressing HA-ALIX, and vectors expressing GST or the indicated GST-DM14 domain fusion proteins. Virion pellets and the cell lysates were analyzed by Western blotting with anti-CA and anti-HA antibodies to detect Gag, Gag cleavage products, and ALIX.

Techniques Used: Binding Assay, Isolation, Western Blot, Staining, Plasmid Preparation, Expressing

26) Product Images from "Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1"

Article Title: Wide-Ranging Effects of the Yeast Ptc1 Protein Phosphatase Acting Through the MAPK Kinase Mkk1

Journal: Genetics

doi: 10.1534/genetics.115.183202

Ptc1 dephosphorylates Mkk1 in vitro . Recombinant GST-Mkk1 bound to glutathione agarose beads was incubated with yeast extract obtained from slt2 cells expressing the hyperactive allele Bck1–20 in the presence of [γ- 32 P]ATP. After extensive washing, phosphorylated Mkk1 was incubated with equivalent amounts of heat-inactivated Ptc1 (Ptc1*), active Ptc1, or Ppz1. The beads were resuspended in sample buffer and boiled, and the supernatant was subjected to 8% SDS-PAGE. Radioactivity was detected by means of a PMI system (BioRad). Recombinant GST was also included in the experiment as a control. Coomassie staining of the relevant section of the gel shows the amount of GST-Mkk1 present in the different lanes.
Figure Legend Snippet: Ptc1 dephosphorylates Mkk1 in vitro . Recombinant GST-Mkk1 bound to glutathione agarose beads was incubated with yeast extract obtained from slt2 cells expressing the hyperactive allele Bck1–20 in the presence of [γ- 32 P]ATP. After extensive washing, phosphorylated Mkk1 was incubated with equivalent amounts of heat-inactivated Ptc1 (Ptc1*), active Ptc1, or Ppz1. The beads were resuspended in sample buffer and boiled, and the supernatant was subjected to 8% SDS-PAGE. Radioactivity was detected by means of a PMI system (BioRad). Recombinant GST was also included in the experiment as a control. Coomassie staining of the relevant section of the gel shows the amount of GST-Mkk1 present in the different lanes.

Techniques Used: In Vitro, Recombinant, Incubation, Expressing, SDS Page, Radioactivity, Staining

27) Product Images from "ProLIF – quantitative integrin protein–protein interactions and synergistic membrane effects on proteoliposomes"

Article Title: ProLIF – quantitative integrin protein–protein interactions and synergistic membrane effects on proteoliposomes

Journal: Journal of Cell Science

doi: 10.1242/jcs.214270

ProLIF is a flow cytometry-based assay for detection of specific protein-lipid interactions. (A) Outline of ProLIF workflow. Step 1: Bio-Beads™ are added to lipids solubilised in Triton X-100 to remove the detergent and obtain liposomes. Step 2: liposomes are incubated with membrane-free cell extract containing the EGFP-tagged protein of interest. Step 3: Streptavidin–Sepharose (SA) beads are added in order to capture the liposomes via interaction with biotinylated lipids present in the liposome membrane. Step 4: SA beads are analysed by flow cytometry (FACS). Red dots and blue dots represent biotinylated lipids and PIs, respectively. Green fragments represent EGFP-tagged proteins from the cell lysate. (B) Biotinylated-lipid-containing liposomes were generated with and without encapsulated Cy5 dye, captured on SA beads in the presence or absence of increasing amounts of free biotin and analysed via FACS. The molar ratio between biotinylated lipids and soluble biotin added in each sample is indicated ( n =1). (C) Scatter plot and fluorescence histogram from SA beads alone incubated with cell lysate from EGFP-transfected cells and analysed by FACS. (D) Biotinylated-lipid-containing liposomes, with the indicated PI content, were incubated with cell lysates from EGFP alone- or BTK-PH–EGFP-transfected cells (equal EGFP concentrations) and then captured by SA beads and analysed by FACS. Shown are representative dot blots, and size gating in FACS, and histograms depicting EGFP fluorescence intensity (FL1) profiles (note that the axis labels are as in C). The red population in the scatter plot was gated for quantification. Data shown represent three individual experiments. (E) Binding of the BTK-PH–EGFP domain (from cell lysate as in D) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to control PI-free liposomes (data are normalised median fluorescence intensities shown as the mean±s.e.m.; n =5 independent experiments). (F) Binding of EGFP-tagged PLC-PH domain (from cell lysate) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to control PI-free liposomes (data are normalised median fluorescence intensities shown as the mean±s.e.m.; n =5 independent experiments). (G) Binding of tandem FYVE-EGFP domains (from cell lysate) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to PI-free liposomes (data are normalised median fluorescence intensities shown as the mean ±s.e.m.; n =6 independent experiments). ** P
Figure Legend Snippet: ProLIF is a flow cytometry-based assay for detection of specific protein-lipid interactions. (A) Outline of ProLIF workflow. Step 1: Bio-Beads™ are added to lipids solubilised in Triton X-100 to remove the detergent and obtain liposomes. Step 2: liposomes are incubated with membrane-free cell extract containing the EGFP-tagged protein of interest. Step 3: Streptavidin–Sepharose (SA) beads are added in order to capture the liposomes via interaction with biotinylated lipids present in the liposome membrane. Step 4: SA beads are analysed by flow cytometry (FACS). Red dots and blue dots represent biotinylated lipids and PIs, respectively. Green fragments represent EGFP-tagged proteins from the cell lysate. (B) Biotinylated-lipid-containing liposomes were generated with and without encapsulated Cy5 dye, captured on SA beads in the presence or absence of increasing amounts of free biotin and analysed via FACS. The molar ratio between biotinylated lipids and soluble biotin added in each sample is indicated ( n =1). (C) Scatter plot and fluorescence histogram from SA beads alone incubated with cell lysate from EGFP-transfected cells and analysed by FACS. (D) Biotinylated-lipid-containing liposomes, with the indicated PI content, were incubated with cell lysates from EGFP alone- or BTK-PH–EGFP-transfected cells (equal EGFP concentrations) and then captured by SA beads and analysed by FACS. Shown are representative dot blots, and size gating in FACS, and histograms depicting EGFP fluorescence intensity (FL1) profiles (note that the axis labels are as in C). The red population in the scatter plot was gated for quantification. Data shown represent three individual experiments. (E) Binding of the BTK-PH–EGFP domain (from cell lysate as in D) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to control PI-free liposomes (data are normalised median fluorescence intensities shown as the mean±s.e.m.; n =5 independent experiments). (F) Binding of EGFP-tagged PLC-PH domain (from cell lysate) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to control PI-free liposomes (data are normalised median fluorescence intensities shown as the mean±s.e.m.; n =5 independent experiments). (G) Binding of tandem FYVE-EGFP domains (from cell lysate) to biotinylated-lipid-containing liposomes, with the indicated PI content, relative to PI-free liposomes (data are normalised median fluorescence intensities shown as the mean ±s.e.m.; n =6 independent experiments). ** P

Techniques Used: Flow Cytometry, Cytometry, Incubation, FACS, Generated, Fluorescence, Transfection, Binding Assay, Planar Chromatography

28) Product Images from "Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import"

Article Title: Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.6.1639-1655.2002

Functional domain mapping of Pex5pL. (A) Schematic representation of Chinese hamster ClPEX5L product constructs. The truncated products of ClPEX5L (the longer isoform of ClPEX5 ) were expressed in E. coli as fusion proteins placed at the C terminus of GST. Numbers, amino acid residues of ClPex5pL; vertical bars, highly conserved pentapeptide WXXXF/Y motifs; hatched bars, Pex5pL-specific 37-amino-acid sequence. (B) Interaction of Chinese hamster Pex5pL variants with other peroxins and PTS1. Binding assays were performed using recombinant fusions of GST to Pex5pL and its variants. GST fusion proteins (5 μg each) were incubated with lysates of 207P7 cells (10 6 ) expressing a high level of Pex7p. Purified Pex13p (0.1 μg) and His 6 -GFP-SKL (5 μg) were separately incubated with glutathione-Sepharose beads conjugated to GST fusion proteins (5 μg each). After a thorough washing, proteins bound to Sepharose beads were analyzed by SDS-PAGE on 12% gels and immunoblotting using antibodies specific for Pex7p, Pex13p, Pex14p, and GFP. (C) Heterologous expression and purification of recombinant PEX5L proteins. GST-Pex5pL variants were expressed in E. coli and purified as described in Materials and Methods. GST fusion proteins (1 μg each) were analyzed by SDS-PAGE and stained with Coomassie blue. (D) GST-Pex5pL(190-233) and GST-Pex5pL(190-233)S214F were likewise incubated with the lysates of 207P7 cells (10 6 ). Pex7p in bound fractions was probed with an anti-Pex7p antibody (top). GST fusion proteins were stained with Coomassie blue (bottom).
Figure Legend Snippet: Functional domain mapping of Pex5pL. (A) Schematic representation of Chinese hamster ClPEX5L product constructs. The truncated products of ClPEX5L (the longer isoform of ClPEX5 ) were expressed in E. coli as fusion proteins placed at the C terminus of GST. Numbers, amino acid residues of ClPex5pL; vertical bars, highly conserved pentapeptide WXXXF/Y motifs; hatched bars, Pex5pL-specific 37-amino-acid sequence. (B) Interaction of Chinese hamster Pex5pL variants with other peroxins and PTS1. Binding assays were performed using recombinant fusions of GST to Pex5pL and its variants. GST fusion proteins (5 μg each) were incubated with lysates of 207P7 cells (10 6 ) expressing a high level of Pex7p. Purified Pex13p (0.1 μg) and His 6 -GFP-SKL (5 μg) were separately incubated with glutathione-Sepharose beads conjugated to GST fusion proteins (5 μg each). After a thorough washing, proteins bound to Sepharose beads were analyzed by SDS-PAGE on 12% gels and immunoblotting using antibodies specific for Pex7p, Pex13p, Pex14p, and GFP. (C) Heterologous expression and purification of recombinant PEX5L proteins. GST-Pex5pL variants were expressed in E. coli and purified as described in Materials and Methods. GST fusion proteins (1 μg each) were analyzed by SDS-PAGE and stained with Coomassie blue. (D) GST-Pex5pL(190-233) and GST-Pex5pL(190-233)S214F were likewise incubated with the lysates of 207P7 cells (10 6 ). Pex7p in bound fractions was probed with an anti-Pex7p antibody (top). GST fusion proteins were stained with Coomassie blue (bottom).

Techniques Used: Functional Assay, Construct, Sequencing, Binding Assay, Recombinant, Incubation, Expressing, Purification, SDS Page, Staining

Interaction of Pex14p and Pex13p with cargo-loaded or unloaded Pex5p. (A) In vitro binding assays were performed using fusion proteins GST-Pex14p (2 μg), GST-Pex13p (2 μg), Pex5pL (2 μg), and His 6 -GFP-SKL (4 μg) (top) or recombinant catalase (4 μg) (bottom). GST pull-down assays were likewise done in the absence of cargoes (top, lanes 6 and 7). Components added to the assay mixtures, including GST in place of GST fusion proteins, are indicated at the top. Pex5pL, His 6 -GFP-SKL, and catalase in fractions bound to GST-Pex14p- and GST-Pex13p-linked Sepharose were detected by immunoblotting using antibodies specific for the respective proteins. (B) Formation of a hetero-oligomeric complex comprising Pex14p, Pex13p, Pex5p, and PTS1 cargo protein. Binding assays were done as for panel A using GST-Pex14p (2 μg), purified recombinant proteins, Pex13p (0.1 μg), Pex5pS (2 μg), Pex5pL (2 μg), and His 6 -GFP-SKL (4 μg). One-tenth aliquots of the input, Pex5pS, Pex13p, and His 6 -GFP-SKL were loaded in lane 10; Pex5pL was in lane 11. Components added to the assay mixtures are indicated at the top. Pex5p, Pex13p, and His 6 -GFP-SKL in fractions bound to GST-Pex14p were detected as for panel A.
Figure Legend Snippet: Interaction of Pex14p and Pex13p with cargo-loaded or unloaded Pex5p. (A) In vitro binding assays were performed using fusion proteins GST-Pex14p (2 μg), GST-Pex13p (2 μg), Pex5pL (2 μg), and His 6 -GFP-SKL (4 μg) (top) or recombinant catalase (4 μg) (bottom). GST pull-down assays were likewise done in the absence of cargoes (top, lanes 6 and 7). Components added to the assay mixtures, including GST in place of GST fusion proteins, are indicated at the top. Pex5pL, His 6 -GFP-SKL, and catalase in fractions bound to GST-Pex14p- and GST-Pex13p-linked Sepharose were detected by immunoblotting using antibodies specific for the respective proteins. (B) Formation of a hetero-oligomeric complex comprising Pex14p, Pex13p, Pex5p, and PTS1 cargo protein. Binding assays were done as for panel A using GST-Pex14p (2 μg), purified recombinant proteins, Pex13p (0.1 μg), Pex5pS (2 μg), Pex5pL (2 μg), and His 6 -GFP-SKL (4 μg). One-tenth aliquots of the input, Pex5pS, Pex13p, and His 6 -GFP-SKL were loaded in lane 10; Pex5pL was in lane 11. Components added to the assay mixtures are indicated at the top. Pex5p, Pex13p, and His 6 -GFP-SKL in fractions bound to GST-Pex14p were detected as for panel A.

Techniques Used: In Vitro, Binding Assay, Recombinant, Protein Binding, Purification

29) Product Images from "The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV ▿The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV ▿ †"

Article Title: The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV ▿The NS Segment of an H5N1 Highly Pathogenic Avian Influenza Virus (HPAIV) Is Sufficient To Alter Replication Efficiency, Cell Tropism, and Host Range of an H7N1 HPAIV ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.01668-09

The NS1 proteins from FPV and GD have similar F2F3-binding capacities. In a GST pulldown assay, in vitro translated and 35 S-labeled NS1 proteins of FPV, GD, or PR8 were mixed with F2F3-GST or H 2 O as a control and precipitated using glutathione-Sepharose
Figure Legend Snippet: The NS1 proteins from FPV and GD have similar F2F3-binding capacities. In a GST pulldown assay, in vitro translated and 35 S-labeled NS1 proteins of FPV, GD, or PR8 were mixed with F2F3-GST or H 2 O as a control and precipitated using glutathione-Sepharose

Techniques Used: Binding Assay, GST Pulldown Assay, In Vitro, Labeling

30) Product Images from "Functional Interaction between Chfr and Kif22 Controls Genomic Stability *Functional Interaction between Chfr and Kif22 Controls Genomic Stability * S⃞"

Article Title: Functional Interaction between Chfr and Kif22 Controls Genomic Stability *Functional Interaction between Chfr and Kif22 Controls Genomic Stability * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M900333200

Chfr interacts with Kif22. A , 293T cells stably expressing triple-tagged Chfr ( SFB-Chfr ) were used in tandem affinity purification, and the associated proteins in the Chfr complex were separated by SDS-PAGE and visualized by Coomassie staining. The presence of Kif22 in the Chfr complex identified by mass spectrometry analysis was indicated. MW , molecular mass standards. B and C , immunoprecipitation ( IP ) using control IgG or Kif22 ( B ) or Kif11 antibody ( C ) was performed using 293T extracts, and the association of endogenous Chfr was evaluated by immunoblotting. D , 293T cells were transfected with plasmids encoding FLAG-tagged Kif22 or Myc-tagged Chfr alone or in combination. The immunoprecipitation was performed using anti-FLAG antibodies, and the associated Chfr was identified by Western blotting ( WB ) using anti-Myc antibody. E , reverse co-immunoprecipitation was performed using anti-Myc antibody, and the presence of Kif22 in Chfr immunocomplex was examined by Western blotting using anti-FLAG antibody. F , either control GST or GST-Chfr fusion proteins immobilized on agarose beads were incubated with extracts prepared from 293T cells expressing FLAG-tagged Kif22, and the interaction of Kif22 with Chfr was assessed by immunoblotting. G , either control GST or GST-Chfr fusion proteins immobilized on agarose beads were incubated with bacterially expressed recombinant MBP-Kif22, and the interaction of Kif22 with Chfr was assessed by immunoblotting with anti-MBP antibody. H , T24 cells were allowed to grow to confluency for 96 h and then trypsinized and released into fresh medium. Samples were taken at the indicated time points and analyzed by fluorescence-activated cell sorter and Western blotting.
Figure Legend Snippet: Chfr interacts with Kif22. A , 293T cells stably expressing triple-tagged Chfr ( SFB-Chfr ) were used in tandem affinity purification, and the associated proteins in the Chfr complex were separated by SDS-PAGE and visualized by Coomassie staining. The presence of Kif22 in the Chfr complex identified by mass spectrometry analysis was indicated. MW , molecular mass standards. B and C , immunoprecipitation ( IP ) using control IgG or Kif22 ( B ) or Kif11 antibody ( C ) was performed using 293T extracts, and the association of endogenous Chfr was evaluated by immunoblotting. D , 293T cells were transfected with plasmids encoding FLAG-tagged Kif22 or Myc-tagged Chfr alone or in combination. The immunoprecipitation was performed using anti-FLAG antibodies, and the associated Chfr was identified by Western blotting ( WB ) using anti-Myc antibody. E , reverse co-immunoprecipitation was performed using anti-Myc antibody, and the presence of Kif22 in Chfr immunocomplex was examined by Western blotting using anti-FLAG antibody. F , either control GST or GST-Chfr fusion proteins immobilized on agarose beads were incubated with extracts prepared from 293T cells expressing FLAG-tagged Kif22, and the interaction of Kif22 with Chfr was assessed by immunoblotting. G , either control GST or GST-Chfr fusion proteins immobilized on agarose beads were incubated with bacterially expressed recombinant MBP-Kif22, and the interaction of Kif22 with Chfr was assessed by immunoblotting with anti-MBP antibody. H , T24 cells were allowed to grow to confluency for 96 h and then trypsinized and released into fresh medium. Samples were taken at the indicated time points and analyzed by fluorescence-activated cell sorter and Western blotting.

Techniques Used: Stable Transfection, Expressing, Affinity Purification, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Western Blot, Incubation, Recombinant, Fluorescence

31) Product Images from "?-Catenin Binds to the Activation Function 2 Region of the Androgen Receptor and Modulates the Effects of the N-Terminal Domain and TIF2 on Ligand-Dependent Transcription"

Article Title: ?-Catenin Binds to the Activation Function 2 Region of the Androgen Receptor and Modulates the Effects of the N-Terminal Domain and TIF2 on Ligand-Dependent Transcription

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.5.1674-1687.2003

(A) Structural arrangement of VP16/β-catenin constructs in this experiment, demonstrating the location of armadillo repeats. (B) CV-1 cells in a mammalian two-hybrid assay were cotransfected with 10 ng of GAL4/AR LBD, 100 ng of pFR-LUC, 10 ng of Renilla null luciferase reporter, and 50 ng of VP16/β-catenin(S33A), VP16/β-cateninΔN, or VP16/β-cateninΔC(S33A). (C) CV-1 cells in a transactivation assay were cotransfected with 10 ng of pCMVhAR507-919, 100 ng of MMTV-LUC, 50 ng of VP16/β-catenin(S33A), VP16/β-cateninΔN, VP16/β-cateninΔC(S33A), or equimolar amounts of VP16 empty vector. (D) In vitro interaction of the androgen receptor LBD with β-catenin. [ 35 S]AR LBD was synthesized in vitro and incubated in the presence or absence of 1 μM R1881, with glutathione-Sepharose beads bound with GST-β-catenin, GST-β-catenin(1-257), or GST-β-catenin(418-781). Control beads contained GST alone. Beads were washed, and bound 35 S-labeled proteins were eluted and analyzed by SDS-PAGE and autoradiography. The input lane contained 20% of the total radioactivity added for each reaction.
Figure Legend Snippet: (A) Structural arrangement of VP16/β-catenin constructs in this experiment, demonstrating the location of armadillo repeats. (B) CV-1 cells in a mammalian two-hybrid assay were cotransfected with 10 ng of GAL4/AR LBD, 100 ng of pFR-LUC, 10 ng of Renilla null luciferase reporter, and 50 ng of VP16/β-catenin(S33A), VP16/β-cateninΔN, or VP16/β-cateninΔC(S33A). (C) CV-1 cells in a transactivation assay were cotransfected with 10 ng of pCMVhAR507-919, 100 ng of MMTV-LUC, 50 ng of VP16/β-catenin(S33A), VP16/β-cateninΔN, VP16/β-cateninΔC(S33A), or equimolar amounts of VP16 empty vector. (D) In vitro interaction of the androgen receptor LBD with β-catenin. [ 35 S]AR LBD was synthesized in vitro and incubated in the presence or absence of 1 μM R1881, with glutathione-Sepharose beads bound with GST-β-catenin, GST-β-catenin(1-257), or GST-β-catenin(418-781). Control beads contained GST alone. Beads were washed, and bound 35 S-labeled proteins were eluted and analyzed by SDS-PAGE and autoradiography. The input lane contained 20% of the total radioactivity added for each reaction.

Techniques Used: Construct, Two Hybrid Assay, Luciferase, Transactivation Assay, Plasmid Preparation, In Vitro, Synthesized, Incubation, Labeling, SDS Page, Autoradiography, Radioactivity

32) Product Images from "Neuronal Ceroid Lipofuscinoses Are Connected at Molecular Level: Interaction of CLN5 Protein with CLN2 and CLN3"

Article Title: Neuronal Ceroid Lipofuscinoses Are Connected at Molecular Level: Interaction of CLN5 Protein with CLN2 and CLN3

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-01-0031

Interaction analyses of CLN-proteins. (A) COS-1 cells were transfected by either WT or mutant CLN5 cDNA constructs, and cell lysates were immunoprecipitated with CLN2-specific antibody. CLN2 was expressed endogenously. Results were obtained by Western blotting with CLN5-specific antibody. (B) COS-1 cells were transfected with CLN3 cDNA construct and with either WT or mutant CLN5 cDNA constructs. Cell lysates were immunoprecipitated with CLN5-specific antibody. Results were obtained by Western blotting with a CLN3-specific antibody. (C) Radiolabeled CLN2 or CLN3, produced by in vitro translation, was coupled with GST or GST-CLN5 produced in E. coli and pulled down with Glutathione-Sepharose. Results were obtained by SDS-PAGE and fluorography. Coupled proteins are indicated above, molecular weights of the marker bands are shown on the left, and the CLN-specific bands on the right. Posit., crude COS-1 cell lysate (A) expressing endogenous CLN2 (B) transfected with CLN5 cDNA.
Figure Legend Snippet: Interaction analyses of CLN-proteins. (A) COS-1 cells were transfected by either WT or mutant CLN5 cDNA constructs, and cell lysates were immunoprecipitated with CLN2-specific antibody. CLN2 was expressed endogenously. Results were obtained by Western blotting with CLN5-specific antibody. (B) COS-1 cells were transfected with CLN3 cDNA construct and with either WT or mutant CLN5 cDNA constructs. Cell lysates were immunoprecipitated with CLN5-specific antibody. Results were obtained by Western blotting with a CLN3-specific antibody. (C) Radiolabeled CLN2 or CLN3, produced by in vitro translation, was coupled with GST or GST-CLN5 produced in E. coli and pulled down with Glutathione-Sepharose. Results were obtained by SDS-PAGE and fluorography. Coupled proteins are indicated above, molecular weights of the marker bands are shown on the left, and the CLN-specific bands on the right. Posit., crude COS-1 cell lysate (A) expressing endogenous CLN2 (B) transfected with CLN5 cDNA.

Techniques Used: Transfection, Mutagenesis, Construct, Immunoprecipitation, Western Blot, Produced, In Vitro, SDS Page, Marker, Expressing

33) Product Images from "Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2"

Article Title: Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms16023677

Glutathione- S -transferase (GST)-pulldown assays of ALG-2 mutants. Endogenous Sec31A and ALIX in ALG-2-knockdown HEK293 cells (HEK293/ALG-2 KD cells) were pulled down with GST-fused wild-type (WT) and mutant ALG-2 proteins of ( A ) EF-hands ( E47A , EF1; E114A , EF3; E47A / E114A , EF1 and EF3) and ( B ) hydrophobic pockets (Pocket 1, Y180A ; Pocket 2, ΔGF122 ; Pocket 3, L52A , S53G , W57A , F85A , W89A , I92A , F148S ). Unfused GST was used as a negative control (Ctrl). Cleared cell lysates were incubated with glutathione Sepharose beads immobilizing GST-fusion proteins in the presence of ( A ) 2 mM EGTA or ( A , B ) 100 μM CaCl 2 , as described in the Experimental Section . Proteins bound to the beads (pulldown) were resolved by SDS-PAGE (7.5% and 10% gels for Sec31A and ALIX analyses, respectively) and transferred to PVDF membranes for Western blotting (WB) with antibodies against Sec31A and ALIX, as indicated. The relative amount of cleared cell lysate proteins (input) used for analysis of pulldown products was 12.5%. Representative data obtained from three independent experiments are shown.
Figure Legend Snippet: Glutathione- S -transferase (GST)-pulldown assays of ALG-2 mutants. Endogenous Sec31A and ALIX in ALG-2-knockdown HEK293 cells (HEK293/ALG-2 KD cells) were pulled down with GST-fused wild-type (WT) and mutant ALG-2 proteins of ( A ) EF-hands ( E47A , EF1; E114A , EF3; E47A / E114A , EF1 and EF3) and ( B ) hydrophobic pockets (Pocket 1, Y180A ; Pocket 2, ΔGF122 ; Pocket 3, L52A , S53G , W57A , F85A , W89A , I92A , F148S ). Unfused GST was used as a negative control (Ctrl). Cleared cell lysates were incubated with glutathione Sepharose beads immobilizing GST-fusion proteins in the presence of ( A ) 2 mM EGTA or ( A , B ) 100 μM CaCl 2 , as described in the Experimental Section . Proteins bound to the beads (pulldown) were resolved by SDS-PAGE (7.5% and 10% gels for Sec31A and ALIX analyses, respectively) and transferred to PVDF membranes for Western blotting (WB) with antibodies against Sec31A and ALIX, as indicated. The relative amount of cleared cell lysate proteins (input) used for analysis of pulldown products was 12.5%. Representative data obtained from three independent experiments are shown.

Techniques Used: Mutagenesis, Negative Control, Incubation, SDS Page, Western Blot

34) Product Images from "The N-Terminal Region of the Human Autophagy Protein ATG16L1 Contains a Domain That Folds into a Helical Structure Consistent with Formation of a Coiled-Coil"

Article Title: The N-Terminal Region of the Human Autophagy Protein ATG16L1 Contains a Domain That Folds into a Helical Structure Consistent with Formation of a Coiled-Coil

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076237

Purification of recombinant human ATG16L1 coiled-coil. (A) GST-CCD1-FLAG-6His expression produces a truncated product (orange arrowhead). (B) Glutathione sepharose purification of GST-CCD2-FLAG-6His. 1 – total cell lysate, 2 – soluble extract, 3 – unbound flow through, 4–7 successive elution fractions. (C) CCD2-FLAG-6His following removal of the GST tag by TEV cleavage (lane 1). (D) Elution fractions of CCD2-FLAG-6His after anion exchange. The position of the truncated protein is marked by an orange arrowhead. (E) CCD3-FLAG-6His (lane 1) is highly pure and shows no evidence of truncation following glutathione sepharose affinity purification, TEV cleavage and hydrophobic interaction chromatography. In all panels the black arrowhead marks the bands representing the expected size of the ATG16L1 construct; M denotes PageRuler™ Plus Prestained protein standards (Thermo Scientific).
Figure Legend Snippet: Purification of recombinant human ATG16L1 coiled-coil. (A) GST-CCD1-FLAG-6His expression produces a truncated product (orange arrowhead). (B) Glutathione sepharose purification of GST-CCD2-FLAG-6His. 1 – total cell lysate, 2 – soluble extract, 3 – unbound flow through, 4–7 successive elution fractions. (C) CCD2-FLAG-6His following removal of the GST tag by TEV cleavage (lane 1). (D) Elution fractions of CCD2-FLAG-6His after anion exchange. The position of the truncated protein is marked by an orange arrowhead. (E) CCD3-FLAG-6His (lane 1) is highly pure and shows no evidence of truncation following glutathione sepharose affinity purification, TEV cleavage and hydrophobic interaction chromatography. In all panels the black arrowhead marks the bands representing the expected size of the ATG16L1 construct; M denotes PageRuler™ Plus Prestained protein standards (Thermo Scientific).

Techniques Used: Purification, Recombinant, Expressing, Flow Cytometry, Affinity Purification, Hydrophobic Interaction Chromatography, Construct

35) Product Images from "Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog"

Article Title: Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog

Journal: Parasites & Vectors

doi: 10.1186/s13071-016-1676-1

Expression and purification of GST-GFP, GST-TgCaMKrk, and GST-TgCaMKrkKA. Proteins were expressed by using a wheat germ cell-free protein synthesis system. Total wheat germ extracts were subjected to affinity chromatography using glutathione-Sepharose beads. The purified proteins were separated on denaturing gels and then ( a ) silver stained or ( b ) transferred to a nitrocellulose sheet and probed with anti-GST antibodies. Lanes 1 and 2, GST-GFP; Lanes 3 and 4, GST-TgCaMKrk; Lanes 5 and 6, GST-TgCaMKrkKA. Lanes 1, 3, and 5, total wheat germ extracts; Lanes 2, 4, and 6, purified proteins. Molecular masses (kDa) are shown on the left
Figure Legend Snippet: Expression and purification of GST-GFP, GST-TgCaMKrk, and GST-TgCaMKrkKA. Proteins were expressed by using a wheat germ cell-free protein synthesis system. Total wheat germ extracts were subjected to affinity chromatography using glutathione-Sepharose beads. The purified proteins were separated on denaturing gels and then ( a ) silver stained or ( b ) transferred to a nitrocellulose sheet and probed with anti-GST antibodies. Lanes 1 and 2, GST-GFP; Lanes 3 and 4, GST-TgCaMKrk; Lanes 5 and 6, GST-TgCaMKrkKA. Lanes 1, 3, and 5, total wheat germ extracts; Lanes 2, 4, and 6, purified proteins. Molecular masses (kDa) are shown on the left

Techniques Used: Expressing, Purification, Affinity Chromatography, Staining

36) Product Images from "Cardiovirus Leader proteins bind exportins: implications for virus replication and nucleocytoplasmic trafficking inhibition"

Article Title: Cardiovirus Leader proteins bind exportins: implications for virus replication and nucleocytoplasmic trafficking inhibition

Journal: Virology

doi: 10.1016/j.virol.2015.10.001

GST-tagged L E mutations. Bait proteins, GST-L E and related mutant derivatives (A) as well as L E -GST, L S -GST and L T -GST (B) were incubated with HeLa cytosol. After glutathione sepharose bead extractions, Western analyses determined the amount of bound
Figure Legend Snippet: GST-tagged L E mutations. Bait proteins, GST-L E and related mutant derivatives (A) as well as L E -GST, L S -GST and L T -GST (B) were incubated with HeLa cytosol. After glutathione sepharose bead extractions, Western analyses determined the amount of bound

Techniques Used: Mutagenesis, Incubation, Western Blot

Crm1 inhibition. (A) Leptomycin B (LMB, 4 nM) was pre-incubated with rCrm1 before the addition of bait proteins (singly phosphorylated GSL-L E or GST) and (putative) auxiliary prey proteins (rRan with RCC1). The complexes were extracted with glutathione-sepharose
Figure Legend Snippet: Crm1 inhibition. (A) Leptomycin B (LMB, 4 nM) was pre-incubated with rCrm1 before the addition of bait proteins (singly phosphorylated GSL-L E or GST) and (putative) auxiliary prey proteins (rRan with RCC1). The complexes were extracted with glutathione-sepharose

Techniques Used: Inhibition, Incubation

37) Product Images from "The p53-induced mouse zinc finger protein wig-1 binds double-stranded RNA with high affinity"

Article Title: The p53-induced mouse zinc finger protein wig-1 binds double-stranded RNA with high affinity

Journal: Nucleic Acids Research

doi:

Wig-1 protein produced in mammalian cells also specifically binds dsRNA. ( A ) NIH 3T3 cells were transfected with the FLAG-tagged mouse wig-1 expression plasmid. After 48 h total protein cell lysates were prepared and 50 µg aliquots were incubated with poly(I·C) immobilized on agarose beads. The incubations were performed in the absence or presence of the competitor poly(C) or poly(I·C). The bound wig-1 protein was analyzed by immunoblotting using anti-FLAG antibodies. ( B ) As in (A) except that NIH 3T3 cells were transfected with a FLAG-tagged wig-1 expression plasmid lacking the first zinc finger domain.
Figure Legend Snippet: Wig-1 protein produced in mammalian cells also specifically binds dsRNA. ( A ) NIH 3T3 cells were transfected with the FLAG-tagged mouse wig-1 expression plasmid. After 48 h total protein cell lysates were prepared and 50 µg aliquots were incubated with poly(I·C) immobilized on agarose beads. The incubations were performed in the absence or presence of the competitor poly(C) or poly(I·C). The bound wig-1 protein was analyzed by immunoblotting using anti-FLAG antibodies. ( B ) As in (A) except that NIH 3T3 cells were transfected with a FLAG-tagged wig-1 expression plasmid lacking the first zinc finger domain.

Techniques Used: Produced, Transfection, Expressing, Plasmid Preparation, Incubation

38) Product Images from "Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-?B activation"

Article Title: Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-?B activation

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

doi:

Specificity of the interaction of tyrosine-phosphorylated IκB-α with the regulatory p85α subunit of PI3-kinase. ( A ) Interaction of tyrosine-phosphorylated IκB-α with the C-terminal SH2 domain of p85α. Two μg of GST-C-SH2 agarose conjugate (lanes 1 and 2) were incubated with whole-cell extracts from control (−) or 200 μM pV-treated (+) Jurkat T cells. The binding of IκB-α to the GST proteins was detected by Western blotting. The analysis of total cell extract by Western blotting for IκB-α is shown in lanes 3 and 4. The positions of IκB-α and its tyrosine-phosphorylated form, as well as that of the GST fusion protein, are indicated by arrows. ( B ) Specificity of interaction of tyrosine-phosphorylated IκB-α with p85α. Whole-cell extracts from control (−) and pV-treated (+) Jurkat T cells were incubated with 6 μM of the indicated peptides. Two μg of GST (lanes 1 and 2) or GST-p85α (lanes 3–10) agarose conjugates were added and the presence of IκB-α in the complexes was analyzed by Western blotting. The position of tyrosine-phosphorylated IκB-α is indicated.
Figure Legend Snippet: Specificity of the interaction of tyrosine-phosphorylated IκB-α with the regulatory p85α subunit of PI3-kinase. ( A ) Interaction of tyrosine-phosphorylated IκB-α with the C-terminal SH2 domain of p85α. Two μg of GST-C-SH2 agarose conjugate (lanes 1 and 2) were incubated with whole-cell extracts from control (−) or 200 μM pV-treated (+) Jurkat T cells. The binding of IκB-α to the GST proteins was detected by Western blotting. The analysis of total cell extract by Western blotting for IκB-α is shown in lanes 3 and 4. The positions of IκB-α and its tyrosine-phosphorylated form, as well as that of the GST fusion protein, are indicated by arrows. ( B ) Specificity of interaction of tyrosine-phosphorylated IκB-α with p85α. Whole-cell extracts from control (−) and pV-treated (+) Jurkat T cells were incubated with 6 μM of the indicated peptides. Two μg of GST (lanes 1 and 2) or GST-p85α (lanes 3–10) agarose conjugates were added and the presence of IκB-α in the complexes was analyzed by Western blotting. The position of tyrosine-phosphorylated IκB-α is indicated.

Techniques Used: Incubation, Binding Assay, Western Blot

39) Product Images from "Dehydrin MtCAS31 promotes autophagic degradation under drought stress"

Article Title: Dehydrin MtCAS31 promotes autophagic degradation under drought stress

Journal: Autophagy

doi: 10.1080/15548627.2019.1643656

MtCAS31 interacts with MtPIP2;7. (A) Interaction of MtCAS31 and MtPIP2;7 in the transformed yeast strain NMY51, determined by a split-ubiquitin yeast two-hybrid assay. MtPIP2;7 was fused with the C terminus of ubiquitin (MtPIP2;7-Cub), and MtCAS31 was fused with the N terminus of mutant ubiquitin (NubG-MtCAS31). MtPIP2;7-Cub/Alg5-NubI was used as the positive control. MtPIP2;7-Cub/Alg5-NubG, MtPIP2;7-Cub/NubG and Alg5-CubI/NubG-MtCAS31 were employed as negative controls. Different co-transformed yeast cells were dropped onto synthetic dropout (SD) medium lacking tryptophan, leucine, adenine, and histidine (SD/-Trp/-Leu/-Ade/-His) and containing 20 mg/mL X-α-gal. (B) GST affinity-isolation assay to identify the interaction between MtPIP2;7 and MtCAS31. MtPIP2;7 was fused with a GST tag (GST-MtPIP2;7), and MtCAS31 was fused with a His tag (MtCAS31-His). GST-MtPIP2;7 or GST alone was precipitated with glutathione Sepharose 4B agarose beads for 3 h and incubated with MtCAS31-His. The precipitates were separated via SDS-PAGE and analyzed by immunoblotting using anti-His and anti-GST antibodies. (C) Interaction of MtCAS31 with MtPIP2;7 as determined by bimolecular fluorescence complementation. MtCAS31 was fused with the N terminus of YFP (MtCAS31-YFP[N]), and MtPIP2;7 was fused with the C terminus of YFP (MtPIP2;7-YFP[C]). Both constructs were driven by CaMV35S . HDEL-RFP, an ER marker, was co-transformed into Arabidopsis protoplasts. Co-transformed protoplasts were incubated for 16 h, and the fluorescence signals were detected by confocal laser scanning microscopy (Olympus FluoView FV1000) with excitation at 488 nm (for GFP fluorescence detection) and 546 nm (for RFP detection). MtCAS31-YFP[N]/Medtr4g415300-YFP[C]/HDEL-RFP and MtCAS31-YFP[N]/Medtr1g095070-YFP[C]/HDEL-RFP were employed as negative controls. YFP, yellow fluorescent protein. HDEL, amino acid sequence for localization to the ER. RFP, red fluorescent protein. Bar: 10 μm. (D) Detection of the MtCAS31-MtPIP2;7 interaction by coimmunoprecipitation (Co-IP). MtPIP2;7 was tagged with MYC (MtPIP2;7-MYC). GFP-FLAG/MtPIP2;7-MYC was employed as the negative control. Total proteins were extracted from the N. benthamiana leaf, which was co-transformed with the indicated constructs and incubated with FLAG beads to immunoprecipitate the target protein. Coprecipitated proteins were analyzed by immunoblotting using anti-FLAG and anti-MYC antibodies.
Figure Legend Snippet: MtCAS31 interacts with MtPIP2;7. (A) Interaction of MtCAS31 and MtPIP2;7 in the transformed yeast strain NMY51, determined by a split-ubiquitin yeast two-hybrid assay. MtPIP2;7 was fused with the C terminus of ubiquitin (MtPIP2;7-Cub), and MtCAS31 was fused with the N terminus of mutant ubiquitin (NubG-MtCAS31). MtPIP2;7-Cub/Alg5-NubI was used as the positive control. MtPIP2;7-Cub/Alg5-NubG, MtPIP2;7-Cub/NubG and Alg5-CubI/NubG-MtCAS31 were employed as negative controls. Different co-transformed yeast cells were dropped onto synthetic dropout (SD) medium lacking tryptophan, leucine, adenine, and histidine (SD/-Trp/-Leu/-Ade/-His) and containing 20 mg/mL X-α-gal. (B) GST affinity-isolation assay to identify the interaction between MtPIP2;7 and MtCAS31. MtPIP2;7 was fused with a GST tag (GST-MtPIP2;7), and MtCAS31 was fused with a His tag (MtCAS31-His). GST-MtPIP2;7 or GST alone was precipitated with glutathione Sepharose 4B agarose beads for 3 h and incubated with MtCAS31-His. The precipitates were separated via SDS-PAGE and analyzed by immunoblotting using anti-His and anti-GST antibodies. (C) Interaction of MtCAS31 with MtPIP2;7 as determined by bimolecular fluorescence complementation. MtCAS31 was fused with the N terminus of YFP (MtCAS31-YFP[N]), and MtPIP2;7 was fused with the C terminus of YFP (MtPIP2;7-YFP[C]). Both constructs were driven by CaMV35S . HDEL-RFP, an ER marker, was co-transformed into Arabidopsis protoplasts. Co-transformed protoplasts were incubated for 16 h, and the fluorescence signals were detected by confocal laser scanning microscopy (Olympus FluoView FV1000) with excitation at 488 nm (for GFP fluorescence detection) and 546 nm (for RFP detection). MtCAS31-YFP[N]/Medtr4g415300-YFP[C]/HDEL-RFP and MtCAS31-YFP[N]/Medtr1g095070-YFP[C]/HDEL-RFP were employed as negative controls. YFP, yellow fluorescent protein. HDEL, amino acid sequence for localization to the ER. RFP, red fluorescent protein. Bar: 10 μm. (D) Detection of the MtCAS31-MtPIP2;7 interaction by coimmunoprecipitation (Co-IP). MtPIP2;7 was tagged with MYC (MtPIP2;7-MYC). GFP-FLAG/MtPIP2;7-MYC was employed as the negative control. Total proteins were extracted from the N. benthamiana leaf, which was co-transformed with the indicated constructs and incubated with FLAG beads to immunoprecipitate the target protein. Coprecipitated proteins were analyzed by immunoblotting using anti-FLAG and anti-MYC antibodies.

Techniques Used: Transformation Assay, Y2H Assay, Mutagenesis, Positive Control, Isolation, Incubation, SDS Page, Fluorescence, Construct, Marker, Confocal Laser Scanning Microscopy, Sequencing, Co-Immunoprecipitation Assay, Negative Control

40) Product Images from "PRAME Is a Golgi-Targeted Protein That Associates with the Elongin BC Complex and Is Upregulated by Interferon-Gamma and Bacterial PAMPs"

Article Title: PRAME Is a Golgi-Targeted Protein That Associates with the Elongin BC Complex and Is Upregulated by Interferon-Gamma and Bacterial PAMPs

Journal: PLoS ONE

doi: 10.1371/journal.pone.0058052

PRAME associates with the Elongin BC complex. ( A ) SDS-PAGE and silver staining showing affinity capture of proteins from HL60 whole cell extracts (WCE) by immobilised GST or GST-PRAME proteins. GST and GST-PRAME proteins are indicated. Putative PRAME-specific bands are indicated and bands of approximately 12 kDa and 17 kDa were excised for mass spectrometry analysis. ( B ) Co-immunoprecipitation of PRAME with Elongin complex components. Whole cell extracts of HEK293 cells transfected with PRAME-FLAG-6xHis (or empty vector control) applied to anti-FLAG sepharose beads as described in Materials and Methods. After extensive washing, co-purified PRAME and E3 ubiquitin ligase complex components were detected by western blotting using specific antibodies as indicated. ( C ) GST-pulldown experiment showing binding of ELB and ELC proteins in HL60 whole cell extracts to GST or GST-PRAME proteins. The top panel is a Coomassie-stained gel showing the input whole cell extract, and the purified GST and GST-PRAME proteins. The lower panels are western blots revealing PRAME, ELB and ELC proteins bound to GST proteins. ( D ) GST-pulldown experiments revealing interactions of 35 [S]-labelled in vitro translated human ELC (hELC), C.elegans ELC (wELC), C.elegans ELC (L47D-L49D-Y88D-Y91D) (wELC mutant) and C.elegans ELB proteins with GST or GST-PRAME. ( E ) Yeast two hybrid assays of LexA-PRAME interactions with GAL4 AD-fused human ELC (hELC) or C.elegans proteins (wELB, wELC, wELC mutant). Western blots of the HA-tagged elongin fusion proteins are also shown. Reporter activity is expressed as β-galactosidase activity normalised to amount of protein in the extracts. ( F ) Immunofluorescence staining showing subcellular localisation of endogenous ELC, ELB and CUL2 proteins in HL60 cells. ( G ) Immunofluorescence staining showing colocalisation of endogenous ELC and PRAME proteins in HL60 cells following treatment with LPS/IFNγ for 4 hours.
Figure Legend Snippet: PRAME associates with the Elongin BC complex. ( A ) SDS-PAGE and silver staining showing affinity capture of proteins from HL60 whole cell extracts (WCE) by immobilised GST or GST-PRAME proteins. GST and GST-PRAME proteins are indicated. Putative PRAME-specific bands are indicated and bands of approximately 12 kDa and 17 kDa were excised for mass spectrometry analysis. ( B ) Co-immunoprecipitation of PRAME with Elongin complex components. Whole cell extracts of HEK293 cells transfected with PRAME-FLAG-6xHis (or empty vector control) applied to anti-FLAG sepharose beads as described in Materials and Methods. After extensive washing, co-purified PRAME and E3 ubiquitin ligase complex components were detected by western blotting using specific antibodies as indicated. ( C ) GST-pulldown experiment showing binding of ELB and ELC proteins in HL60 whole cell extracts to GST or GST-PRAME proteins. The top panel is a Coomassie-stained gel showing the input whole cell extract, and the purified GST and GST-PRAME proteins. The lower panels are western blots revealing PRAME, ELB and ELC proteins bound to GST proteins. ( D ) GST-pulldown experiments revealing interactions of 35 [S]-labelled in vitro translated human ELC (hELC), C.elegans ELC (wELC), C.elegans ELC (L47D-L49D-Y88D-Y91D) (wELC mutant) and C.elegans ELB proteins with GST or GST-PRAME. ( E ) Yeast two hybrid assays of LexA-PRAME interactions with GAL4 AD-fused human ELC (hELC) or C.elegans proteins (wELB, wELC, wELC mutant). Western blots of the HA-tagged elongin fusion proteins are also shown. Reporter activity is expressed as β-galactosidase activity normalised to amount of protein in the extracts. ( F ) Immunofluorescence staining showing subcellular localisation of endogenous ELC, ELB and CUL2 proteins in HL60 cells. ( G ) Immunofluorescence staining showing colocalisation of endogenous ELC and PRAME proteins in HL60 cells following treatment with LPS/IFNγ for 4 hours.

Techniques Used: SDS Page, Silver Staining, Mass Spectrometry, Immunoprecipitation, Transfection, Plasmid Preparation, Purification, Western Blot, Binding Assay, Staining, In Vitro, Mutagenesis, Activity Assay, Immunofluorescence

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Protease Inhibitor:

Article Title: The Interaction between Rice ERF3 and WOX11 Promotes Crown Root Development by Regulating Gene Expression Involved in Cytokinin Signaling [OPEN]
Article Snippet: .. Pull-down was performed as described ( ) with the following modifications: Equal volumes of GST or WOX3-His, and ERF3-GST or WOX11-His recombinant proteins were incubated for 6 h at 4°C with 400 μL of GST (GE Healthcare; 17-5132-01) or His (Promega; REF V8500) resin in a total volume of 1 mL of GST or His binding buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% Lgepal CA-630, and protease inhibitor) for 2 to 3 h at 4°C, and the binding reaction was washed five times (10 min each time at 4°C) by the binding buffer. .. After extensive washing, the pulled down proteins were eluted by boiling, separated on 12% SDS-PAGE, and detected by immunoblots using an anti-GST antibody (abcam; ab19256) and anti-His antibody (abcam; ab9108), respectively.

Purification:

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    GE Healthcare glutathione sepharose beads
    PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After <t>glutathione-agarose</t> pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.
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    Abundance, overexpression, and activation of Rho GTPases in GM/IFN-γ/LPS– and M/IL4-cultured macrophages. (A) GM/IFN-γ/LPS– and M/IL4-cultured macrophages were lysed, separated by 10% SDS–PAGE, and subjected to immunoblotting with anti-Rac1, anti-RhoA, anti-RhoG, or anti-Cdc42 antibodies. Intensities of the bands were normalized to β-actin, and then plotted as a ratio relative to GM/IFN-γ/LPS–cultured macrophages in each experiment for each GTPase. Data are means ± SEM of three to four independent experiments. Typical immunoblots are shown in Supplemental Figure S3A. (B) GM/IFN-γ/LPS– and M/IL4-cultured macrophages were lysed, followed immediately by measurement of the GTP-bound form of Rac1, RhoA, and Cdc42 using the respective colorimetric G-LISA kit. The measured absorbance at 490 nm was normalized to total protein levels and plotted as a ratio relative to GM/IFN-γ/LPS–cultured macrophages in each experiment for each GTPase. Data are means ± SEM of three to four independent experiments. Levels of GTP-bound RhoG in lysed GM/IFN-γ/LPS– and M/IL4-cultured macrophages were measured using a pull-down assay with a recombinant <t>ELMO-GST</t> loaded onto glutathione-sepharose beads, followed by 10% SDS–PAGE and immunoblotting with an anti-RhoG antibody. Levels of RhoG.GTP were normalized relative to the level in GM/IFN-γ/LPS–cultured macrophages in each experiment. A typical immunoblot for the RhoG.GTP pull-down assay is shown in Supplemental Figure S3B. Data are means ± SEM from four independent experiments. C. difficile toxin B treatment (3 h in serum-free medium) was used to inhibit all four GTPases, i.e., as a negative control, in all G-LISA and RhoG.GTP pull-down assays. (C–E) GM/IFN-γ/LPS–cultured macrophages were transfected with fluorescently tagged constructs of either wild-type (C) or constitutively active Rac1, RhoA, RhoG, or Cdc42 (D, E), or of the Rac1 and RhoG GEFs Tiam1 and sGEF, as indicated (D, E). The specific constructs used were Rac1-GFP, RhoA-GFP, RhoG-CFP, Cdc42-GFP, Rac1-Q61L-GFP, RhoA-Q63L-GFP, RhoG-G12V-CFP, Cdc42-G12V-YFP, Tiam1-GFP, and sGEF-GFP. GM/IFN-γ/LPS–cultured macrophages transfected with GFP alone were used as a negative control, while M/IL4-cultured macrophages transfected with GFP alone were used as a positive control. After 24-h transfection, the cells were incubated with fluorescently labeled 70 kDa dextran (TMR-dextran, 125 µg/ml) for 15 min at 37°C, and washed, fixed, and imaged immediately (D); only transfected cells were selected for measurements of macropinocytosis, which was quantified (C, E) as the total volume of TMR-positive vacuoles per cell from 3D stacks using 3D particle analysis in ImageJ software, applying a lower particle volume threshold of 0.26 µm 3 . Typical images (D) and quantifications (C, E; means ± SEM) are representative of 20–50 cells from three to five independent experiments using blood from at least two separate donors. Scale bars, 15 µm.
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    PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After glutathione-agarose pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation *

    doi: 10.1074/jbc.M116.729699

    Figure Lengend Snippet: PARP1 ADP-ribosylates, whereas PARG de-ADP-ribosylates Smad1 and Smad5. A , in vitro ADP-ribosylation assay of Smad1, Smad5, Smad4, and Smad3. GST-Smad proteins were incubated with 32 P-β-NAD + and recombinant PARP1. After glutathione-agarose pulldown, ADP-ribosylated GST-Smad1/5/4/3 were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows GST-Smad proteins stained with Coomassie Brilliant Blue after SDS-PAGE. M , molecular size marker. A representative autoradiogram of four assays is shown. Molecular size markers in kDa are also marked. B , in vitro de-PARylation of GST-Smad1 and GST-Smad5. PARG or vehicle were incubated with equal amounts of GST-Smad1/5, 32 P-β-NAD + , and recombinant PARP1 for 30 min at 37 °C. ADP-ribosylated proteins were imaged by autoradiography. The radioactive protein bands of PARP1 and GST-Smads are marked. The lower panel shows total GST proteins stained with Coomassie Brilliant Blue. M , molecular size marker. A representative autoradiogram of five assays is shown. Molecular size markers in kDa are also marked. C , immunoblot of endogenous PARP1 from HEK293T cell extracts bound to the indicated GST-Smad1 MH1 domain mutants. TCL shows the levels of endogenous PARP1. Total GST-Smad1 mutant proteins used for immunoblotting of endogenous PARP1 are stained with Coomassie Brilliant Blue in the middle panel . The Smad1 sequence motif that was mutated ( red letters ) and that represents a genuine ADP-ribosylation target sequence is shown in the bottom panel . A representative immunoblot of three repeats is shown. Molecular size markers in kDa are also marked. D , in vitro ADP-ribosylation assay of GST-Smad1-MH1 domain mutants. Control GST, beads, WT-Smad1-MH1 domain, and three mutants (as shown in C ) were incubated with 32 P-β-NAD + and recombinant PARP1. ADP-ribosylated proteins were imaged via autoradiography. The radioactive protein bands of PARP1 and GST-Smad1-MH1 are marked. Total GST proteins were checked by Coomassie Brilliant Blue staining. Lane 1/3 WT indicates a reaction where one-third of the GST-Smad1-MH1 protein was used compared with the WT lanes. A representative autoradiogram of two assays is shown. Molecular size markers in kDa are also marked. E , immunoblot of recombinant PARP1 (20 ng) bound to the indicated GST-Smad1 MH1 domain mutants. The experiment is a repeat of the ribosylation assay of Fig. 8 D , except that only cold β-NAD + was used during incubation, followed by pulldown and immunoblotting. On the side, increasing amounts of recombinant PARP1 along with TCL from HEK293T cells show the levels of recombinant PARP1 used in the assay relative to endogenous PARP1. Total GST-Smad1 mutant proteins checked by Coomassie Brilliant Blue staining, used for immunoblotting of recombinant PARP1. A representative immunoblot of two repeats is shown. Molecular size markers in kDa are also marked. F , molecular model adapted to a detail from the crystal structure of two Smad3 MH1 domains bound to the Smad-binding DNA element (PDB code 1mhd ). Shown is a ribbon diagram of the whole Smad3 MH1 domain with colored amino acids and the acceptor glutamate ( red ) and lysine ( blue ) residues drawn as stick and ball structures on the bottom side of the surface of the regulatory α-helix of one Smad3 MH1 subunit ( white arrow ). The β-hairpin that contacts DNA is also indicated ( white arrow ). WB , Western blotting.

    Article Snippet: Then proteins were extracted from bacteria using a Triton X-100 containing lysis buffer (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 100 mm NaCl, 5% glycerol, 0.5% Triton X-100), supplemented with 1 mm DTT and protease inhibitors, and incubated end over end at 4 °C, overnight, with glutathione-Sepharose beads (catalog no. 17-5132-01, lot no. 10172617; GE Healthcare).

    Techniques: In Vitro, Incubation, Recombinant, Autoradiography, Staining, SDS Page, Marker, Mutagenesis, Sequencing, Binding Assay, Western Blot

    Purification of recombinant NECs. (A) Schematic diagrams of the wild-type and recombinant HSV-1 U L 31 and U L 34 viral proteins used in this study. Line 1, wild-type HSV-1 NEC U L 31 and U L 34; line 2, GST-NEC 185-Δ50 fusion proteins; lines 3 and 4, GST-NEC 185-Δ50 fusion proteins carrying a single substitution mutation in residue K279 (line 3) or a double mutation in residues R281 and D282 (line 4) of U L 31 Δ50 . (B) GST, GST-NEC 185-Δ50 , and the two GST-NEC 185-Δ50 mutants were expressed in E. coli , lysed, and precipitated using glutathione-Sepharose beads. The lysates and beads were analyzed by electrophoresis in a denaturing gel and then immunoblotted with anti-U L 31 and anti-U L 34 antisera or stained with CBB.

    Journal: Journal of Virology

    Article Title: Identification of the Capsid Binding Site in the Herpes Simplex Virus 1 Nuclear Egress Complex and Its Role in Viral Primary Envelopment and Replication

    doi: 10.1128/JVI.01290-19

    Figure Lengend Snippet: Purification of recombinant NECs. (A) Schematic diagrams of the wild-type and recombinant HSV-1 U L 31 and U L 34 viral proteins used in this study. Line 1, wild-type HSV-1 NEC U L 31 and U L 34; line 2, GST-NEC 185-Δ50 fusion proteins; lines 3 and 4, GST-NEC 185-Δ50 fusion proteins carrying a single substitution mutation in residue K279 (line 3) or a double mutation in residues R281 and D282 (line 4) of U L 31 Δ50 . (B) GST, GST-NEC 185-Δ50 , and the two GST-NEC 185-Δ50 mutants were expressed in E. coli , lysed, and precipitated using glutathione-Sepharose beads. The lysates and beads were analyzed by electrophoresis in a denaturing gel and then immunoblotted with anti-U L 31 and anti-U L 34 antisera or stained with CBB.

    Article Snippet: The other part of each fraction, containing C capsids, was incubated with GST proteins immobilized on glutathione-Sepharose beads as described above for 1 h at 4°C.

    Techniques: Purification, Recombinant, Mutagenesis, Electrophoresis, Staining

    Effect of the mutations in U L 31 R281/D282 on binding of recombinant NEC to capsid proteins from cells expressing each capsid protein. (A to D) 293FT cells were transfected with plasmids expressing either Flag-U L 25 (A), Flag-VP23 (B), Flag-U L 17 (C), or Flag-VP5 (D) for 24 h. These cells then were lysed and reacted with GST, GST-NEC 185-Δ50 , or GST-NEC 185-Δ50 R281A/D282A 31 that was immobilized on glutathione-Sepharose beads for 1 h at 4°C. After extensive washing, the beads were divided into two parts. One part was analyzed by electrophoresis in a denaturing gel and immunoblotted with anti-Flag antibody (top gels), and the other part was analyzed by electrophoresis in a denaturing gel and stained with CBB (bottom gels).

    Journal: Journal of Virology

    Article Title: Identification of the Capsid Binding Site in the Herpes Simplex Virus 1 Nuclear Egress Complex and Its Role in Viral Primary Envelopment and Replication

    doi: 10.1128/JVI.01290-19

    Figure Lengend Snippet: Effect of the mutations in U L 31 R281/D282 on binding of recombinant NEC to capsid proteins from cells expressing each capsid protein. (A to D) 293FT cells were transfected with plasmids expressing either Flag-U L 25 (A), Flag-VP23 (B), Flag-U L 17 (C), or Flag-VP5 (D) for 24 h. These cells then were lysed and reacted with GST, GST-NEC 185-Δ50 , or GST-NEC 185-Δ50 R281A/D282A 31 that was immobilized on glutathione-Sepharose beads for 1 h at 4°C. After extensive washing, the beads were divided into two parts. One part was analyzed by electrophoresis in a denaturing gel and immunoblotted with anti-Flag antibody (top gels), and the other part was analyzed by electrophoresis in a denaturing gel and stained with CBB (bottom gels).

    Article Snippet: The other part of each fraction, containing C capsids, was incubated with GST proteins immobilized on glutathione-Sepharose beads as described above for 1 h at 4°C.

    Techniques: Binding Assay, Recombinant, Expressing, Transfection, Electrophoresis, Staining

    Effect of the U L 31 R281 and D282 mutations on recombinant NEC binding to nucleocapsids. (A) Vero cells were infected with YK497 (U L 17-Myc/Flag-U L 25) at an MOI of 3 and harvested at 18 h postinfection. Nuclear lysates were isolated and layered onto 20% to 50% sucrose gradients and ultracentrifuged. The positions of type A, B, and C capsid bands in the sucrose gradient are indicated. (B) Proteins in the gradient fractions, shown in panel A, containing type A, B, or C capsids were analyzed by immunoblotting with the indicated antibodies. (C) Fractions containing C capsids were reacted with GST, GST-NEC 185-Δ50 , or GST-NEC 185-Δ50 R281A/D282A 31 immobilized on glutathione-Sepharose beads for 1 h at 4°C. Beads were then extensively washed and divided into two parts. One part was analyzed by electrophoresis in a denaturing gel and immunoblotted with anti-VP5, anti-Myc, anti-Flag, and anti-VP23 antibodies (top gels), and the other was analyzed by electrophoresis in a denaturing gel and stained with CBB (bottom gel).

    Journal: Journal of Virology

    Article Title: Identification of the Capsid Binding Site in the Herpes Simplex Virus 1 Nuclear Egress Complex and Its Role in Viral Primary Envelopment and Replication

    doi: 10.1128/JVI.01290-19

    Figure Lengend Snippet: Effect of the U L 31 R281 and D282 mutations on recombinant NEC binding to nucleocapsids. (A) Vero cells were infected with YK497 (U L 17-Myc/Flag-U L 25) at an MOI of 3 and harvested at 18 h postinfection. Nuclear lysates were isolated and layered onto 20% to 50% sucrose gradients and ultracentrifuged. The positions of type A, B, and C capsid bands in the sucrose gradient are indicated. (B) Proteins in the gradient fractions, shown in panel A, containing type A, B, or C capsids were analyzed by immunoblotting with the indicated antibodies. (C) Fractions containing C capsids were reacted with GST, GST-NEC 185-Δ50 , or GST-NEC 185-Δ50 R281A/D282A 31 immobilized on glutathione-Sepharose beads for 1 h at 4°C. Beads were then extensively washed and divided into two parts. One part was analyzed by electrophoresis in a denaturing gel and immunoblotted with anti-VP5, anti-Myc, anti-Flag, and anti-VP23 antibodies (top gels), and the other was analyzed by electrophoresis in a denaturing gel and stained with CBB (bottom gel).

    Article Snippet: The other part of each fraction, containing C capsids, was incubated with GST proteins immobilized on glutathione-Sepharose beads as described above for 1 h at 4°C.

    Techniques: Recombinant, Binding Assay, Infection, Isolation, Electrophoresis, Staining

    Effect of the U L 31 R281/D282 mutations on U L 31 interaction with U L 34 and capsid proteins in HSV-1-infected cells. Vero cells infected with YK735 (Strep-U L 34), YK736 (Strep-U L 34/U L 31-R281A/D282A), or YK727 (Strep-U L 34/U L 31-R281A/D282A-repair) at an MOI of 5 for 18 h were lysed, precipitated with Strep-Tactin Sepharose beads, and analyzed by immunoblotting with the indicated antibodies.

    Journal: Journal of Virology

    Article Title: Identification of the Capsid Binding Site in the Herpes Simplex Virus 1 Nuclear Egress Complex and Its Role in Viral Primary Envelopment and Replication

    doi: 10.1128/JVI.01290-19

    Figure Lengend Snippet: Effect of the U L 31 R281/D282 mutations on U L 31 interaction with U L 34 and capsid proteins in HSV-1-infected cells. Vero cells infected with YK735 (Strep-U L 34), YK736 (Strep-U L 34/U L 31-R281A/D282A), or YK727 (Strep-U L 34/U L 31-R281A/D282A-repair) at an MOI of 5 for 18 h were lysed, precipitated with Strep-Tactin Sepharose beads, and analyzed by immunoblotting with the indicated antibodies.

    Article Snippet: The other part of each fraction, containing C capsids, was incubated with GST proteins immobilized on glutathione-Sepharose beads as described above for 1 h at 4°C.

    Techniques: Infection

    Abundance, overexpression, and activation of Rho GTPases in GM/IFN-γ/LPS– and M/IL4-cultured macrophages. (A) GM/IFN-γ/LPS– and M/IL4-cultured macrophages were lysed, separated by 10% SDS–PAGE, and subjected to immunoblotting with anti-Rac1, anti-RhoA, anti-RhoG, or anti-Cdc42 antibodies. Intensities of the bands were normalized to β-actin, and then plotted as a ratio relative to GM/IFN-γ/LPS–cultured macrophages in each experiment for each GTPase. Data are means ± SEM of three to four independent experiments. Typical immunoblots are shown in Supplemental Figure S3A. (B) GM/IFN-γ/LPS– and M/IL4-cultured macrophages were lysed, followed immediately by measurement of the GTP-bound form of Rac1, RhoA, and Cdc42 using the respective colorimetric G-LISA kit. The measured absorbance at 490 nm was normalized to total protein levels and plotted as a ratio relative to GM/IFN-γ/LPS–cultured macrophages in each experiment for each GTPase. Data are means ± SEM of three to four independent experiments. Levels of GTP-bound RhoG in lysed GM/IFN-γ/LPS– and M/IL4-cultured macrophages were measured using a pull-down assay with a recombinant ELMO-GST loaded onto glutathione-sepharose beads, followed by 10% SDS–PAGE and immunoblotting with an anti-RhoG antibody. Levels of RhoG.GTP were normalized relative to the level in GM/IFN-γ/LPS–cultured macrophages in each experiment. A typical immunoblot for the RhoG.GTP pull-down assay is shown in Supplemental Figure S3B. Data are means ± SEM from four independent experiments. C. difficile toxin B treatment (3 h in serum-free medium) was used to inhibit all four GTPases, i.e., as a negative control, in all G-LISA and RhoG.GTP pull-down assays. (C–E) GM/IFN-γ/LPS–cultured macrophages were transfected with fluorescently tagged constructs of either wild-type (C) or constitutively active Rac1, RhoA, RhoG, or Cdc42 (D, E), or of the Rac1 and RhoG GEFs Tiam1 and sGEF, as indicated (D, E). The specific constructs used were Rac1-GFP, RhoA-GFP, RhoG-CFP, Cdc42-GFP, Rac1-Q61L-GFP, RhoA-Q63L-GFP, RhoG-G12V-CFP, Cdc42-G12V-YFP, Tiam1-GFP, and sGEF-GFP. GM/IFN-γ/LPS–cultured macrophages transfected with GFP alone were used as a negative control, while M/IL4-cultured macrophages transfected with GFP alone were used as a positive control. After 24-h transfection, the cells were incubated with fluorescently labeled 70 kDa dextran (TMR-dextran, 125 µg/ml) for 15 min at 37°C, and washed, fixed, and imaged immediately (D); only transfected cells were selected for measurements of macropinocytosis, which was quantified (C, E) as the total volume of TMR-positive vacuoles per cell from 3D stacks using 3D particle analysis in ImageJ software, applying a lower particle volume threshold of 0.26 µm 3 . Typical images (D) and quantifications (C, E; means ± SEM) are representative of 20–50 cells from three to five independent experiments using blood from at least two separate donors. Scale bars, 15 µm.

    Journal: Molecular Biology of the Cell

    Article Title: Differential ability of proinflammatory and anti-inflammatory macrophages to perform macropinocytosis

    doi: 10.1091/mbc.E17-06-0419

    Figure Lengend Snippet: Abundance, overexpression, and activation of Rho GTPases in GM/IFN-γ/LPS– and M/IL4-cultured macrophages. (A) GM/IFN-γ/LPS– and M/IL4-cultured macrophages were lysed, separated by 10% SDS–PAGE, and subjected to immunoblotting with anti-Rac1, anti-RhoA, anti-RhoG, or anti-Cdc42 antibodies. Intensities of the bands were normalized to β-actin, and then plotted as a ratio relative to GM/IFN-γ/LPS–cultured macrophages in each experiment for each GTPase. Data are means ± SEM of three to four independent experiments. Typical immunoblots are shown in Supplemental Figure S3A. (B) GM/IFN-γ/LPS– and M/IL4-cultured macrophages were lysed, followed immediately by measurement of the GTP-bound form of Rac1, RhoA, and Cdc42 using the respective colorimetric G-LISA kit. The measured absorbance at 490 nm was normalized to total protein levels and plotted as a ratio relative to GM/IFN-γ/LPS–cultured macrophages in each experiment for each GTPase. Data are means ± SEM of three to four independent experiments. Levels of GTP-bound RhoG in lysed GM/IFN-γ/LPS– and M/IL4-cultured macrophages were measured using a pull-down assay with a recombinant ELMO-GST loaded onto glutathione-sepharose beads, followed by 10% SDS–PAGE and immunoblotting with an anti-RhoG antibody. Levels of RhoG.GTP were normalized relative to the level in GM/IFN-γ/LPS–cultured macrophages in each experiment. A typical immunoblot for the RhoG.GTP pull-down assay is shown in Supplemental Figure S3B. Data are means ± SEM from four independent experiments. C. difficile toxin B treatment (3 h in serum-free medium) was used to inhibit all four GTPases, i.e., as a negative control, in all G-LISA and RhoG.GTP pull-down assays. (C–E) GM/IFN-γ/LPS–cultured macrophages were transfected with fluorescently tagged constructs of either wild-type (C) or constitutively active Rac1, RhoA, RhoG, or Cdc42 (D, E), or of the Rac1 and RhoG GEFs Tiam1 and sGEF, as indicated (D, E). The specific constructs used were Rac1-GFP, RhoA-GFP, RhoG-CFP, Cdc42-GFP, Rac1-Q61L-GFP, RhoA-Q63L-GFP, RhoG-G12V-CFP, Cdc42-G12V-YFP, Tiam1-GFP, and sGEF-GFP. GM/IFN-γ/LPS–cultured macrophages transfected with GFP alone were used as a negative control, while M/IL4-cultured macrophages transfected with GFP alone were used as a positive control. After 24-h transfection, the cells were incubated with fluorescently labeled 70 kDa dextran (TMR-dextran, 125 µg/ml) for 15 min at 37°C, and washed, fixed, and imaged immediately (D); only transfected cells were selected for measurements of macropinocytosis, which was quantified (C, E) as the total volume of TMR-positive vacuoles per cell from 3D stacks using 3D particle analysis in ImageJ software, applying a lower particle volume threshold of 0.26 µm 3 . Typical images (D) and quantifications (C, E; means ± SEM) are representative of 20–50 cells from three to five independent experiments using blood from at least two separate donors. Scale bars, 15 µm.

    Article Snippet: Lysates were cleared at 14,000 × g for 10 min. Supernatants were rotated for 30 min with ≈10 μg ELMO-GST (GST fusion protein containing the full-length RhoG effector ELMO) conjugated to glutathione-Sepharose beads (GE Healthcare).

    Techniques: Over Expression, Activation Assay, Cell Culture, SDS Page, Western Blot, Pull Down Assay, Recombinant, Negative Control, Transfection, Construct, Positive Control, Incubation, Labeling, Software