ezview red anti flag m2 affinity gel  (Millipore)


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    Name:
    EZview Red ANTI FLAG M2 Affinity Gel
    Description:
    EZ view Red Anti FLAG M2 Affinity Gel is a resin that consists of Anti FLAG M2 antibody covalently bonded to 4 Agarose beads The affinity gel is used to bind FLAG fusion proteins to samples such as cell lysates and tissue for purification of FLAG tagged proteins in preparation of immunoprecipitation assays Red dye enhances visability for more efficient results Agarose beads bind at N terminal Met N terminal and C terminal FLAG fusion proteins 3xFLAG tagged fusion proteins
    Catalog Number:
    f2426
    Price:
    None
    Applications:
    Immunoprecipitation (IP) of FLAG- and 3xFLAG-tagged fusion proteins.Elution - FLAG peptide, Glycine, pH 3.5, 3x FLAG peptideBrowse additional application references in our FLAG(R) Literature portal.
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    Structured Review

    Millipore ezview red anti flag m2 affinity gel
    Cys-replacement stabilizes FAT10 and its conjugates in vivo. a HEK293 cells expressing WT or Cys-free <t>FLAG-tagged</t> FAT10 were treated for 0.5, 1, or 2.5 h with 50 µg/mL CHX to monitor degradation rates. In addition, cells were treated with 10 µM proteasome inhibitor MG132 for 3.5 h, as indicated. Cells were harvested, lysed, and subjected to immunoprecipitation using <t>EZview</t> Red <t>anti-FLAG-M2</t> affinity gel. Proteins were separated on 4–12% NUPAGE gradient gels and subjected to western blot analysis using a directly peroxidase-labeled, monoclonal FLAG-reactive antibody (clone M2). β-actin was used as loading control. One representative experiment out of four independent experiments with similar outcomes is shown. b ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated WT or Cys-free FLAG-FAT10 normalized to the amount of β-actin in the lysate. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Shown are the values of four independent experiments with similar outcomes as means ± s.e.m. c Molecular dynamics (MD) simulations of the N- (left) and C-domain (right) of human FAT10. Top panels: three of the most prominent structures obtained by root mean square deviation clustering from each MD simulation in cartoon representation (WT: white, Cys-free: blue). Bottom panels: Residue-wise root mean square fluctuation (RMSF) values. Arrows in the graph show position of cysteine mutations. N- and C-terminal tails are flexible. Moreover, flexibility is decreased in the N-domain in Cys-free mutants while the overall structure of both domains is preserved
    EZ view Red Anti FLAG M2 Affinity Gel is a resin that consists of Anti FLAG M2 antibody covalently bonded to 4 Agarose beads The affinity gel is used to bind FLAG fusion proteins to samples such as cell lysates and tissue for purification of FLAG tagged proteins in preparation of immunoprecipitation assays Red dye enhances visability for more efficient results Agarose beads bind at N terminal Met N terminal and C terminal FLAG fusion proteins 3xFLAG tagged fusion proteins
    https://www.bioz.com/result/ezview red anti flag m2 affinity gel/product/Millipore
    Average 99 stars, based on 250 article reviews
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    ezview red anti flag m2 affinity gel - by Bioz Stars, 2021-01
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    Images

    1) Product Images from "The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation"

    Article Title: The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05776-3

    Cys-replacement stabilizes FAT10 and its conjugates in vivo. a HEK293 cells expressing WT or Cys-free FLAG-tagged FAT10 were treated for 0.5, 1, or 2.5 h with 50 µg/mL CHX to monitor degradation rates. In addition, cells were treated with 10 µM proteasome inhibitor MG132 for 3.5 h, as indicated. Cells were harvested, lysed, and subjected to immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. Proteins were separated on 4–12% NUPAGE gradient gels and subjected to western blot analysis using a directly peroxidase-labeled, monoclonal FLAG-reactive antibody (clone M2). β-actin was used as loading control. One representative experiment out of four independent experiments with similar outcomes is shown. b ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated WT or Cys-free FLAG-FAT10 normalized to the amount of β-actin in the lysate. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Shown are the values of four independent experiments with similar outcomes as means ± s.e.m. c Molecular dynamics (MD) simulations of the N- (left) and C-domain (right) of human FAT10. Top panels: three of the most prominent structures obtained by root mean square deviation clustering from each MD simulation in cartoon representation (WT: white, Cys-free: blue). Bottom panels: Residue-wise root mean square fluctuation (RMSF) values. Arrows in the graph show position of cysteine mutations. N- and C-terminal tails are flexible. Moreover, flexibility is decreased in the N-domain in Cys-free mutants while the overall structure of both domains is preserved
    Figure Legend Snippet: Cys-replacement stabilizes FAT10 and its conjugates in vivo. a HEK293 cells expressing WT or Cys-free FLAG-tagged FAT10 were treated for 0.5, 1, or 2.5 h with 50 µg/mL CHX to monitor degradation rates. In addition, cells were treated with 10 µM proteasome inhibitor MG132 for 3.5 h, as indicated. Cells were harvested, lysed, and subjected to immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. Proteins were separated on 4–12% NUPAGE gradient gels and subjected to western blot analysis using a directly peroxidase-labeled, monoclonal FLAG-reactive antibody (clone M2). β-actin was used as loading control. One representative experiment out of four independent experiments with similar outcomes is shown. b ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated WT or Cys-free FLAG-FAT10 normalized to the amount of β-actin in the lysate. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Shown are the values of four independent experiments with similar outcomes as means ± s.e.m. c Molecular dynamics (MD) simulations of the N- (left) and C-domain (right) of human FAT10. Top panels: three of the most prominent structures obtained by root mean square deviation clustering from each MD simulation in cartoon representation (WT: white, Cys-free: blue). Bottom panels: Residue-wise root mean square fluctuation (RMSF) values. Arrows in the graph show position of cysteine mutations. N- and C-terminal tails are flexible. Moreover, flexibility is decreased in the N-domain in Cys-free mutants while the overall structure of both domains is preserved

    Techniques Used: In Vivo, Expressing, Immunoprecipitation, Western Blot, Labeling

    The FAT10 UBDs are loosely folded and degraded less efficiently when replaced by Ub. a . b Schematic presentation of WT FLAG-FAT10 and the two FAT10-ubiquitin hybrids (FLAG-FAT10-Ub and FLAG-Ub-FAT10), where either the N- or the C-domain of FAT10 was exchanged with lysine-less Ub (K0) to prevent Ub chain formation. To suppress cleavage of Ub(K0) from the FAT10 C-domain in Ub-FAT10, Ub(K0) was expressed without the di-glycine motif. c Western blots showing bulk conjugates of WT FAT10, Ub-FAT10 or FAT10-Ub proteins, and USE1 auto-FAT10ylation. d Degradation of monomeric WT FAT10, Ub-FAT10, and FAT10-Ub was monitored by transient transfection of HEK293 cells with the indicated expression constructs. Cells were treated for 2.5 or 5 h with 50 µg/mL CHX to inhibit de novo protein synthesis. Where indicated, cells were additionally treated with 10 µM proteasome inhibitor MG132 for 6 h. c , d HEK293 cells were harvested and lysed 24 h after transfection. Cleared protein lysates were used for immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. The upper panels show the bulk conjugates in the immunoprecipitation, lower panels show the expression of the proteins in the lysates. β-actin was used as loading control. One representative experiment out of three experiments with similar outcomes is shown. e ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated FLAG-FAT10 and FLAG-tagged FAT10-Ub chimeras in the cells. The ECL signals were quantified with Image Lab 4.1. software (BioRad) and normalized to signals of the loading control β-actin. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Shown are the values of three independent experiments with similar outcomes as means ± s.e.m.
    Figure Legend Snippet: The FAT10 UBDs are loosely folded and degraded less efficiently when replaced by Ub. a . b Schematic presentation of WT FLAG-FAT10 and the two FAT10-ubiquitin hybrids (FLAG-FAT10-Ub and FLAG-Ub-FAT10), where either the N- or the C-domain of FAT10 was exchanged with lysine-less Ub (K0) to prevent Ub chain formation. To suppress cleavage of Ub(K0) from the FAT10 C-domain in Ub-FAT10, Ub(K0) was expressed without the di-glycine motif. c Western blots showing bulk conjugates of WT FAT10, Ub-FAT10 or FAT10-Ub proteins, and USE1 auto-FAT10ylation. d Degradation of monomeric WT FAT10, Ub-FAT10, and FAT10-Ub was monitored by transient transfection of HEK293 cells with the indicated expression constructs. Cells were treated for 2.5 or 5 h with 50 µg/mL CHX to inhibit de novo protein synthesis. Where indicated, cells were additionally treated with 10 µM proteasome inhibitor MG132 for 6 h. c , d HEK293 cells were harvested and lysed 24 h after transfection. Cleared protein lysates were used for immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. The upper panels show the bulk conjugates in the immunoprecipitation, lower panels show the expression of the proteins in the lysates. β-actin was used as loading control. One representative experiment out of three experiments with similar outcomes is shown. e ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated FLAG-FAT10 and FLAG-tagged FAT10-Ub chimeras in the cells. The ECL signals were quantified with Image Lab 4.1. software (BioRad) and normalized to signals of the loading control β-actin. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Shown are the values of three independent experiments with similar outcomes as means ± s.e.m.

    Techniques Used: Western Blot, Transfection, Expressing, Construct, Immunoprecipitation, Software

    The presence of the FAT10 linker is crucial for FAT10 conjugation. a Scheme showing the amino acid substitutions or deletion of the WT FAT10 linker sequence (KPSDE). The different linker mutants 5x Ala, 5x Pro, 5x Gly, and Δ-linker were generated by site-directed mutagenesis of the expression plasmid pcDNA3.1-His-3xFLAG-FAT10. The His-3xFLAG tag is referred to as FLAG in the text as well as in the figures. b . c The degradation rate of monomeric FAT10 in the lysate as well as of FAT10 conjugates was monitored in HEK293 cells expressing the different FLAG-tagged FAT10 linker variants. Cells were treated for 2.5 or 5 h with 50 µg/mL CHX to inhibit de novo protein synthesis. Where indicated, cells were additionally treated with 10 µM proteasome inhibitor MG132 for 6 h. b , c HEK293 cells were harvested and lysed 24 h after transfection. Cleared protein lysates were subjected to immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. Proteins were separated on 12.5% Laemmli gels and visualized by western blot analysis with a monoclonal FLAG-reactive antibody (clone M2) or a USE1-reactive polyclonal antibody, as indicated. β-actin was used as loading control. One experiment out of three experiments with similar outcomes is shown. d Quantification of the amount of monomeric and of conjugated FLAG-FAT10 linker variants in c . The enhanced chemiluminescence (ECL) signals were quantified with Image Lab 4.1. software (BioRad) and normalized to signals of the loading control β-actin. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Values are shown for three independent experiments with similar outcomes as means ± s.e.m.
    Figure Legend Snippet: The presence of the FAT10 linker is crucial for FAT10 conjugation. a Scheme showing the amino acid substitutions or deletion of the WT FAT10 linker sequence (KPSDE). The different linker mutants 5x Ala, 5x Pro, 5x Gly, and Δ-linker were generated by site-directed mutagenesis of the expression plasmid pcDNA3.1-His-3xFLAG-FAT10. The His-3xFLAG tag is referred to as FLAG in the text as well as in the figures. b . c The degradation rate of monomeric FAT10 in the lysate as well as of FAT10 conjugates was monitored in HEK293 cells expressing the different FLAG-tagged FAT10 linker variants. Cells were treated for 2.5 or 5 h with 50 µg/mL CHX to inhibit de novo protein synthesis. Where indicated, cells were additionally treated with 10 µM proteasome inhibitor MG132 for 6 h. b , c HEK293 cells were harvested and lysed 24 h after transfection. Cleared protein lysates were subjected to immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. Proteins were separated on 12.5% Laemmli gels and visualized by western blot analysis with a monoclonal FLAG-reactive antibody (clone M2) or a USE1-reactive polyclonal antibody, as indicated. β-actin was used as loading control. One experiment out of three experiments with similar outcomes is shown. d Quantification of the amount of monomeric and of conjugated FLAG-FAT10 linker variants in c . The enhanced chemiluminescence (ECL) signals were quantified with Image Lab 4.1. software (BioRad) and normalized to signals of the loading control β-actin. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Values are shown for three independent experiments with similar outcomes as means ± s.e.m.

    Techniques Used: Conjugation Assay, Sequencing, Generated, Mutagenesis, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Software

    2) Product Images from "MARINOBUFAGENIN INTERFERES WITH THE FUNCTION OF THE MINERALOCORTICOID RECEPTOR"

    Article Title: MARINOBUFAGENIN INTERFERES WITH THE FUNCTION OF THE MINERALOCORTICOID RECEPTOR

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2007.03.085

    MBG reduces interaction between MR and SRC-3. Cos1 cells were transfected with expression vectors for Flag-SRC-3 and MR and 24h later treated with either 0.1% ethanol (V), 10 −8 M aldosterone (Ald) or 10 −8 M aldosterone with 10 −6 marinobufagenin (Ald + MBG) for 30 min. Cells were lysed and aliquots of input extracts (lanes 1-3) as well as material immunoprecipitated with EZview™ Red ANTI-FLAG® M2 Affinity Gel (lanes 4-6) or IgG (lane 7) were subjected to Western blot analyses for SRC-3 ( top ), MR ( middle ) and actin ( bottom ). The blot is representative of four experiments.
    Figure Legend Snippet: MBG reduces interaction between MR and SRC-3. Cos1 cells were transfected with expression vectors for Flag-SRC-3 and MR and 24h later treated with either 0.1% ethanol (V), 10 −8 M aldosterone (Ald) or 10 −8 M aldosterone with 10 −6 marinobufagenin (Ald + MBG) for 30 min. Cells were lysed and aliquots of input extracts (lanes 1-3) as well as material immunoprecipitated with EZview™ Red ANTI-FLAG® M2 Affinity Gel (lanes 4-6) or IgG (lane 7) were subjected to Western blot analyses for SRC-3 ( top ), MR ( middle ) and actin ( bottom ). The blot is representative of four experiments.

    Techniques Used: Transfection, Expressing, Immunoprecipitation, Western Blot

    3) Product Images from "ZASP Interacts with the Mechanosensing Protein Ankrd2 and p53 in the Signalling Network of Striated Muscle"

    Article Title: ZASP Interacts with the Mechanosensing Protein Ankrd2 and p53 in the Signalling Network of Striated Muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092259

    PDZ and ZM1 regions of ZASP6 directly bind Ankrd2. A) Co-IP of ZASP6 and Ankrd2 using lysates of COS-7 cells transfected with the indicated combination of pCMV vectors expressing cMyc-Ankrd2 and FLAG-ZASP6. Immunoprecipitation was performed with anti-FLAG antibody. The presence of Ankrd2 in the immune complex was confirmed by probing the membrane with mouse polyclonal anti-Ankrd2 antibody (upper panel). As controls, the cell lysates were tested with polyclonal antibodies to anti-Ankrd2 (middle panel) and anti-ZASP6 (bottom panel). B) Co-IP of PDZ-ZASP6 and Ankrd2 using lysates of COS-7 cells transfected with cMyc-Ankrd2 and pEGFP-PDZ as indicated. The cell lysates were immunoprecipitated with an anti-cMyc monoclonal antibody and probed with a polyclonal goat anti-GFP antibody conjugated to HRP (upper panel). Comparable expression levels of Ankrd2 and PDZ-ZASP6, weredemonstrated by probing the membrane with a mouse anti-Ankrd2 polyclonal antibody (middle panel) and rabbit anti-GFP polyclonal antibody (lower panel). C) Co-IP of ZM1-ZASP6 and Ankrd2 using lysates of COS-7 cells transfected with cMyc-Ankrd2 and FLAG-ZM1 as indicated. Ankrd2 was immunoprecipitated with anti-FLAG EZview resin and detected with an anti-Ankrd2 mouse polyclonal antibody (upper panel). As controls, cell lysates were tested with an anti-Ankrd2 mouse polyclonal antibody (middle panel) and with an anti-FLAG rabbit polyclonal antibody (bottom panel). All secondary antibodies were conjugated with HRP. D) GST-overlay assay with purified recombinant proteins expressed in E. coli . His-Ankrd2 (4 μg) was separated by SDS-PAGE, blotted and membrane strips were incubated with 4 μg of either GST-ZASP6, GST-PDZ, GST-ZM1 or GST alone, washed and then probed with anti-GST goat polyclonal antibody. As a control, an anti-histidine antibody was used to probe membrane strips identical to those used for the overlay to show that the His-Ankrd2 protein was equally loaded (lower panel). E) A schematic diagram of ZASP6 showing the positions of the PDZ domain (aa 11–84) and ZM-motif (aa 148–173). In this study the expression vectors with constructs for PDZ and ZM1 (ZASP6 without the PDZ domain) expressed respectively 85 and 197 amino acid fragments of ZASP6.
    Figure Legend Snippet: PDZ and ZM1 regions of ZASP6 directly bind Ankrd2. A) Co-IP of ZASP6 and Ankrd2 using lysates of COS-7 cells transfected with the indicated combination of pCMV vectors expressing cMyc-Ankrd2 and FLAG-ZASP6. Immunoprecipitation was performed with anti-FLAG antibody. The presence of Ankrd2 in the immune complex was confirmed by probing the membrane with mouse polyclonal anti-Ankrd2 antibody (upper panel). As controls, the cell lysates were tested with polyclonal antibodies to anti-Ankrd2 (middle panel) and anti-ZASP6 (bottom panel). B) Co-IP of PDZ-ZASP6 and Ankrd2 using lysates of COS-7 cells transfected with cMyc-Ankrd2 and pEGFP-PDZ as indicated. The cell lysates were immunoprecipitated with an anti-cMyc monoclonal antibody and probed with a polyclonal goat anti-GFP antibody conjugated to HRP (upper panel). Comparable expression levels of Ankrd2 and PDZ-ZASP6, weredemonstrated by probing the membrane with a mouse anti-Ankrd2 polyclonal antibody (middle panel) and rabbit anti-GFP polyclonal antibody (lower panel). C) Co-IP of ZM1-ZASP6 and Ankrd2 using lysates of COS-7 cells transfected with cMyc-Ankrd2 and FLAG-ZM1 as indicated. Ankrd2 was immunoprecipitated with anti-FLAG EZview resin and detected with an anti-Ankrd2 mouse polyclonal antibody (upper panel). As controls, cell lysates were tested with an anti-Ankrd2 mouse polyclonal antibody (middle panel) and with an anti-FLAG rabbit polyclonal antibody (bottom panel). All secondary antibodies were conjugated with HRP. D) GST-overlay assay with purified recombinant proteins expressed in E. coli . His-Ankrd2 (4 μg) was separated by SDS-PAGE, blotted and membrane strips were incubated with 4 μg of either GST-ZASP6, GST-PDZ, GST-ZM1 or GST alone, washed and then probed with anti-GST goat polyclonal antibody. As a control, an anti-histidine antibody was used to probe membrane strips identical to those used for the overlay to show that the His-Ankrd2 protein was equally loaded (lower panel). E) A schematic diagram of ZASP6 showing the positions of the PDZ domain (aa 11–84) and ZM-motif (aa 148–173). In this study the expression vectors with constructs for PDZ and ZM1 (ZASP6 without the PDZ domain) expressed respectively 85 and 197 amino acid fragments of ZASP6.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Overlay Assay, Purification, Recombinant, SDS Page, Incubation, Construct

    4) Product Images from "MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling"

    Article Title: MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI97712

    The molecular basis for MAPK4 binding to AKT, part 1. ( A ) The domain architecture of human MAPK4. MAPK4 contains an N-terminal kinase domain (aa20 to aa312), a conserved C34 (aa313 to aa462) motif shared between MAPK4 (Erk4) and MAPK6 (Erk3), and a C-terminal tail. Asterisk indicates phosphorylation site. ( B ) HEK293T cells were transfected with FLAG-tagged AKT1 and HA-tagged WT or C-terminally truncated MAPK4 including N462, N312, and N185. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-HA antibody. The kinase domain of MAPK4 binds to AKT1 and the aa186–aa312 fragment is essential for this interaction. ( C ) His-tagged AKT1 along with FLAG-tagged MAPK4 and MAPK4 S186A mutant were reconstructed into the DLD1 AKT1/2 -KO cells that lack all 3 isoforms of AKT. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots for phosphorylation of AKT and total AKT. WCL: whole-cell lysate. ( D ) HA-tagged AKT2 and AKT3 were reconstructed into the DLD1 AKT1/2 -KO cells along FLAG-tagged MAPK4 or MAPK4 S186A mutant. Western blots using Phospho-AKT T308 antibody were used to detect the phosphorylation of AKT2 at T309 and AKT3 at T305, respectively. ( E ) FLAG-tagged MAPK4 and MAPK4 S186A mutant were transduced into the H157 MAPK4 -KO cells. Western blots were used to determine AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.
    Figure Legend Snippet: The molecular basis for MAPK4 binding to AKT, part 1. ( A ) The domain architecture of human MAPK4. MAPK4 contains an N-terminal kinase domain (aa20 to aa312), a conserved C34 (aa313 to aa462) motif shared between MAPK4 (Erk4) and MAPK6 (Erk3), and a C-terminal tail. Asterisk indicates phosphorylation site. ( B ) HEK293T cells were transfected with FLAG-tagged AKT1 and HA-tagged WT or C-terminally truncated MAPK4 including N462, N312, and N185. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-HA antibody. The kinase domain of MAPK4 binds to AKT1 and the aa186–aa312 fragment is essential for this interaction. ( C ) His-tagged AKT1 along with FLAG-tagged MAPK4 and MAPK4 S186A mutant were reconstructed into the DLD1 AKT1/2 -KO cells that lack all 3 isoforms of AKT. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots for phosphorylation of AKT and total AKT. WCL: whole-cell lysate. ( D ) HA-tagged AKT2 and AKT3 were reconstructed into the DLD1 AKT1/2 -KO cells along FLAG-tagged MAPK4 or MAPK4 S186A mutant. Western blots using Phospho-AKT T308 antibody were used to detect the phosphorylation of AKT2 at T309 and AKT3 at T305, respectively. ( E ) FLAG-tagged MAPK4 and MAPK4 S186A mutant were transduced into the H157 MAPK4 -KO cells. Western blots were used to determine AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.

    Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Mutagenesis

    MAPK4 interacts with AKT and is a novel T308 kinase. ( A ) FLAG/His-tagged MAPK4 and the MAPK4 2A mutant were overexpressed in PNT1A cells and purified using Ni-NTA column followed by immunoprecipitation using anti-FLAG M2 affinity gel. In vitro kinase assay was performed using a commercially available recombinant AKT1. AKT phosphorylation was detected by Western blots. MAPK4 2A carries KVAVAA in place of the KVAVKK ATP binding motif. Coomassie blue staining reveals that the purified FLAG/His-tagged MAPK4 (with a calculated molecular weight of 69.6 kDa) contains one major band around 70 kDa. F/H: 2× FLAG and 10× His tag. ( B ) MAPK4 and the MAPK4 2A and MAPK4 5A mutants were overexpressed in HCT116 PDK1 -KO cells. AKT phosphorylation was detected by Western blots. MAPK4 5A carries AAAAAA in place of the KVAVKK ATP binding motif. Coimmunoprecipitation (co-IP) assays reveal ( C ) ectopically overexpressed AKT1 binding to ectopically overexpressed MAPK4 in HEK293T cells, and ( D ) endogenous AKT1 binding to endogenous MAPK4 in VCaP cells. ( E ) Pull-down assay on the binding between purified GST-MAPK4 protein and purified AKT1 as well as endogenous AKT in the HEK293T, H1299, and HepG2 cell lysates. GST was used as control. GST-MAPK4 and GST were overexpressed in E . coli and purified using the glutathione sepharose beads. Coomassie blue staining confirms major bands of around 90 kDa and 26 kDa in the purified GST-MAPK4 and GST, respectively. ( F ) BiFC assay for AKT association with MAPK4 in cytoplasm. Hela cells were cotransfected with YN-MAPK4 and YC-AKT1 or YC control vectors. Forty-eight hours later, the cells were fixed, counterstained with DAPI, and imaged for YFP fluorescence to indicate MAPK4-AKT1 interaction. Original magnification: ×400. ( G ) Three different MK5 siRNAs were transfected into control and MAPK4-overexpressing HEK293T cells. Western blots were used to confirm MK5 knockdown and compare AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.
    Figure Legend Snippet: MAPK4 interacts with AKT and is a novel T308 kinase. ( A ) FLAG/His-tagged MAPK4 and the MAPK4 2A mutant were overexpressed in PNT1A cells and purified using Ni-NTA column followed by immunoprecipitation using anti-FLAG M2 affinity gel. In vitro kinase assay was performed using a commercially available recombinant AKT1. AKT phosphorylation was detected by Western blots. MAPK4 2A carries KVAVAA in place of the KVAVKK ATP binding motif. Coomassie blue staining reveals that the purified FLAG/His-tagged MAPK4 (with a calculated molecular weight of 69.6 kDa) contains one major band around 70 kDa. F/H: 2× FLAG and 10× His tag. ( B ) MAPK4 and the MAPK4 2A and MAPK4 5A mutants were overexpressed in HCT116 PDK1 -KO cells. AKT phosphorylation was detected by Western blots. MAPK4 5A carries AAAAAA in place of the KVAVKK ATP binding motif. Coimmunoprecipitation (co-IP) assays reveal ( C ) ectopically overexpressed AKT1 binding to ectopically overexpressed MAPK4 in HEK293T cells, and ( D ) endogenous AKT1 binding to endogenous MAPK4 in VCaP cells. ( E ) Pull-down assay on the binding between purified GST-MAPK4 protein and purified AKT1 as well as endogenous AKT in the HEK293T, H1299, and HepG2 cell lysates. GST was used as control. GST-MAPK4 and GST were overexpressed in E . coli and purified using the glutathione sepharose beads. Coomassie blue staining confirms major bands of around 90 kDa and 26 kDa in the purified GST-MAPK4 and GST, respectively. ( F ) BiFC assay for AKT association with MAPK4 in cytoplasm. Hela cells were cotransfected with YN-MAPK4 and YC-AKT1 or YC control vectors. Forty-eight hours later, the cells were fixed, counterstained with DAPI, and imaged for YFP fluorescence to indicate MAPK4-AKT1 interaction. Original magnification: ×400. ( G ) Three different MK5 siRNAs were transfected into control and MAPK4-overexpressing HEK293T cells. Western blots were used to confirm MK5 knockdown and compare AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.

    Techniques Used: Mutagenesis, Purification, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Western Blot, Binding Assay, Staining, Molecular Weight, Co-Immunoprecipitation Assay, Pull Down Assay, Bimolecular Fluorescence Complementation Assay, Fluorescence, Transfection

    The molecular basis for MAPK4 binding to AKT, part 2. ( A ) The FLAG-tagged MAPK4 and MAPK4 E/D5A (5A-FLAG) mutant were transduced into HEK293T cells with or without cotransfection of AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. The MAPK4 E/D5A (5A-FLAG) mutant carries AAAKAA in place of the potential CD motif EEDKDE. ( B ) FLAG-tagged WT MAPK4 and the MAPK4 E250A , MAPK4 E251A , MAPK4 D252A , MAPK4 D254A , MAPK4 E255A mutants were transfected into HEK293T cells together with AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( C ) FLAG-tagged WT MAPK4 (WT) and the MAPK4 D254A , MAPK4 E255A mutants were transfected into the HCT116 MAPK4 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( D ). ( E ) Alignment of MAPK4 protein sequence around the CD motif across species. ( F ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transduced into HCT116 cells together with FLAG-tagged MAPK4. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( G ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transfected into the HCT116 PDK1 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( H ) The domain architecture of human AKT1 and the sequence alignment of human AKT1, AKT2, and AKT3 proteins. ( I ) Alignment of AKT1 protein sequence around the D-motif across species.
    Figure Legend Snippet: The molecular basis for MAPK4 binding to AKT, part 2. ( A ) The FLAG-tagged MAPK4 and MAPK4 E/D5A (5A-FLAG) mutant were transduced into HEK293T cells with or without cotransfection of AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. The MAPK4 E/D5A (5A-FLAG) mutant carries AAAKAA in place of the potential CD motif EEDKDE. ( B ) FLAG-tagged WT MAPK4 and the MAPK4 E250A , MAPK4 E251A , MAPK4 D252A , MAPK4 D254A , MAPK4 E255A mutants were transfected into HEK293T cells together with AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( C ) FLAG-tagged WT MAPK4 (WT) and the MAPK4 D254A , MAPK4 E255A mutants were transfected into the HCT116 MAPK4 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( D ). ( E ) Alignment of MAPK4 protein sequence around the CD motif across species. ( F ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transduced into HCT116 cells together with FLAG-tagged MAPK4. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( G ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transfected into the HCT116 PDK1 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( H ) The domain architecture of human AKT1 and the sequence alignment of human AKT1, AKT2, and AKT3 proteins. ( I ) Alignment of AKT1 protein sequence around the D-motif across species.

    Techniques Used: Binding Assay, Mutagenesis, Cotransfection, Immunoprecipitation, Western Blot, Transfection, Sequencing

    5) Product Images from "MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling"

    Article Title: MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI97712

    The molecular basis for MAPK4 binding to AKT, part 1. ( A ) The domain architecture of human MAPK4. MAPK4 contains an N-terminal kinase domain (aa20 to aa312), a conserved C34 (aa313 to aa462) motif shared between MAPK4 (Erk4) and MAPK6 (Erk3), and a C-terminal tail. Asterisk indicates phosphorylation site. ( B ) HEK293T cells were transfected with FLAG-tagged AKT1 and HA-tagged WT or C-terminally truncated MAPK4 including N462, N312, and N185. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-HA antibody. The kinase domain of MAPK4 binds to AKT1 and the aa186–aa312 fragment is essential for this interaction. ( C ) His-tagged AKT1 along with FLAG-tagged MAPK4 and MAPK4 S186A mutant were reconstructed into the DLD1 AKT1/2 -KO cells that lack all 3 isoforms of AKT. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots for phosphorylation of AKT and total AKT. WCL: whole-cell lysate. ( D ) HA-tagged AKT2 and AKT3 were reconstructed into the DLD1 AKT1/2 -KO cells along FLAG-tagged MAPK4 or MAPK4 S186A mutant. Western blots using Phospho-AKT T308 antibody were used to detect the phosphorylation of AKT2 at T309 and AKT3 at T305, respectively. ( E ) FLAG-tagged MAPK4 and MAPK4 S186A mutant were transduced into the H157 MAPK4 -KO cells. Western blots were used to determine AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.
    Figure Legend Snippet: The molecular basis for MAPK4 binding to AKT, part 1. ( A ) The domain architecture of human MAPK4. MAPK4 contains an N-terminal kinase domain (aa20 to aa312), a conserved C34 (aa313 to aa462) motif shared between MAPK4 (Erk4) and MAPK6 (Erk3), and a C-terminal tail. Asterisk indicates phosphorylation site. ( B ) HEK293T cells were transfected with FLAG-tagged AKT1 and HA-tagged WT or C-terminally truncated MAPK4 including N462, N312, and N185. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-HA antibody. The kinase domain of MAPK4 binds to AKT1 and the aa186–aa312 fragment is essential for this interaction. ( C ) His-tagged AKT1 along with FLAG-tagged MAPK4 and MAPK4 S186A mutant were reconstructed into the DLD1 AKT1/2 -KO cells that lack all 3 isoforms of AKT. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots for phosphorylation of AKT and total AKT. WCL: whole-cell lysate. ( D ) HA-tagged AKT2 and AKT3 were reconstructed into the DLD1 AKT1/2 -KO cells along FLAG-tagged MAPK4 or MAPK4 S186A mutant. Western blots using Phospho-AKT T308 antibody were used to detect the phosphorylation of AKT2 at T309 and AKT3 at T305, respectively. ( E ) FLAG-tagged MAPK4 and MAPK4 S186A mutant were transduced into the H157 MAPK4 -KO cells. Western blots were used to determine AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.

    Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Mutagenesis

    MAPK4 interacts with AKT and is a novel T308 kinase. ( A ) FLAG/His-tagged MAPK4 and the MAPK4 2A mutant were overexpressed in PNT1A cells and purified using Ni-NTA column followed by immunoprecipitation using anti-FLAG M2 affinity gel. In vitro kinase assay was performed using a commercially available recombinant AKT1. AKT phosphorylation was detected by Western blots. MAPK4 2A carries KVAVAA in place of the KVAVKK ATP binding motif. Coomassie blue staining reveals that the purified FLAG/His-tagged MAPK4 (with a calculated molecular weight of 69.6 kDa) contains one major band around 70 kDa. F/H: 2× FLAG and 10× His tag. ( B ) MAPK4 and the MAPK4 2A and MAPK4 5A mutants were overexpressed in HCT116 PDK1 -KO cells. AKT phosphorylation was detected by Western blots. MAPK4 5A carries AAAAAA in place of the KVAVKK ATP binding motif. Coimmunoprecipitation (co-IP) assays reveal ( C ) ectopically overexpressed AKT1 binding to ectopically overexpressed MAPK4 in HEK293T cells, and ( D ) endogenous AKT1 binding to endogenous MAPK4 in VCaP cells. ( E ) Pull-down assay on the binding between purified GST-MAPK4 protein and purified AKT1 as well as endogenous AKT in the HEK293T, H1299, and HepG2 cell lysates. GST was used as control. GST-MAPK4 and GST were overexpressed in E . coli and purified using the glutathione sepharose beads. Coomassie blue staining confirms major bands of around 90 kDa and 26 kDa in the purified GST-MAPK4 and GST, respectively. ( F ) BiFC assay for AKT association with MAPK4 in cytoplasm. Hela cells were cotransfected with YN-MAPK4 and YC-AKT1 or YC control vectors. Forty-eight hours later, the cells were fixed, counterstained with DAPI, and imaged for YFP fluorescence to indicate MAPK4-AKT1 interaction. Original magnification: ×400. ( G ) Three different MK5 siRNAs were transfected into control and MAPK4-overexpressing HEK293T cells. Western blots were used to confirm MK5 knockdown and compare AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.
    Figure Legend Snippet: MAPK4 interacts with AKT and is a novel T308 kinase. ( A ) FLAG/His-tagged MAPK4 and the MAPK4 2A mutant were overexpressed in PNT1A cells and purified using Ni-NTA column followed by immunoprecipitation using anti-FLAG M2 affinity gel. In vitro kinase assay was performed using a commercially available recombinant AKT1. AKT phosphorylation was detected by Western blots. MAPK4 2A carries KVAVAA in place of the KVAVKK ATP binding motif. Coomassie blue staining reveals that the purified FLAG/His-tagged MAPK4 (with a calculated molecular weight of 69.6 kDa) contains one major band around 70 kDa. F/H: 2× FLAG and 10× His tag. ( B ) MAPK4 and the MAPK4 2A and MAPK4 5A mutants were overexpressed in HCT116 PDK1 -KO cells. AKT phosphorylation was detected by Western blots. MAPK4 5A carries AAAAAA in place of the KVAVKK ATP binding motif. Coimmunoprecipitation (co-IP) assays reveal ( C ) ectopically overexpressed AKT1 binding to ectopically overexpressed MAPK4 in HEK293T cells, and ( D ) endogenous AKT1 binding to endogenous MAPK4 in VCaP cells. ( E ) Pull-down assay on the binding between purified GST-MAPK4 protein and purified AKT1 as well as endogenous AKT in the HEK293T, H1299, and HepG2 cell lysates. GST was used as control. GST-MAPK4 and GST were overexpressed in E . coli and purified using the glutathione sepharose beads. Coomassie blue staining confirms major bands of around 90 kDa and 26 kDa in the purified GST-MAPK4 and GST, respectively. ( F ) BiFC assay for AKT association with MAPK4 in cytoplasm. Hela cells were cotransfected with YN-MAPK4 and YC-AKT1 or YC control vectors. Forty-eight hours later, the cells were fixed, counterstained with DAPI, and imaged for YFP fluorescence to indicate MAPK4-AKT1 interaction. Original magnification: ×400. ( G ) Three different MK5 siRNAs were transfected into control and MAPK4-overexpressing HEK293T cells. Western blots were used to confirm MK5 knockdown and compare AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.

    Techniques Used: Mutagenesis, Purification, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Western Blot, Binding Assay, Staining, Molecular Weight, Co-Immunoprecipitation Assay, Pull Down Assay, Bimolecular Fluorescence Complementation Assay, Fluorescence, Transfection

    The molecular basis for MAPK4 binding to AKT, part 2. ( A ) The FLAG-tagged MAPK4 and MAPK4 E/D5A (5A-FLAG) mutant were transduced into HEK293T cells with or without cotransfection of AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. The MAPK4 E/D5A (5A-FLAG) mutant carries AAAKAA in place of the potential CD motif EEDKDE. ( B ) FLAG-tagged WT MAPK4 and the MAPK4 E250A , MAPK4 E251A , MAPK4 D252A , MAPK4 D254A , MAPK4 E255A mutants were transfected into HEK293T cells together with AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( C ) FLAG-tagged WT MAPK4 (WT) and the MAPK4 D254A , MAPK4 E255A mutants were transfected into the HCT116 MAPK4 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( D ). ( E ) Alignment of MAPK4 protein sequence around the CD motif across species. ( F ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transduced into HCT116 cells together with FLAG-tagged MAPK4. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( G ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transfected into the HCT116 PDK1 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( H ) The domain architecture of human AKT1 and the sequence alignment of human AKT1, AKT2, and AKT3 proteins. ( I ) Alignment of AKT1 protein sequence around the D-motif across species.
    Figure Legend Snippet: The molecular basis for MAPK4 binding to AKT, part 2. ( A ) The FLAG-tagged MAPK4 and MAPK4 E/D5A (5A-FLAG) mutant were transduced into HEK293T cells with or without cotransfection of AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. The MAPK4 E/D5A (5A-FLAG) mutant carries AAAKAA in place of the potential CD motif EEDKDE. ( B ) FLAG-tagged WT MAPK4 and the MAPK4 E250A , MAPK4 E251A , MAPK4 D252A , MAPK4 D254A , MAPK4 E255A mutants were transfected into HEK293T cells together with AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( C ) FLAG-tagged WT MAPK4 (WT) and the MAPK4 D254A , MAPK4 E255A mutants were transfected into the HCT116 MAPK4 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( D ). ( E ) Alignment of MAPK4 protein sequence around the CD motif across species. ( F ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transduced into HCT116 cells together with FLAG-tagged MAPK4. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( G ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transfected into the HCT116 PDK1 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( H ) The domain architecture of human AKT1 and the sequence alignment of human AKT1, AKT2, and AKT3 proteins. ( I ) Alignment of AKT1 protein sequence around the D-motif across species.

    Techniques Used: Binding Assay, Mutagenesis, Cotransfection, Immunoprecipitation, Western Blot, Transfection, Sequencing

    6) Product Images from "Human Apolipoprotein A-I Is Associated with Dengue Virus and Enhances Virus Infection through SR-BI"

    Article Title: Human Apolipoprotein A-I Is Associated with Dengue Virus and Enhances Virus Infection through SR-BI

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070390

    Co-precipitation of DV with ApoA-I. (A) Vero cells were infected with DV at a MOI of 1 and culture medium were changed to DMEM with 10% human serum HS (HSM) at 2 dpi. The mock-infected cells by DMEM was used as a control and also subjected to the same medium change. Culture supernatants were harvested at 7 dpi and purified by sucrose cushion ultracentrifugation (UC). The virus pellets were resuspended in serum-free DMEM. Presence of ApoA-I was analyzed by Western blotting using anti-ApoA-I antibody. (B) Human serum was added into DV/SFM to a final concentration of 10% and the mixture was incubated at 4° for 1 hour, followed by sucrose cushion ultracentrifugation. The pellets were analyzed by Western blotting using anti-ApoA-I and anti-E antibodies respectively. (C) Co-immunoprecipitation of ApoA-I with DV. AD-293 cells were transfected with a plasmid expressing FLAG-tagged ApoA-I (pApoAI-FLAG) and cultured in serum-free DMEM. At 3 dpt, secreted ApoA-I in the culture supernatant was purified with anti-FLAG M2 Affinity Gel. The resulting ApoAI-FLAG/M2 beads were washed twice with 1×TBS and incubated with DV/SFM at 4°C for over night. The co-immunoprecipitates were eluted and detected by Western blotting with anti-E and anti-FLAG antibodies. As a control, co-immunoprecipitation was also performed using the supernatant from cells transfected with empty vector p3×FLAG-CMV-14 (pFLAG). M, pre-stained protein marker.
    Figure Legend Snippet: Co-precipitation of DV with ApoA-I. (A) Vero cells were infected with DV at a MOI of 1 and culture medium were changed to DMEM with 10% human serum HS (HSM) at 2 dpi. The mock-infected cells by DMEM was used as a control and also subjected to the same medium change. Culture supernatants were harvested at 7 dpi and purified by sucrose cushion ultracentrifugation (UC). The virus pellets were resuspended in serum-free DMEM. Presence of ApoA-I was analyzed by Western blotting using anti-ApoA-I antibody. (B) Human serum was added into DV/SFM to a final concentration of 10% and the mixture was incubated at 4° for 1 hour, followed by sucrose cushion ultracentrifugation. The pellets were analyzed by Western blotting using anti-ApoA-I and anti-E antibodies respectively. (C) Co-immunoprecipitation of ApoA-I with DV. AD-293 cells were transfected with a plasmid expressing FLAG-tagged ApoA-I (pApoAI-FLAG) and cultured in serum-free DMEM. At 3 dpt, secreted ApoA-I in the culture supernatant was purified with anti-FLAG M2 Affinity Gel. The resulting ApoAI-FLAG/M2 beads were washed twice with 1×TBS and incubated with DV/SFM at 4°C for over night. The co-immunoprecipitates were eluted and detected by Western blotting with anti-E and anti-FLAG antibodies. As a control, co-immunoprecipitation was also performed using the supernatant from cells transfected with empty vector p3×FLAG-CMV-14 (pFLAG). M, pre-stained protein marker.

    Techniques Used: Infection, Purification, Western Blot, Concentration Assay, Incubation, Immunoprecipitation, Transfection, Plasmid Preparation, Expressing, Cell Culture, Staining, Marker

    7) Product Images from "MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling"

    Article Title: MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI97712

    The molecular basis for MAPK4 binding to AKT, part 1. ( A ) The domain architecture of human MAPK4. MAPK4 contains an N-terminal kinase domain (aa20 to aa312), a conserved C34 (aa313 to aa462) motif shared between MAPK4 (Erk4) and MAPK6 (Erk3), and a C-terminal tail. Asterisk indicates phosphorylation site. ( B ) HEK293T cells were transfected with FLAG-tagged AKT1 and HA-tagged WT or C-terminally truncated MAPK4 including N462, N312, and N185. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-HA antibody. The kinase domain of MAPK4 binds to AKT1 and the aa186–aa312 fragment is essential for this interaction. ( C ) His-tagged AKT1 along with FLAG-tagged MAPK4 and MAPK4 S186A mutant were reconstructed into the DLD1 AKT1/2 -KO cells that lack all 3 isoforms of AKT. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots for phosphorylation of AKT and total AKT. WCL: whole-cell lysate. ( D ) HA-tagged AKT2 and AKT3 were reconstructed into the DLD1 AKT1/2 -KO cells along FLAG-tagged MAPK4 or MAPK4 S186A mutant. Western blots using Phospho-AKT T308 antibody were used to detect the phosphorylation of AKT2 at T309 and AKT3 at T305, respectively. ( E ) FLAG-tagged MAPK4 and MAPK4 S186A mutant were transduced into the H157 MAPK4 -KO cells. Western blots were used to determine AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.
    Figure Legend Snippet: The molecular basis for MAPK4 binding to AKT, part 1. ( A ) The domain architecture of human MAPK4. MAPK4 contains an N-terminal kinase domain (aa20 to aa312), a conserved C34 (aa313 to aa462) motif shared between MAPK4 (Erk4) and MAPK6 (Erk3), and a C-terminal tail. Asterisk indicates phosphorylation site. ( B ) HEK293T cells were transfected with FLAG-tagged AKT1 and HA-tagged WT or C-terminally truncated MAPK4 including N462, N312, and N185. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-HA antibody. The kinase domain of MAPK4 binds to AKT1 and the aa186–aa312 fragment is essential for this interaction. ( C ) His-tagged AKT1 along with FLAG-tagged MAPK4 and MAPK4 S186A mutant were reconstructed into the DLD1 AKT1/2 -KO cells that lack all 3 isoforms of AKT. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots for phosphorylation of AKT and total AKT. WCL: whole-cell lysate. ( D ) HA-tagged AKT2 and AKT3 were reconstructed into the DLD1 AKT1/2 -KO cells along FLAG-tagged MAPK4 or MAPK4 S186A mutant. Western blots using Phospho-AKT T308 antibody were used to detect the phosphorylation of AKT2 at T309 and AKT3 at T305, respectively. ( E ) FLAG-tagged MAPK4 and MAPK4 S186A mutant were transduced into the H157 MAPK4 -KO cells. Western blots were used to determine AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.

    Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Mutagenesis

    MAPK4 interacts with AKT and is a novel T308 kinase. ( A ) FLAG/His-tagged MAPK4 and the MAPK4 2A mutant were overexpressed in PNT1A cells and purified using Ni-NTA column followed by immunoprecipitation using anti-FLAG M2 affinity gel. In vitro kinase assay was performed using a commercially available recombinant AKT1. AKT phosphorylation was detected by Western blots. MAPK4 2A carries KVAVAA in place of the KVAVKK ATP binding motif. Coomassie blue staining reveals that the purified FLAG/His-tagged MAPK4 (with a calculated molecular weight of 69.6 kDa) contains one major band around 70 kDa. F/H: 2× FLAG and 10× His tag. ( B ) MAPK4 and the MAPK4 2A and MAPK4 5A mutants were overexpressed in HCT116 PDK1 -KO cells. AKT phosphorylation was detected by Western blots. MAPK4 5A carries AAAAAA in place of the KVAVKK ATP binding motif. Coimmunoprecipitation (co-IP) assays reveal ( C ) ectopically overexpressed AKT1 binding to ectopically overexpressed MAPK4 in HEK293T cells, and ( D ) endogenous AKT1 binding to endogenous MAPK4 in VCaP cells. ( E ) Pull-down assay on the binding between purified GST-MAPK4 protein and purified AKT1 as well as endogenous AKT in the HEK293T, H1299, and HepG2 cell lysates. GST was used as control. GST-MAPK4 and GST were overexpressed in E . coli and purified using the glutathione sepharose beads. Coomassie blue staining confirms major bands of around 90 kDa and 26 kDa in the purified GST-MAPK4 and GST, respectively. ( F ) BiFC assay for AKT association with MAPK4 in cytoplasm. Hela cells were cotransfected with YN-MAPK4 and YC-AKT1 or YC control vectors. Forty-eight hours later, the cells were fixed, counterstained with DAPI, and imaged for YFP fluorescence to indicate MAPK4-AKT1 interaction. Original magnification: ×400. ( G ) Three different MK5 siRNAs were transfected into control and MAPK4-overexpressing HEK293T cells. Western blots were used to confirm MK5 knockdown and compare AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.
    Figure Legend Snippet: MAPK4 interacts with AKT and is a novel T308 kinase. ( A ) FLAG/His-tagged MAPK4 and the MAPK4 2A mutant were overexpressed in PNT1A cells and purified using Ni-NTA column followed by immunoprecipitation using anti-FLAG M2 affinity gel. In vitro kinase assay was performed using a commercially available recombinant AKT1. AKT phosphorylation was detected by Western blots. MAPK4 2A carries KVAVAA in place of the KVAVKK ATP binding motif. Coomassie blue staining reveals that the purified FLAG/His-tagged MAPK4 (with a calculated molecular weight of 69.6 kDa) contains one major band around 70 kDa. F/H: 2× FLAG and 10× His tag. ( B ) MAPK4 and the MAPK4 2A and MAPK4 5A mutants were overexpressed in HCT116 PDK1 -KO cells. AKT phosphorylation was detected by Western blots. MAPK4 5A carries AAAAAA in place of the KVAVKK ATP binding motif. Coimmunoprecipitation (co-IP) assays reveal ( C ) ectopically overexpressed AKT1 binding to ectopically overexpressed MAPK4 in HEK293T cells, and ( D ) endogenous AKT1 binding to endogenous MAPK4 in VCaP cells. ( E ) Pull-down assay on the binding between purified GST-MAPK4 protein and purified AKT1 as well as endogenous AKT in the HEK293T, H1299, and HepG2 cell lysates. GST was used as control. GST-MAPK4 and GST were overexpressed in E . coli and purified using the glutathione sepharose beads. Coomassie blue staining confirms major bands of around 90 kDa and 26 kDa in the purified GST-MAPK4 and GST, respectively. ( F ) BiFC assay for AKT association with MAPK4 in cytoplasm. Hela cells were cotransfected with YN-MAPK4 and YC-AKT1 or YC control vectors. Forty-eight hours later, the cells were fixed, counterstained with DAPI, and imaged for YFP fluorescence to indicate MAPK4-AKT1 interaction. Original magnification: ×400. ( G ) Three different MK5 siRNAs were transfected into control and MAPK4-overexpressing HEK293T cells. Western blots were used to confirm MK5 knockdown and compare AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.

    Techniques Used: Mutagenesis, Purification, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Western Blot, Binding Assay, Staining, Molecular Weight, Co-Immunoprecipitation Assay, Pull Down Assay, Bimolecular Fluorescence Complementation Assay, Fluorescence, Transfection

    The molecular basis for MAPK4 binding to AKT, part 2. ( A ) The FLAG-tagged MAPK4 and MAPK4 E/D5A (5A-FLAG) mutant were transduced into HEK293T cells with or without cotransfection of AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. The MAPK4 E/D5A (5A-FLAG) mutant carries AAAKAA in place of the potential CD motif EEDKDE. ( B ) FLAG-tagged WT MAPK4 and the MAPK4 E250A , MAPK4 E251A , MAPK4 D252A , MAPK4 D254A , MAPK4 E255A mutants were transfected into HEK293T cells together with AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( C ) FLAG-tagged WT MAPK4 (WT) and the MAPK4 D254A , MAPK4 E255A mutants were transfected into the HCT116 MAPK4 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( D ). ( E ) Alignment of MAPK4 protein sequence around the CD motif across species. ( F ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transduced into HCT116 cells together with FLAG-tagged MAPK4. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( G ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transfected into the HCT116 PDK1 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( H ) The domain architecture of human AKT1 and the sequence alignment of human AKT1, AKT2, and AKT3 proteins. ( I ) Alignment of AKT1 protein sequence around the D-motif across species.
    Figure Legend Snippet: The molecular basis for MAPK4 binding to AKT, part 2. ( A ) The FLAG-tagged MAPK4 and MAPK4 E/D5A (5A-FLAG) mutant were transduced into HEK293T cells with or without cotransfection of AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. The MAPK4 E/D5A (5A-FLAG) mutant carries AAAKAA in place of the potential CD motif EEDKDE. ( B ) FLAG-tagged WT MAPK4 and the MAPK4 E250A , MAPK4 E251A , MAPK4 D252A , MAPK4 D254A , MAPK4 E255A mutants were transfected into HEK293T cells together with AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( C ) FLAG-tagged WT MAPK4 (WT) and the MAPK4 D254A , MAPK4 E255A mutants were transfected into the HCT116 MAPK4 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( D ). ( E ) Alignment of MAPK4 protein sequence around the CD motif across species. ( F ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transduced into HCT116 cells together with FLAG-tagged MAPK4. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( G ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transfected into the HCT116 PDK1 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( H ) The domain architecture of human AKT1 and the sequence alignment of human AKT1, AKT2, and AKT3 proteins. ( I ) Alignment of AKT1 protein sequence around the D-motif across species.

    Techniques Used: Binding Assay, Mutagenesis, Cotransfection, Immunoprecipitation, Western Blot, Transfection, Sequencing

    8) Product Images from "Ubiquitin mediates the physical and functional interaction between human DNA polymerases ? and ?"

    Article Title: Ubiquitin mediates the physical and functional interaction between human DNA polymerases ? and ?

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1277

    Interactions between a polι-Ub chimera and polη. ( A ) Cartoon of the polι-Ub chimera with the I44A substitution indicated. Yeast two-hybrid analysis of interactions between polι, polι-Ub, polι-Ub-I44A, and polι-F507S-P680A-Ub and wild-type polη, PCNA and ubiquitin (Ub). Saccharomyces cerevisiae strain AH109 was transformed separately with the GAL4-AD expression vectors pACT2 (control), pACT2-polι wild type (pAR116), pACT2-polι-Ub (pJRM127), polι-Ub_I44A (pJRM150) and polι-F507S/P680A-Ub (pJRM151) as indicated, in combination with each of the following GAL4-BD expression vectors: pGBKT7 and pGBKT7-polη_wild type, (pAVR65), pGBKT7-PCNA (pAVR18) and pGBKT7-Ub (pBP129) as indicated. Several colonies from each transformation were grown overnight at 30°C in selective medium, and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 30°C for 4 days. Images were taken after 2 days of growth ( 2 ) or 4 days of growth ( 4 ). ( B ) FLAG-pull-down assay demonstrating interactions between polι and polη (lane 2), and polι-Ub and polη (lane 4). Extracts from HEK293T cells transfected with plasmids encoding FLAG-tagged wild-type polι (pJRM46) or a polι-Ub fusion (pJRM140) and HA-tagged wild-type polη (pJRM56) were incubated overnight at 4°C with 20 µl of EZview Red ANTI-FLAG M2 Affinity Gel, washed three times and analyzed directly by SDS–PAGE and Western blot with respective antibodies. Lanes 1 and 3 represent 10% of corresponding extracts used for each pull-down reaction. ( C ) FLAG-pull-down assay demonstrating the strength of interactions between PCNA and polι (lane 2), or polι-Ub (lane 4). Extracts from HEK293T cells transfected with plasmids encoding FLAG-tagged wild-type polι (pJRM46) or a polι-Ub fusion (pJRM140) were incubated overnight at 4°C with 20 µl of EZview Red ANTI-FLAG M2 Affinity Gel, washed three times and analyzed directly by SDS–PAGE and Western blot with respective antibodies. Lanes 1 and 3 represent 10% of corresponding extracts used for each pull-down reaction. ( D ) MRC5 human cells were transfected with plasmids encoding eCFP-polι wild type (peCFP-C1-polι) and eCFP-polι-Ub (pJRM128). Twenty hours after post-transfection, the cells were irradiated with UV (7 J/m 2 ). After 6 h, cells were fixed and the presence of foci examined. The histogram represents the mean and standard deviation calculated after counting 200 cells from three independent experiments with each construct.
    Figure Legend Snippet: Interactions between a polι-Ub chimera and polη. ( A ) Cartoon of the polι-Ub chimera with the I44A substitution indicated. Yeast two-hybrid analysis of interactions between polι, polι-Ub, polι-Ub-I44A, and polι-F507S-P680A-Ub and wild-type polη, PCNA and ubiquitin (Ub). Saccharomyces cerevisiae strain AH109 was transformed separately with the GAL4-AD expression vectors pACT2 (control), pACT2-polι wild type (pAR116), pACT2-polι-Ub (pJRM127), polι-Ub_I44A (pJRM150) and polι-F507S/P680A-Ub (pJRM151) as indicated, in combination with each of the following GAL4-BD expression vectors: pGBKT7 and pGBKT7-polη_wild type, (pAVR65), pGBKT7-PCNA (pAVR18) and pGBKT7-Ub (pBP129) as indicated. Several colonies from each transformation were grown overnight at 30°C in selective medium, and a sample was spotted on to a DOBA-Trp-Leu-His-Ade plate and incubated at 30°C for 4 days. Images were taken after 2 days of growth ( 2 ) or 4 days of growth ( 4 ). ( B ) FLAG-pull-down assay demonstrating interactions between polι and polη (lane 2), and polι-Ub and polη (lane 4). Extracts from HEK293T cells transfected with plasmids encoding FLAG-tagged wild-type polι (pJRM46) or a polι-Ub fusion (pJRM140) and HA-tagged wild-type polη (pJRM56) were incubated overnight at 4°C with 20 µl of EZview Red ANTI-FLAG M2 Affinity Gel, washed three times and analyzed directly by SDS–PAGE and Western blot with respective antibodies. Lanes 1 and 3 represent 10% of corresponding extracts used for each pull-down reaction. ( C ) FLAG-pull-down assay demonstrating the strength of interactions between PCNA and polι (lane 2), or polι-Ub (lane 4). Extracts from HEK293T cells transfected with plasmids encoding FLAG-tagged wild-type polι (pJRM46) or a polι-Ub fusion (pJRM140) were incubated overnight at 4°C with 20 µl of EZview Red ANTI-FLAG M2 Affinity Gel, washed three times and analyzed directly by SDS–PAGE and Western blot with respective antibodies. Lanes 1 and 3 represent 10% of corresponding extracts used for each pull-down reaction. ( D ) MRC5 human cells were transfected with plasmids encoding eCFP-polι wild type (peCFP-C1-polι) and eCFP-polι-Ub (pJRM128). Twenty hours after post-transfection, the cells were irradiated with UV (7 J/m 2 ). After 6 h, cells were fixed and the presence of foci examined. The histogram represents the mean and standard deviation calculated after counting 200 cells from three independent experiments with each construct.

    Techniques Used: Transformation Assay, Expressing, Incubation, Pull Down Assay, Transfection, SDS Page, Western Blot, Irradiation, Standard Deviation, Construct

    9) Product Images from "The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation"

    Article Title: The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05776-3

    Cys-replacement stabilizes FAT10 and its conjugates in vivo. a HEK293 cells expressing WT or Cys-free FLAG-tagged FAT10 were treated for 0.5, 1, or 2.5 h with 50 µg/mL CHX to monitor degradation rates. In addition, cells were treated with 10 µM proteasome inhibitor MG132 for 3.5 h, as indicated. Cells were harvested, lysed, and subjected to immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. Proteins were separated on 4–12% NUPAGE gradient gels and subjected to western blot analysis using a directly peroxidase-labeled, monoclonal FLAG-reactive antibody (clone M2). β-actin was used as loading control. One representative experiment out of four independent experiments with similar outcomes is shown. b ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated WT or Cys-free FLAG-FAT10 normalized to the amount of β-actin in the lysate. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Shown are the values of four independent experiments with similar outcomes as means ± s.e.m. c Molecular dynamics (MD) simulations of the N- (left) and C-domain (right) of human FAT10. Top panels: three of the most prominent structures obtained by root mean square deviation clustering from each MD simulation in cartoon representation (WT: white, Cys-free: blue). Bottom panels: Residue-wise root mean square fluctuation (RMSF) values. Arrows in the graph show position of cysteine mutations. N- and C-terminal tails are flexible. Moreover, flexibility is decreased in the N-domain in Cys-free mutants while the overall structure of both domains is preserved
    Figure Legend Snippet: Cys-replacement stabilizes FAT10 and its conjugates in vivo. a HEK293 cells expressing WT or Cys-free FLAG-tagged FAT10 were treated for 0.5, 1, or 2.5 h with 50 µg/mL CHX to monitor degradation rates. In addition, cells were treated with 10 µM proteasome inhibitor MG132 for 3.5 h, as indicated. Cells were harvested, lysed, and subjected to immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. Proteins were separated on 4–12% NUPAGE gradient gels and subjected to western blot analysis using a directly peroxidase-labeled, monoclonal FLAG-reactive antibody (clone M2). β-actin was used as loading control. One representative experiment out of four independent experiments with similar outcomes is shown. b ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated WT or Cys-free FLAG-FAT10 normalized to the amount of β-actin in the lysate. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Shown are the values of four independent experiments with similar outcomes as means ± s.e.m. c Molecular dynamics (MD) simulations of the N- (left) and C-domain (right) of human FAT10. Top panels: three of the most prominent structures obtained by root mean square deviation clustering from each MD simulation in cartoon representation (WT: white, Cys-free: blue). Bottom panels: Residue-wise root mean square fluctuation (RMSF) values. Arrows in the graph show position of cysteine mutations. N- and C-terminal tails are flexible. Moreover, flexibility is decreased in the N-domain in Cys-free mutants while the overall structure of both domains is preserved

    Techniques Used: In Vivo, Expressing, Immunoprecipitation, Western Blot, Labeling

    The FAT10 UBDs are loosely folded and degraded less efficiently when replaced by Ub. a Melting temperatures are plotted in a bar diagram for WT ΔN FAT10, the C0 ΔN FAT10 mutant, and ubiquitin and were obtained from the respective maxima of the first derivative curves of three independent differential scanning fluorimetry measurements where the temperature was varied in 0.036 °C steps from 20 to 95 °C. The data represent mean ± standard deviations (s.d.). The individual data points are shown in Supplementary Fig. 7a . b Schematic presentation of WT FLAG-FAT10 and the two FAT10-ubiquitin hybrids (FLAG-FAT10-Ub and FLAG-Ub-FAT10), where either the N- or the C-domain of FAT10 was exchanged with lysine-less Ub (K0) to prevent Ub chain formation. To suppress cleavage of Ub(K0) from the FAT10 C-domain in Ub-FAT10, Ub(K0) was expressed without the di-glycine motif. c Western blots showing bulk conjugates of WT FAT10, Ub-FAT10 or FAT10-Ub proteins, and USE1 auto-FAT10ylation. d Degradation of monomeric WT FAT10, Ub-FAT10, and FAT10-Ub was monitored by transient transfection of HEK293 cells with the indicated expression constructs. Cells were treated for 2.5 or 5 h with 50 µg/mL CHX to inhibit de novo protein synthesis. Where indicated, cells were additionally treated with 10 µM proteasome inhibitor MG132 for 6 h. c , d HEK293 cells were harvested and lysed 24 h after transfection. Cleared protein lysates were used for immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. The upper panels show the bulk conjugates in the immunoprecipitation, lower panels show the expression of the proteins in the lysates. β-actin was used as loading control. One representative experiment out of three experiments with similar outcomes is shown. e ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated FLAG-FAT10 and FLAG-tagged FAT10-Ub chimeras in the cells. The ECL signals were quantified with Image Lab 4.1. software (BioRad) and normalized to signals of the loading control β-actin. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Shown are the values of three independent experiments with similar outcomes as means ± s.e.m.
    Figure Legend Snippet: The FAT10 UBDs are loosely folded and degraded less efficiently when replaced by Ub. a Melting temperatures are plotted in a bar diagram for WT ΔN FAT10, the C0 ΔN FAT10 mutant, and ubiquitin and were obtained from the respective maxima of the first derivative curves of three independent differential scanning fluorimetry measurements where the temperature was varied in 0.036 °C steps from 20 to 95 °C. The data represent mean ± standard deviations (s.d.). The individual data points are shown in Supplementary Fig. 7a . b Schematic presentation of WT FLAG-FAT10 and the two FAT10-ubiquitin hybrids (FLAG-FAT10-Ub and FLAG-Ub-FAT10), where either the N- or the C-domain of FAT10 was exchanged with lysine-less Ub (K0) to prevent Ub chain formation. To suppress cleavage of Ub(K0) from the FAT10 C-domain in Ub-FAT10, Ub(K0) was expressed without the di-glycine motif. c Western blots showing bulk conjugates of WT FAT10, Ub-FAT10 or FAT10-Ub proteins, and USE1 auto-FAT10ylation. d Degradation of monomeric WT FAT10, Ub-FAT10, and FAT10-Ub was monitored by transient transfection of HEK293 cells with the indicated expression constructs. Cells were treated for 2.5 or 5 h with 50 µg/mL CHX to inhibit de novo protein synthesis. Where indicated, cells were additionally treated with 10 µM proteasome inhibitor MG132 for 6 h. c , d HEK293 cells were harvested and lysed 24 h after transfection. Cleared protein lysates were used for immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. The upper panels show the bulk conjugates in the immunoprecipitation, lower panels show the expression of the proteins in the lysates. β-actin was used as loading control. One representative experiment out of three experiments with similar outcomes is shown. e ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated FLAG-FAT10 and FLAG-tagged FAT10-Ub chimeras in the cells. The ECL signals were quantified with Image Lab 4.1. software (BioRad) and normalized to signals of the loading control β-actin. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Shown are the values of three independent experiments with similar outcomes as means ± s.e.m.

    Techniques Used: Mutagenesis, Western Blot, Transfection, Expressing, Construct, Immunoprecipitation, Software

    The presence of the FAT10 linker is crucial for FAT10 conjugation. a Scheme showing the amino acid substitutions or deletion of the WT FAT10 linker sequence (KPSDE). The different linker mutants 5x Ala, 5x Pro, 5x Gly, and Δ-linker were generated by site-directed mutagenesis of the expression plasmid pcDNA3.1-His-3xFLAG-FAT10. The His-3xFLAG tag is referred to as FLAG in the text as well as in the figures. b HEK293 cells were transiently transfected with expression constructs for the indicated proteins to monitor proteome-wide and USE1-specific conjugation of WT as well as of mutant FAT10. Uncropped blots are shown in Supplementary Figure 8 . c The degradation rate of monomeric FAT10 in the lysate as well as of FAT10 conjugates was monitored in HEK293 cells expressing the different FLAG-tagged FAT10 linker variants. Cells were treated for 2.5 or 5 h with 50 µg/mL CHX to inhibit de novo protein synthesis. Where indicated, cells were additionally treated with 10 µM proteasome inhibitor MG132 for 6 h. b , c HEK293 cells were harvested and lysed 24 h after transfection. Cleared protein lysates were subjected to immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. Proteins were separated on 12.5% Laemmli gels and visualized by western blot analysis with a monoclonal FLAG-reactive antibody (clone M2) or a USE1-reactive polyclonal antibody, as indicated. β-actin was used as loading control. One experiment out of three experiments with similar outcomes is shown. d Quantification of the amount of monomeric and of conjugated FLAG-FAT10 linker variants in c . The enhanced chemiluminescence (ECL) signals were quantified with Image Lab 4.1. software (BioRad) and normalized to signals of the loading control β-actin. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Values are shown for three independent experiments with similar outcomes as means ± s.e.m.
    Figure Legend Snippet: The presence of the FAT10 linker is crucial for FAT10 conjugation. a Scheme showing the amino acid substitutions or deletion of the WT FAT10 linker sequence (KPSDE). The different linker mutants 5x Ala, 5x Pro, 5x Gly, and Δ-linker were generated by site-directed mutagenesis of the expression plasmid pcDNA3.1-His-3xFLAG-FAT10. The His-3xFLAG tag is referred to as FLAG in the text as well as in the figures. b HEK293 cells were transiently transfected with expression constructs for the indicated proteins to monitor proteome-wide and USE1-specific conjugation of WT as well as of mutant FAT10. Uncropped blots are shown in Supplementary Figure 8 . c The degradation rate of monomeric FAT10 in the lysate as well as of FAT10 conjugates was monitored in HEK293 cells expressing the different FLAG-tagged FAT10 linker variants. Cells were treated for 2.5 or 5 h with 50 µg/mL CHX to inhibit de novo protein synthesis. Where indicated, cells were additionally treated with 10 µM proteasome inhibitor MG132 for 6 h. b , c HEK293 cells were harvested and lysed 24 h after transfection. Cleared protein lysates were subjected to immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. Proteins were separated on 12.5% Laemmli gels and visualized by western blot analysis with a monoclonal FLAG-reactive antibody (clone M2) or a USE1-reactive polyclonal antibody, as indicated. β-actin was used as loading control. One experiment out of three experiments with similar outcomes is shown. d Quantification of the amount of monomeric and of conjugated FLAG-FAT10 linker variants in c . The enhanced chemiluminescence (ECL) signals were quantified with Image Lab 4.1. software (BioRad) and normalized to signals of the loading control β-actin. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Values are shown for three independent experiments with similar outcomes as means ± s.e.m.

    Techniques Used: Conjugation Assay, Sequencing, Generated, Mutagenesis, Expressing, Plasmid Preparation, Transfection, Construct, Immunoprecipitation, Western Blot, Software

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    Immunoprecipitation:

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    Article Snippet: .. The mixture of protein extracts was immunoprecipitated with anti-Myc-conjugated agarose beads (Sigma, F-2426) and co-immunoprecipitated GST-HY5 proteins were detected with anti-GST antibody (Abcam, ab19256). .. In vivo Sumoylation Assay To determine the sumoylation status of COP1, proteins were extracted in a buffer composed of 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM DTT, 20 mM NEM, 1% TritonX-100, and 1× complete protease inhibitor mixture (Roche, 04693159001).

    Article Title: A Conserved Cys-loop Receptor Aspartate Residue in the M3-M4 Cytoplasmic Loop Is Required for GABAA Receptor Assembly * Receptor Assembly * S⃞
    Article Snippet: .. Immunoprecipitation —Protein complexes containing FLAG-tagged GABAA receptor subunits were immunoprecipitated using EZview Red anti-FLAG M2 beads (Sigma) at 4 °C overnight. .. After three washes with extracting RIPA buffer, protein complexes were eluted with 100 μg/ml FLAG peptide (Sigma).

    Article Title: The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation
    Article Snippet: .. Cleared lysates were subjected to anti-FLAG immunoprecipitation using EZview Red Anti-FLAG-M2 Affinity Gel (Sigma) for at least two hours at 4 °C or incubated over night with a FAT10-reactive monoclonal antibody (clone 4F1 ) bound to protein A sepharose. ..

    Incubation:

    Article Title: MARINOBUFAGENIN INTERFERES WITH THE FUNCTION OF THE MINERALOCORTICOID RECEPTOR
    Article Snippet: .. The resulting lysate was incubated with rotation with 20 μl EZview™ Red ANTI-FLAG® M2 Affinity Gel (Sigma) or 3 μg normal mouse IgG (Santa Cruz) for 2 hours at 4°C, prior to the addition of 25 μl of a 50% slurry of prewashed protein G agarose beads and an additional 2 h incubation with rotation. .. The immunocomplex was washed 4 times with lyses buffer at 4°C and subsequently heated to 95°C for 10 min in 25 μl of 1x Laemmli buffer and resolved by a NuPAGE Novex 3-8% Tris-Acetate gel (Invitrogen).

    Article Title: The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation
    Article Snippet: .. Cleared lysates were subjected to anti-FLAG immunoprecipitation using EZview Red Anti-FLAG-M2 Affinity Gel (Sigma) for at least two hours at 4 °C or incubated over night with a FAT10-reactive monoclonal antibody (clone 4F1 ) bound to protein A sepharose. ..

    other:

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    Article Snippet: Mouse monoclonal EZview™ red anti-FLAG M2 affinity gel was from Sigma–Aldrich (catalogue number F2426).

    Construct:

    Article Title: Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase
    Article Snippet: .. For the Bcl-xL -synthase interaction study, the human ORF constructs for alpha, beta, b, c, delta, d, epsilon, gamma and OSCP ATP-synthase subunits, tagged with Myc and DDK (Flag), from Origene Technologies (Rockville, MD), were expressed in 293T cells and purified, using the EZview^™ Red ANTI-FLAG^® M2 Affinity Gel (Sigma, USA), according to the manufacturer’s protocol. .. The expression was verified and the binding of Bcl-xL to individual subunits was assessed by immunoblot analysis, using the mouse anti-Myc and Rabbit anti-Bcl-xL antibodies (Cell signaling Technology), respectively.

    Purification:

    Article Title: Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase
    Article Snippet: .. For the Bcl-xL -synthase interaction study, the human ORF constructs for alpha, beta, b, c, delta, d, epsilon, gamma and OSCP ATP-synthase subunits, tagged with Myc and DDK (Flag), from Origene Technologies (Rockville, MD), were expressed in 293T cells and purified, using the EZview^™ Red ANTI-FLAG^® M2 Affinity Gel (Sigma, USA), according to the manufacturer’s protocol. .. The expression was verified and the binding of Bcl-xL to individual subunits was assessed by immunoblot analysis, using the mouse anti-Myc and Rabbit anti-Bcl-xL antibodies (Cell signaling Technology), respectively.

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    • ezview red anti flag m2 affinity gel  (Millipore)
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    Millipore ezview red anti flag m2 affinity gel
    MAPK6 phosphorylates AKT at S473 independent of mTORC2. ( A ) Coomassie blue staining of the purified MAPK6 protein from HEK293T cells. MW: molecular weight. IgG-H: heavy chain of IgG. IgG-L: light chain of IgG. ( B ) Western blots on the products from the in vitro kinase assays assessing MAPK6 phosphorylation of AKT. MAPK6 proteins were overexpressed and purified using the <t>EZview</t> Red anti-FLAG M2 affinity gel from the HEK293T cells transiently transfected with the pRK5-MAPK6-HF plasmid (left panel) and the engineered PC3 cells overexpressing MAPK6-FLAG (right panel). HF: 10 x His and 2 x FLAG tag. ( C ) In vitro kinase assays assessing AKT phosphorylation by the MAPK6-f (MAPK6-FLAG) proteins purified from the engineered Rictor +/+ and Rictor −/− MEF cells with 0.5 µg/ml Dox-induced expression of MAPK6-f or control (iCtrl). Shown are Western blots. ( D ) Western blots on the H1299 cells transfected with siRNA targeting MAPK6 (siMAPK6 #A4 and #A6) or control (siLuc) for 72 h followed by treatments using 1 µM PP242 or DMSO for 18h. ( E ) Proliferation assays on the H1299, PC3, and SUM159 cells with siRNA mediated knockdown of MAPK6 (siMAPK6 #A4 and #A6) or control (siLuc), treated with PP242 or DMSO. 48h after siRNA transfection, the cells were plated into 24-well plate and treated with PP242 (0.5 μM for H1299 and 1 μM for PC3 and SUM159 cells). Data are representative of at least 3 independent experiments.
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    MAPK6 phosphorylates AKT at S473 independent of mTORC2. ( A ) Coomassie blue staining of the purified MAPK6 protein from HEK293T cells. MW: molecular weight. IgG-H: heavy chain of IgG. IgG-L: light chain of IgG. ( B ) Western blots on the products from the in vitro kinase assays assessing MAPK6 phosphorylation of AKT. MAPK6 proteins were overexpressed and purified using the EZview Red anti-FLAG M2 affinity gel from the HEK293T cells transiently transfected with the pRK5-MAPK6-HF plasmid (left panel) and the engineered PC3 cells overexpressing MAPK6-FLAG (right panel). HF: 10 x His and 2 x FLAG tag. ( C ) In vitro kinase assays assessing AKT phosphorylation by the MAPK6-f (MAPK6-FLAG) proteins purified from the engineered Rictor +/+ and Rictor −/− MEF cells with 0.5 µg/ml Dox-induced expression of MAPK6-f or control (iCtrl). Shown are Western blots. ( D ) Western blots on the H1299 cells transfected with siRNA targeting MAPK6 (siMAPK6 #A4 and #A6) or control (siLuc) for 72 h followed by treatments using 1 µM PP242 or DMSO for 18h. ( E ) Proliferation assays on the H1299, PC3, and SUM159 cells with siRNA mediated knockdown of MAPK6 (siMAPK6 #A4 and #A6) or control (siLuc), treated with PP242 or DMSO. 48h after siRNA transfection, the cells were plated into 24-well plate and treated with PP242 (0.5 μM for H1299 and 1 μM for PC3 and SUM159 cells). Data are representative of at least 3 independent experiments.

    Journal: bioRxiv

    Article Title: Oncogenic Activation of AKT by MAPK6

    doi: 10.1101/2020.09.23.309518

    Figure Lengend Snippet: MAPK6 phosphorylates AKT at S473 independent of mTORC2. ( A ) Coomassie blue staining of the purified MAPK6 protein from HEK293T cells. MW: molecular weight. IgG-H: heavy chain of IgG. IgG-L: light chain of IgG. ( B ) Western blots on the products from the in vitro kinase assays assessing MAPK6 phosphorylation of AKT. MAPK6 proteins were overexpressed and purified using the EZview Red anti-FLAG M2 affinity gel from the HEK293T cells transiently transfected with the pRK5-MAPK6-HF plasmid (left panel) and the engineered PC3 cells overexpressing MAPK6-FLAG (right panel). HF: 10 x His and 2 x FLAG tag. ( C ) In vitro kinase assays assessing AKT phosphorylation by the MAPK6-f (MAPK6-FLAG) proteins purified from the engineered Rictor +/+ and Rictor −/− MEF cells with 0.5 µg/ml Dox-induced expression of MAPK6-f or control (iCtrl). Shown are Western blots. ( D ) Western blots on the H1299 cells transfected with siRNA targeting MAPK6 (siMAPK6 #A4 and #A6) or control (siLuc) for 72 h followed by treatments using 1 µM PP242 or DMSO for 18h. ( E ) Proliferation assays on the H1299, PC3, and SUM159 cells with siRNA mediated knockdown of MAPK6 (siMAPK6 #A4 and #A6) or control (siLuc), treated with PP242 or DMSO. 48h after siRNA transfection, the cells were plated into 24-well plate and treated with PP242 (0.5 μM for H1299 and 1 μM for PC3 and SUM159 cells). Data are representative of at least 3 independent experiments.

    Article Snippet: The supernatant was mixed with EZview Red ANTI-FLAG M2 Affinity Gel (Millipore Sigma) and rotated for 90 minutes at 4°C.

    Techniques: Staining, Purification, Molecular Weight, Western Blot, In Vitro, Transfection, Plasmid Preparation, FLAG-tag, Expressing

    The molecular basis for MAPK4 binding to AKT, part 1. ( A ) The domain architecture of human MAPK4. MAPK4 contains an N-terminal kinase domain (aa20 to aa312), a conserved C34 (aa313 to aa462) motif shared between MAPK4 (Erk4) and MAPK6 (Erk3), and a C-terminal tail. Asterisk indicates phosphorylation site. ( B ) HEK293T cells were transfected with FLAG-tagged AKT1 and HA-tagged WT or C-terminally truncated MAPK4 including N462, N312, and N185. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-HA antibody. The kinase domain of MAPK4 binds to AKT1 and the aa186–aa312 fragment is essential for this interaction. ( C ) His-tagged AKT1 along with FLAG-tagged MAPK4 and MAPK4 S186A mutant were reconstructed into the DLD1 AKT1/2 -KO cells that lack all 3 isoforms of AKT. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots for phosphorylation of AKT and total AKT. WCL: whole-cell lysate. ( D ) HA-tagged AKT2 and AKT3 were reconstructed into the DLD1 AKT1/2 -KO cells along FLAG-tagged MAPK4 or MAPK4 S186A mutant. Western blots using Phospho-AKT T308 antibody were used to detect the phosphorylation of AKT2 at T309 and AKT3 at T305, respectively. ( E ) FLAG-tagged MAPK4 and MAPK4 S186A mutant were transduced into the H157 MAPK4 -KO cells. Western blots were used to determine AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling

    doi: 10.1172/JCI97712

    Figure Lengend Snippet: The molecular basis for MAPK4 binding to AKT, part 1. ( A ) The domain architecture of human MAPK4. MAPK4 contains an N-terminal kinase domain (aa20 to aa312), a conserved C34 (aa313 to aa462) motif shared between MAPK4 (Erk4) and MAPK6 (Erk3), and a C-terminal tail. Asterisk indicates phosphorylation site. ( B ) HEK293T cells were transfected with FLAG-tagged AKT1 and HA-tagged WT or C-terminally truncated MAPK4 including N462, N312, and N185. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-HA antibody. The kinase domain of MAPK4 binds to AKT1 and the aa186–aa312 fragment is essential for this interaction. ( C ) His-tagged AKT1 along with FLAG-tagged MAPK4 and MAPK4 S186A mutant were reconstructed into the DLD1 AKT1/2 -KO cells that lack all 3 isoforms of AKT. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots for phosphorylation of AKT and total AKT. WCL: whole-cell lysate. ( D ) HA-tagged AKT2 and AKT3 were reconstructed into the DLD1 AKT1/2 -KO cells along FLAG-tagged MAPK4 or MAPK4 S186A mutant. Western blots using Phospho-AKT T308 antibody were used to detect the phosphorylation of AKT2 at T309 and AKT3 at T305, respectively. ( E ) FLAG-tagged MAPK4 and MAPK4 S186A mutant were transduced into the H157 MAPK4 -KO cells. Western blots were used to determine AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.

    Article Snippet: For immunoprecipitation using FLAG antibody, EZview Red ANTI-FLAG M2 Affinity Gel (MilliporeSigma) was washed with RIPA buffer (100 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 1% Triton X-100 with 100× protease inhibitors cocktail solution from GenDEPOT [P3100-005] and the phosphatase inhibitors NaF [10 mM] and Na3 VO4 [20 mM]) 3 times and then applied into the cell lysate.

    Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Mutagenesis

    MAPK4 interacts with AKT and is a novel T308 kinase. ( A ) FLAG/His-tagged MAPK4 and the MAPK4 2A mutant were overexpressed in PNT1A cells and purified using Ni-NTA column followed by immunoprecipitation using anti-FLAG M2 affinity gel. In vitro kinase assay was performed using a commercially available recombinant AKT1. AKT phosphorylation was detected by Western blots. MAPK4 2A carries KVAVAA in place of the KVAVKK ATP binding motif. Coomassie blue staining reveals that the purified FLAG/His-tagged MAPK4 (with a calculated molecular weight of 69.6 kDa) contains one major band around 70 kDa. F/H: 2× FLAG and 10× His tag. ( B ) MAPK4 and the MAPK4 2A and MAPK4 5A mutants were overexpressed in HCT116 PDK1 -KO cells. AKT phosphorylation was detected by Western blots. MAPK4 5A carries AAAAAA in place of the KVAVKK ATP binding motif. Coimmunoprecipitation (co-IP) assays reveal ( C ) ectopically overexpressed AKT1 binding to ectopically overexpressed MAPK4 in HEK293T cells, and ( D ) endogenous AKT1 binding to endogenous MAPK4 in VCaP cells. ( E ) Pull-down assay on the binding between purified GST-MAPK4 protein and purified AKT1 as well as endogenous AKT in the HEK293T, H1299, and HepG2 cell lysates. GST was used as control. GST-MAPK4 and GST were overexpressed in E . coli and purified using the glutathione sepharose beads. Coomassie blue staining confirms major bands of around 90 kDa and 26 kDa in the purified GST-MAPK4 and GST, respectively. ( F ) BiFC assay for AKT association with MAPK4 in cytoplasm. Hela cells were cotransfected with YN-MAPK4 and YC-AKT1 or YC control vectors. Forty-eight hours later, the cells were fixed, counterstained with DAPI, and imaged for YFP fluorescence to indicate MAPK4-AKT1 interaction. Original magnification: ×400. ( G ) Three different MK5 siRNAs were transfected into control and MAPK4-overexpressing HEK293T cells. Western blots were used to confirm MK5 knockdown and compare AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.

    Journal: The Journal of Clinical Investigation

    Article Title: MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling

    doi: 10.1172/JCI97712

    Figure Lengend Snippet: MAPK4 interacts with AKT and is a novel T308 kinase. ( A ) FLAG/His-tagged MAPK4 and the MAPK4 2A mutant were overexpressed in PNT1A cells and purified using Ni-NTA column followed by immunoprecipitation using anti-FLAG M2 affinity gel. In vitro kinase assay was performed using a commercially available recombinant AKT1. AKT phosphorylation was detected by Western blots. MAPK4 2A carries KVAVAA in place of the KVAVKK ATP binding motif. Coomassie blue staining reveals that the purified FLAG/His-tagged MAPK4 (with a calculated molecular weight of 69.6 kDa) contains one major band around 70 kDa. F/H: 2× FLAG and 10× His tag. ( B ) MAPK4 and the MAPK4 2A and MAPK4 5A mutants were overexpressed in HCT116 PDK1 -KO cells. AKT phosphorylation was detected by Western blots. MAPK4 5A carries AAAAAA in place of the KVAVKK ATP binding motif. Coimmunoprecipitation (co-IP) assays reveal ( C ) ectopically overexpressed AKT1 binding to ectopically overexpressed MAPK4 in HEK293T cells, and ( D ) endogenous AKT1 binding to endogenous MAPK4 in VCaP cells. ( E ) Pull-down assay on the binding between purified GST-MAPK4 protein and purified AKT1 as well as endogenous AKT in the HEK293T, H1299, and HepG2 cell lysates. GST was used as control. GST-MAPK4 and GST were overexpressed in E . coli and purified using the glutathione sepharose beads. Coomassie blue staining confirms major bands of around 90 kDa and 26 kDa in the purified GST-MAPK4 and GST, respectively. ( F ) BiFC assay for AKT association with MAPK4 in cytoplasm. Hela cells were cotransfected with YN-MAPK4 and YC-AKT1 or YC control vectors. Forty-eight hours later, the cells were fixed, counterstained with DAPI, and imaged for YFP fluorescence to indicate MAPK4-AKT1 interaction. Original magnification: ×400. ( G ) Three different MK5 siRNAs were transfected into control and MAPK4-overexpressing HEK293T cells. Western blots were used to confirm MK5 knockdown and compare AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.

    Article Snippet: For immunoprecipitation using FLAG antibody, EZview Red ANTI-FLAG M2 Affinity Gel (MilliporeSigma) was washed with RIPA buffer (100 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 1% Triton X-100 with 100× protease inhibitors cocktail solution from GenDEPOT [P3100-005] and the phosphatase inhibitors NaF [10 mM] and Na3 VO4 [20 mM]) 3 times and then applied into the cell lysate.

    Techniques: Mutagenesis, Purification, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Western Blot, Binding Assay, Staining, Molecular Weight, Co-Immunoprecipitation Assay, Pull Down Assay, Bimolecular Fluorescence Complementation Assay, Fluorescence, Transfection

    The molecular basis for MAPK4 binding to AKT, part 2. ( A ) The FLAG-tagged MAPK4 and MAPK4 E/D5A (5A-FLAG) mutant were transduced into HEK293T cells with or without cotransfection of AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. The MAPK4 E/D5A (5A-FLAG) mutant carries AAAKAA in place of the potential CD motif EEDKDE. ( B ) FLAG-tagged WT MAPK4 and the MAPK4 E250A , MAPK4 E251A , MAPK4 D252A , MAPK4 D254A , MAPK4 E255A mutants were transfected into HEK293T cells together with AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( C ) FLAG-tagged WT MAPK4 (WT) and the MAPK4 D254A , MAPK4 E255A mutants were transfected into the HCT116 MAPK4 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( D ). ( E ) Alignment of MAPK4 protein sequence around the CD motif across species. ( F ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transduced into HCT116 cells together with FLAG-tagged MAPK4. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( G ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transfected into the HCT116 PDK1 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( H ) The domain architecture of human AKT1 and the sequence alignment of human AKT1, AKT2, and AKT3 proteins. ( I ) Alignment of AKT1 protein sequence around the D-motif across species.

    Journal: The Journal of Clinical Investigation

    Article Title: MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling

    doi: 10.1172/JCI97712

    Figure Lengend Snippet: The molecular basis for MAPK4 binding to AKT, part 2. ( A ) The FLAG-tagged MAPK4 and MAPK4 E/D5A (5A-FLAG) mutant were transduced into HEK293T cells with or without cotransfection of AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. The MAPK4 E/D5A (5A-FLAG) mutant carries AAAKAA in place of the potential CD motif EEDKDE. ( B ) FLAG-tagged WT MAPK4 and the MAPK4 E250A , MAPK4 E251A , MAPK4 D252A , MAPK4 D254A , MAPK4 E255A mutants were transfected into HEK293T cells together with AKT1. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( C ) FLAG-tagged WT MAPK4 (WT) and the MAPK4 D254A , MAPK4 E255A mutants were transfected into the HCT116 MAPK4 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( D ). ( E ) Alignment of MAPK4 protein sequence around the CD motif across species. ( F ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transduced into HCT116 cells together with FLAG-tagged MAPK4. Immunoprecipitation was performed using anti-FLAG M2 affinity gel followed by Western blots using anti-AKT antibody. ( G ) WT AKT1 and the AKT1 K385A , AKT1 K386A , and AKT1 K389A mutants were transfected into the HCT116 PDK1 -KO cells. Phosphorylation of AKT was detected with Western blots. Data are representative of at least 3 independent experiments. ( H ) The domain architecture of human AKT1 and the sequence alignment of human AKT1, AKT2, and AKT3 proteins. ( I ) Alignment of AKT1 protein sequence around the D-motif across species.

    Article Snippet: For immunoprecipitation using FLAG antibody, EZview Red ANTI-FLAG M2 Affinity Gel (MilliporeSigma) was washed with RIPA buffer (100 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0, 1% Triton X-100 with 100× protease inhibitors cocktail solution from GenDEPOT [P3100-005] and the phosphatase inhibitors NaF [10 mM] and Na3 VO4 [20 mM]) 3 times and then applied into the cell lysate.

    Techniques: Binding Assay, Mutagenesis, Cotransfection, Immunoprecipitation, Western Blot, Transfection, Sequencing