Structured Review

GE Healthcare gst vector control
<t>Drp1</t> binds to the Arp2/3 complex in a p-Drp1S600–dependent manner. ( A ) Cultured podocytes with empty vector, FLAG-tagged WT Drp1 (WT), FLAG-tagged Drp1S600A (SA), and FLAG-tagged Drp1S600D (SD) were used. Cells were also transiently transfected with GFP-Arp3. Top panels show anti-FLAG IP material and immunoblotting against GFP and FLAG. Bottom panels show the WCLs. ( B ) Bacterially expressed <t>GST,</t> GST-Drp1S600A, GST-S600D, and GST-S600 WT proteins on GST-sepharose were mixed with purified Arp2/3 complex in the GST-pulldown assay. Coomassie staining of SDS-PAGE gel is shown on the right. Top 2 left blots show recovered materials that were immunoblotted to detect the binding of Arp2 and Arp3 to Drp1. Third blot on the left shows immunoblotting with p-Drp1S600 (p-Drp1), illustrating good mimicry of the phosphorylation epitope by the aspartate mutation. The bottom blot on the left shows immunoblotting for the total level of input Drp1 from the GST-pulldown assay. ( C ) Top panels show control podocyte cells cultured under HG conditions after being treated with vehicle, nontargeting (NT) shRNA, shRNA-1 against Arp3, or shRNA-2 against Arp3. Cells were fixed and stained for mitochondria with an antibody against Tomm20. Mitochondria are shown in grayscale. Bottom panels show podocytes expressing Drp1S600D cultured under NG conditions after being treated as indicated above and stained for mitochondria as before. Mitochondria are shown in grayscale. Scale bars: 25 μm. ( D ) Quantification of mitochondrial length and AR for native podocytes for the images shown in C (top). ( E ) Quantification of mitochondrial length and AR for podocytes stably expressing Drp1S600D for the images shown in C (bottom). Representative images are from a sampling of 3 to 5 separate cell cultures. **** P
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1) Product Images from "Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice"

Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI127277

Drp1 binds to the Arp2/3 complex in a p-Drp1S600–dependent manner. ( A ) Cultured podocytes with empty vector, FLAG-tagged WT Drp1 (WT), FLAG-tagged Drp1S600A (SA), and FLAG-tagged Drp1S600D (SD) were used. Cells were also transiently transfected with GFP-Arp3. Top panels show anti-FLAG IP material and immunoblotting against GFP and FLAG. Bottom panels show the WCLs. ( B ) Bacterially expressed GST, GST-Drp1S600A, GST-S600D, and GST-S600 WT proteins on GST-sepharose were mixed with purified Arp2/3 complex in the GST-pulldown assay. Coomassie staining of SDS-PAGE gel is shown on the right. Top 2 left blots show recovered materials that were immunoblotted to detect the binding of Arp2 and Arp3 to Drp1. Third blot on the left shows immunoblotting with p-Drp1S600 (p-Drp1), illustrating good mimicry of the phosphorylation epitope by the aspartate mutation. The bottom blot on the left shows immunoblotting for the total level of input Drp1 from the GST-pulldown assay. ( C ) Top panels show control podocyte cells cultured under HG conditions after being treated with vehicle, nontargeting (NT) shRNA, shRNA-1 against Arp3, or shRNA-2 against Arp3. Cells were fixed and stained for mitochondria with an antibody against Tomm20. Mitochondria are shown in grayscale. Bottom panels show podocytes expressing Drp1S600D cultured under NG conditions after being treated as indicated above and stained for mitochondria as before. Mitochondria are shown in grayscale. Scale bars: 25 μm. ( D ) Quantification of mitochondrial length and AR for native podocytes for the images shown in C (top). ( E ) Quantification of mitochondrial length and AR for podocytes stably expressing Drp1S600D for the images shown in C (bottom). Representative images are from a sampling of 3 to 5 separate cell cultures. **** P
Figure Legend Snippet: Drp1 binds to the Arp2/3 complex in a p-Drp1S600–dependent manner. ( A ) Cultured podocytes with empty vector, FLAG-tagged WT Drp1 (WT), FLAG-tagged Drp1S600A (SA), and FLAG-tagged Drp1S600D (SD) were used. Cells were also transiently transfected with GFP-Arp3. Top panels show anti-FLAG IP material and immunoblotting against GFP and FLAG. Bottom panels show the WCLs. ( B ) Bacterially expressed GST, GST-Drp1S600A, GST-S600D, and GST-S600 WT proteins on GST-sepharose were mixed with purified Arp2/3 complex in the GST-pulldown assay. Coomassie staining of SDS-PAGE gel is shown on the right. Top 2 left blots show recovered materials that were immunoblotted to detect the binding of Arp2 and Arp3 to Drp1. Third blot on the left shows immunoblotting with p-Drp1S600 (p-Drp1), illustrating good mimicry of the phosphorylation epitope by the aspartate mutation. The bottom blot on the left shows immunoblotting for the total level of input Drp1 from the GST-pulldown assay. ( C ) Top panels show control podocyte cells cultured under HG conditions after being treated with vehicle, nontargeting (NT) shRNA, shRNA-1 against Arp3, or shRNA-2 against Arp3. Cells were fixed and stained for mitochondria with an antibody against Tomm20. Mitochondria are shown in grayscale. Bottom panels show podocytes expressing Drp1S600D cultured under NG conditions after being treated as indicated above and stained for mitochondria as before. Mitochondria are shown in grayscale. Scale bars: 25 μm. ( D ) Quantification of mitochondrial length and AR for native podocytes for the images shown in C (top). ( E ) Quantification of mitochondrial length and AR for podocytes stably expressing Drp1S600D for the images shown in C (bottom). Representative images are from a sampling of 3 to 5 separate cell cultures. **** P

Techniques Used: Cell Culture, Plasmid Preparation, Transfection, Purification, GST Pulldown Assay, Staining, SDS Page, Binding Assay, Mutagenesis, shRNA, Expressing, Stable Transfection, Sampling

Related Articles

GST Pulldown Assay:

Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice
Article Snippet: Paragraph title: GST-pulldown assay. ... Briefly, GST-tagged Drp1 isoforms (WT, S600A, and S600D) or GST vector control (pGEX-2T, GE Healthcare) were transformed into BL21 (DE3) (New England BioLabs) and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (MilliporeSigma, catalog I6758) at 25°C overnight (18 h).

Purification:

Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice
Article Snippet: Briefly, GST-tagged Drp1 isoforms (WT, S600A, and S600D) or GST vector control (pGEX-2T, GE Healthcare) were transformed into BL21 (DE3) (New England BioLabs) and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (MilliporeSigma, catalog I6758) at 25°C overnight (18 h). .. Immobilized GST proteins were purified according to the manufacturer’s protocol.

Incubation:

Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice
Article Snippet: Briefly, GST-tagged Drp1 isoforms (WT, S600A, and S600D) or GST vector control (pGEX-2T, GE Healthcare) were transformed into BL21 (DE3) (New England BioLabs) and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (MilliporeSigma, catalog I6758) at 25°C overnight (18 h). .. GST proteins (2 μg) were incubated with 5 μg purified Arp2/3 complex (Cytoskeleton Inc., catalog RP01P-A) in 1 ml GST-binding buffer (20 mM Tris-HCl at pH 7.5, 25 mM KCl, 1 mM MgCl2 , 50 mM NaCl) at 4°C for 2 hours.

Polyacrylamide Gel Electrophoresis:

Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice
Article Snippet: Briefly, GST-tagged Drp1 isoforms (WT, S600A, and S600D) or GST vector control (pGEX-2T, GE Healthcare) were transformed into BL21 (DE3) (New England BioLabs) and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (MilliporeSigma, catalog I6758) at 25°C overnight (18 h). .. Beads were washed 5 times with binding buffer, boiled in SDS sample buffer, and subjected to 4% to 15% PAGE (Bio-Rad, 4561086).

Transformation Assay:

Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice
Article Snippet: .. Briefly, GST-tagged Drp1 isoforms (WT, S600A, and S600D) or GST vector control (pGEX-2T, GE Healthcare) were transformed into BL21 (DE3) (New England BioLabs) and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (MilliporeSigma, catalog I6758) at 25°C overnight (18 h). .. Immobilized GST proteins were purified according to the manufacturer’s protocol.

Binding Assay:

Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice
Article Snippet: Briefly, GST-tagged Drp1 isoforms (WT, S600A, and S600D) or GST vector control (pGEX-2T, GE Healthcare) were transformed into BL21 (DE3) (New England BioLabs) and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (MilliporeSigma, catalog I6758) at 25°C overnight (18 h). .. Beads were washed 5 times with binding buffer, boiled in SDS sample buffer, and subjected to 4% to 15% PAGE (Bio-Rad, 4561086).

Plasmid Preparation:

Article Title: Drp1S600 phosphorylation regulates mitochondrial fission and progression of nephropathy in diabetic mice
Article Snippet: .. Briefly, GST-tagged Drp1 isoforms (WT, S600A, and S600D) or GST vector control (pGEX-2T, GE Healthcare) were transformed into BL21 (DE3) (New England BioLabs) and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (MilliporeSigma, catalog I6758) at 25°C overnight (18 h). .. Immobilized GST proteins were purified according to the manufacturer’s protocol.

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    GE Healthcare gst znf32 fusion protein full length znf32
    <t>ZNF32</t> directly binds the SOX2 promoter to suppress SOX2 transcription A. The DNA binding sites were identified by CASTing. The DNA sequences of the 22 clones are shown. B. Analyses of the sequences of the 22 clones revealed the ZNF32-DNA binding site as 5′- g(a/c/t)attt -32. C. The 2-kb region upstream of the human SOX2 promoter was from the NCBI web server. Blue characters mark the DNA binding site, 5′- gcattt -32, located at -1545. D. EMSA revealed that the SOX2 probe was preferentially bound by the ZNF32 protein compared with <t>GST,</t> and the binding capacity was weakened as competitive probe was added in vitro . The band shift revealed the protein-DNA binding capacity. E. EMSA showing that protein-probe binding corresponded to nuclear ZNF32 protein expression. Nuclear proteins were isolated from the BE(2)-C stable cell lines lv-NC (lane 1), lv-ZNF32 (lane 2), LV6-NC (lane 3), LV6-ZNF32 (lane 4). The band shift changed with the ZNF32 expression level. F. GFP-ChIP PCR results indicated that ZNF32 over-expression increased the protein-DNA binding ability, suggesting that ZNF32 specifically binds the SOX2 promoter sequence. IgG was used as a negative control.
    Gst Znf32 Fusion Protein Full Length Znf32, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    GE Healthcare gsh sepharose 4b
    Determination of the TGF-β receptor-interacting domain in TMED10. A , illustration of TMED10 and its mutants. SP , signal peptide; TM , transmembrane. B , interaction of TMED10 or its mutants with ALK5. COS7 cells were transfected with the indicated plasmids and harvested for co-IP experiments. The interaction between ALK5 and either TMED10 or its mutants is shown in the top panel . Total expressions of ALK5ca/V5 and TMED10/FLAG (or its mutants) are shown in the middle and bottom panel , respectively. C , non-necessity of both the transmembrane and the intracellular domains of TMED10 to interact with ALK5. The COS7 cells were transfected with the indicated plasmids. Section I , after cell lysis, anti-V5 antibodies were used for immunoprecipitation. The immunoprecipitates were separated by SDS-PAGE followed by Western blotting analysis ( WB ) using anti-FLAG antibody to detect their interaction ( top panel ). Total expressions of ALK5ca/FLAG and TMED10/V5 (or its mutants) in cells are shown in the second and third panel , respectively. Section II , because TMED10Δ(V 186 -E 219 ) seemed to be a secretory protein, immunoprecipitation of TMED10Δ(V 186 -E 219 ) into the media in which the cells were cultured was performed to detect secretory TMED10Δ(V 186 -E 219 ) ( bottom panel ). D , interaction of TMED10 or its mutants with TβRII. Co-IP was performed as described in B above. The interaction between TβRII and TMED10 or it mutants is shown in the top panel . Total expressions of TβRII and TMED10 or its mutants were detected using anti-HA12CA5 ( middle panel ) and anti-FLAG antibodies ( bottom panel ), respectively. E , schematic presentation of GST-TMED10 fusion proteins. The region from Gly 81 to Val 130 in TMED10 was split into two pieces to make GST fusion proteins. F , requirement of the region consisting of 30 amino acids from Gly 81 to Glu 110 within TMED10 to interact with ALK5. A GST pulldown assay was performed using the GST fusion proteins described in E above. ALK5 ectopically expressed in COS7 cells was mixed with GST fusion protein. After loading the protein(s) bound to <t>GSH-Sepharose</t> 4B in SDS-PAGE, Western blotting analysis was performed using anti-ALK5 (V-22) antibody ( top panel ). The cell lysates from COS7 cells transfected with pcDNA3 were used as the negative control. As loading controls, GST and GST fusion proteins were visible with Ponceau S staining ( bottom panel ). G , illustration of GST-TMED10 fusion proteins. The region from Gly 81 to Glu 110 in TMED10 was divided into two pieces to make GST fusion proteins. H , determination of the ALK5-binding region in TMED10. A GST pulldown assay was carried out as described in F above. The top and bottom panels show the interaction of ALK5 with GST fusion proteins and the loading controls with Ponceau S staining. I , requirement of a 20-amino acid-long region in TMED10 to associate with TβRII. A GST pulldown assay was carried out as described in F above. The upper and lower panels show the interaction of TβRII with GST fusion proteins and the loading controls with Ponceau S staining.
    Gsh Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 98/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare gst recombinant proteins
    IbbHLH4 interacts with <t>IbbHLH3</t> to repress the transcriptional activation function. (A) Bimolecular fluorescence complementation (BiFC) assay to detect the interactions of IbbHLH3 and IbbHLH4. The NLS-mCherry construct was co-expressed into protoplasts as a nuclei marker. The empty vectors of nYFP and cYFP interacted with IbbHLH3 and IbbHLH4, respectively, as negative controls. GUS protein was used as negative interaction protein of BiFC. (B) BiFC assay to detect the protein interaction between IbbHLH3 and IbbHLH4. (C) Co-IP assay to verify the interaction of IbbHLH3 with IbbHLH4 in plant. The <t>35S::GST-IbbHLH4</t> (GST-IbbHLH4) was co-expressed with 35S::GFP-IbbHLH3 (GFP-IbbHLH3) or 35S::GFP (GFP, control) in tobacco leaves. The total protein from the tobacco leaves were immunoprecipitated with the GST resin, and were further analyzed by western blot using anti-GST antibody and anti-GFP antibody. 35S::GST (GST) was used as negative control for IbbHLH3 interaction. (D) Transactivation assay using the GAL4DB-based expression system. The activation of Gal4BS by BD-IbbHLH3 binding was suppressed by IbbHLH4, while IbbHLH4 could not affect the activity of Gal4BS. RLUC driven by the 35S promoter was used as internal control to normalize the transfection efficiency. Error bars mean standard deviations (SDs) (n = 10). Different letters mean statistically significance determined by one-way ANOVA (P
    Gst Recombinant Proteins, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare gst fused full length ebp50
    RSK1 binds to <t>EBP50</t> through a PDZ binding motif and PDZ domain interaction which is subjective to signaling regulation A. The C-terminal sequences of all four RSK isoforms contain the putative PDZ binding motif, (S/T)XL, for the tandem repeated PDZ domains of EBP50. B. Cell lysates from HEK293 cells transiently expressing Myc-tagged full-length RSK1 (RSK1) or a PDZ binding motif deletion mutant (RSK1deSTTL) were subjected for in vitro pull-down assay with <t>GST-fused</t> full-length (FL), first PDZ domain (Z1) or second PDZ domain (Z2) EBP50 construct. The pulled down materials were separated by SDS-PAGE and analyzed by Western blotting using anti-Myc antibody. The coomassie blue-stained gel showed the expression and comparable loading of each recombinant protein used in the assay. Molecular marker ( Mr ): kDa. C. Endogenous EBP50 was immunoprecipitated (IP) from HeLa cells using two independent anti-EBP50 antibodies (K31 and O40), and co-immunoprecipitated RSK1 was detected by Western blotting using anti-RSK1 antibody. K31 and O40 were our in-house anti-EBP50 antibodies. They were generated by immunizing rabbits with a synthetic peptide (MARERAHQKRSSKRC for K31 and LQKLGVPVREELLRAQC for O40) and further purified using affinity chromatography. Immunoprecipitation using the antigenic peptide reabsorbed O40 immune serum was included as a negative control. D. EBP50-knocked down HeLa cells that stably re-expressed EGFP-EBP50 or empty vector were serum starved for 20 h, and stimulated with EGF (50 ng/ml) for the indicated intervals. Then, RSK1 was immunoprecipitated from the harvested lysates followed by Western blotting using anti-GFP antibody.
    Gst Fused Full Length Ebp50, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ZNF32 directly binds the SOX2 promoter to suppress SOX2 transcription A. The DNA binding sites were identified by CASTing. The DNA sequences of the 22 clones are shown. B. Analyses of the sequences of the 22 clones revealed the ZNF32-DNA binding site as 5′- g(a/c/t)attt -32. C. The 2-kb region upstream of the human SOX2 promoter was from the NCBI web server. Blue characters mark the DNA binding site, 5′- gcattt -32, located at -1545. D. EMSA revealed that the SOX2 probe was preferentially bound by the ZNF32 protein compared with GST, and the binding capacity was weakened as competitive probe was added in vitro . The band shift revealed the protein-DNA binding capacity. E. EMSA showing that protein-probe binding corresponded to nuclear ZNF32 protein expression. Nuclear proteins were isolated from the BE(2)-C stable cell lines lv-NC (lane 1), lv-ZNF32 (lane 2), LV6-NC (lane 3), LV6-ZNF32 (lane 4). The band shift changed with the ZNF32 expression level. F. GFP-ChIP PCR results indicated that ZNF32 over-expression increased the protein-DNA binding ability, suggesting that ZNF32 specifically binds the SOX2 promoter sequence. IgG was used as a negative control.

    Journal: Oncotarget

    Article Title: Loss of ZNF32 augments the regeneration of nervous lateral line system through negative regulation of SOX2 transcription

    doi: 10.18632/oncotarget.11895

    Figure Lengend Snippet: ZNF32 directly binds the SOX2 promoter to suppress SOX2 transcription A. The DNA binding sites were identified by CASTing. The DNA sequences of the 22 clones are shown. B. Analyses of the sequences of the 22 clones revealed the ZNF32-DNA binding site as 5′- g(a/c/t)attt -32. C. The 2-kb region upstream of the human SOX2 promoter was from the NCBI web server. Blue characters mark the DNA binding site, 5′- gcattt -32, located at -1545. D. EMSA revealed that the SOX2 probe was preferentially bound by the ZNF32 protein compared with GST, and the binding capacity was weakened as competitive probe was added in vitro . The band shift revealed the protein-DNA binding capacity. E. EMSA showing that protein-probe binding corresponded to nuclear ZNF32 protein expression. Nuclear proteins were isolated from the BE(2)-C stable cell lines lv-NC (lane 1), lv-ZNF32 (lane 2), LV6-NC (lane 3), LV6-ZNF32 (lane 4). The band shift changed with the ZNF32 expression level. F. GFP-ChIP PCR results indicated that ZNF32 over-expression increased the protein-DNA binding ability, suggesting that ZNF32 specifically binds the SOX2 promoter sequence. IgG was used as a negative control.

    Article Snippet: Expression, purification and identification of the GST-ZNF32 fusion protein Full-length ZNF32 was cloned into the pGEX-5X-3 vector (GE Healthcare, United Kindom) and transformed into E.coli BL21(DE3) star competent cells.

    Techniques: Binding Assay, Clone Assay, In Vitro, Electrophoretic Mobility Shift Assay, Expressing, Isolation, Stable Transfection, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Over Expression, Sequencing, Negative Control

    Determination of the TGF-β receptor-interacting domain in TMED10. A , illustration of TMED10 and its mutants. SP , signal peptide; TM , transmembrane. B , interaction of TMED10 or its mutants with ALK5. COS7 cells were transfected with the indicated plasmids and harvested for co-IP experiments. The interaction between ALK5 and either TMED10 or its mutants is shown in the top panel . Total expressions of ALK5ca/V5 and TMED10/FLAG (or its mutants) are shown in the middle and bottom panel , respectively. C , non-necessity of both the transmembrane and the intracellular domains of TMED10 to interact with ALK5. The COS7 cells were transfected with the indicated plasmids. Section I , after cell lysis, anti-V5 antibodies were used for immunoprecipitation. The immunoprecipitates were separated by SDS-PAGE followed by Western blotting analysis ( WB ) using anti-FLAG antibody to detect their interaction ( top panel ). Total expressions of ALK5ca/FLAG and TMED10/V5 (or its mutants) in cells are shown in the second and third panel , respectively. Section II , because TMED10Δ(V 186 -E 219 ) seemed to be a secretory protein, immunoprecipitation of TMED10Δ(V 186 -E 219 ) into the media in which the cells were cultured was performed to detect secretory TMED10Δ(V 186 -E 219 ) ( bottom panel ). D , interaction of TMED10 or its mutants with TβRII. Co-IP was performed as described in B above. The interaction between TβRII and TMED10 or it mutants is shown in the top panel . Total expressions of TβRII and TMED10 or its mutants were detected using anti-HA12CA5 ( middle panel ) and anti-FLAG antibodies ( bottom panel ), respectively. E , schematic presentation of GST-TMED10 fusion proteins. The region from Gly 81 to Val 130 in TMED10 was split into two pieces to make GST fusion proteins. F , requirement of the region consisting of 30 amino acids from Gly 81 to Glu 110 within TMED10 to interact with ALK5. A GST pulldown assay was performed using the GST fusion proteins described in E above. ALK5 ectopically expressed in COS7 cells was mixed with GST fusion protein. After loading the protein(s) bound to GSH-Sepharose 4B in SDS-PAGE, Western blotting analysis was performed using anti-ALK5 (V-22) antibody ( top panel ). The cell lysates from COS7 cells transfected with pcDNA3 were used as the negative control. As loading controls, GST and GST fusion proteins were visible with Ponceau S staining ( bottom panel ). G , illustration of GST-TMED10 fusion proteins. The region from Gly 81 to Glu 110 in TMED10 was divided into two pieces to make GST fusion proteins. H , determination of the ALK5-binding region in TMED10. A GST pulldown assay was carried out as described in F above. The top and bottom panels show the interaction of ALK5 with GST fusion proteins and the loading controls with Ponceau S staining. I , requirement of a 20-amino acid-long region in TMED10 to associate with TβRII. A GST pulldown assay was carried out as described in F above. The upper and lower panels show the interaction of TβRII with GST fusion proteins and the loading controls with Ponceau S staining.

    Journal: The Journal of Biological Chemistry

    Article Title: TMED10 Protein Interferes with Transforming Growth Factor (TGF)-β Signaling by Disrupting TGF-β Receptor Complex Formation *

    doi: 10.1074/jbc.M116.769109

    Figure Lengend Snippet: Determination of the TGF-β receptor-interacting domain in TMED10. A , illustration of TMED10 and its mutants. SP , signal peptide; TM , transmembrane. B , interaction of TMED10 or its mutants with ALK5. COS7 cells were transfected with the indicated plasmids and harvested for co-IP experiments. The interaction between ALK5 and either TMED10 or its mutants is shown in the top panel . Total expressions of ALK5ca/V5 and TMED10/FLAG (or its mutants) are shown in the middle and bottom panel , respectively. C , non-necessity of both the transmembrane and the intracellular domains of TMED10 to interact with ALK5. The COS7 cells were transfected with the indicated plasmids. Section I , after cell lysis, anti-V5 antibodies were used for immunoprecipitation. The immunoprecipitates were separated by SDS-PAGE followed by Western blotting analysis ( WB ) using anti-FLAG antibody to detect their interaction ( top panel ). Total expressions of ALK5ca/FLAG and TMED10/V5 (or its mutants) in cells are shown in the second and third panel , respectively. Section II , because TMED10Δ(V 186 -E 219 ) seemed to be a secretory protein, immunoprecipitation of TMED10Δ(V 186 -E 219 ) into the media in which the cells were cultured was performed to detect secretory TMED10Δ(V 186 -E 219 ) ( bottom panel ). D , interaction of TMED10 or its mutants with TβRII. Co-IP was performed as described in B above. The interaction between TβRII and TMED10 or it mutants is shown in the top panel . Total expressions of TβRII and TMED10 or its mutants were detected using anti-HA12CA5 ( middle panel ) and anti-FLAG antibodies ( bottom panel ), respectively. E , schematic presentation of GST-TMED10 fusion proteins. The region from Gly 81 to Val 130 in TMED10 was split into two pieces to make GST fusion proteins. F , requirement of the region consisting of 30 amino acids from Gly 81 to Glu 110 within TMED10 to interact with ALK5. A GST pulldown assay was performed using the GST fusion proteins described in E above. ALK5 ectopically expressed in COS7 cells was mixed with GST fusion protein. After loading the protein(s) bound to GSH-Sepharose 4B in SDS-PAGE, Western blotting analysis was performed using anti-ALK5 (V-22) antibody ( top panel ). The cell lysates from COS7 cells transfected with pcDNA3 were used as the negative control. As loading controls, GST and GST fusion proteins were visible with Ponceau S staining ( bottom panel ). G , illustration of GST-TMED10 fusion proteins. The region from Gly 81 to Glu 110 in TMED10 was divided into two pieces to make GST fusion proteins. H , determination of the ALK5-binding region in TMED10. A GST pulldown assay was carried out as described in F above. The top and bottom panels show the interaction of ALK5 with GST fusion proteins and the loading controls with Ponceau S staining. I , requirement of a 20-amino acid-long region in TMED10 to associate with TβRII. A GST pulldown assay was carried out as described in F above. The upper and lower panels show the interaction of TβRII with GST fusion proteins and the loading controls with Ponceau S staining.

    Article Snippet: Cell lysates were prepared from COS7 cells transfected with ALK5, TβRII/FLAG, or an empty vector precleared with GST immobilized to GSH-Sepharose 4B (GE Healthcare) for 30 min at 4 °C.

    Techniques: Transfection, Co-Immunoprecipitation Assay, Lysis, Immunoprecipitation, SDS Page, Western Blot, Cell Culture, GST Pulldown Assay, Negative Control, Staining, Binding Assay

    Inhibitory action of TMED10 on TGF-β signaling. A , dose-dependent inhibition of TGF-β-driven reporter activity by TMED10. HepG2 cells were transfected with (SBE) 4 ), pCH110, and the indicated plasmids at two different doses. Twenty-four hours later, the cells were stimulated with 5 ng/ml TGF-β for 18 h. Significant differences from the control in the presence of TGF-β are indicated with asterisks. B , effect of TMED10Δ(I 32 -A 80 ) on the activity of TGF-β-driven transcriptional reporter. The experiments were performed as described in A . The combined total amount of TMED10 with TMED10Δ(I 32 -A 80 ) was the same in all columns. Significant differences from the control in the presence of TGF-β are indicated with asterisks. C , inhibitory ability of TMED10 on BMP signaling. The experiments were performed as described in A except for the addition of 25 ng/ml BMP-6. Significant differences from the control in the presence of TGF-β or BMP are indicated with asterisks. D , illustration of C-terminal deletion of TMED10. SP , signal peptide; TM , transmembrane E , dispensation of the C-terminal end of TMED10 for its inhibitory action. The experiments were performed as described in A . Significant differences from the control in the presence of TGF-β are indicated with asterisks. F , inhibition of Smad2 phosphorylation by TMED10 in HaCaT cells. HaCaT cells carrying TMED10/FLAG by the method of lentiviral gene transfer were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-phosphorylated Smad2 ( PS2 ) ( top panel ), anti-Smad2 ( second panel ), anti-FLAG ( third panel ), and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses ( WB ). The expression of phosphorylated Smad2 upon TGF-β stimulation was normalized using the intensity of the band corresponding to Smad2. Relative expression was calculated relative to the value for pLV-CMV-IRES-Puro-infected cells in the absence of TGF-β. G , overexpression of TMED10/FLAG by adenoviral transfer. NMuMG cells were infected with TMED10/FLAG-expressing adenovirus. After preparation of the cell lysates, anti-FLAG ( top panel ) or anti-β-actin antibody ( bottom panel ) was used. H , extension of E-cadherin expression in NMuMG cells expressing TMED10/FLAG upon TGF-β stimulation. TMED10 were introduced into NMuMG cells by an adenoviral gene transfer system as described in G . Forty hours after infection, the cells were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-E-cadherin ( top panel ) and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses. The expression of E-cadherin upon TGF-β stimulation was normalized using the intensity of the band corresponding to β-actin. Relative expression was calculated relative to the value for control cells in the absence of TGF-β. I , inhibition of N-cadherin expression by TMED10 in NMuMG cells. After gene transfer of TMED10/FLAG by adenovirus, the cells were cultured for 40 h. Subsequently, the cells were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-N-cadherin ( top panel ) and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses. The expression of N-cadherin upon TGF-β stimulation was normalized using the intensity of the band corresponding to β-actin. Relative expression was calculated relative to the value for control cells in the absence of TGF-β. J , expression of TMED10 mRNA upon TGF-β stimulation. HepG2 cells were stimulated with 5 ng/ml TGF-β at the different time points. After preparation of total RNA from the cells, PCR was carried out using specific primer sets. As the positive control, the TMEPAI gene, which is well known as a TGF-β target gene, was used. Before qPCR, the amplified PCR product using each primer set could be seen in the agarose gel as a single band. Significant differences from the cells without the treatment of TGF-β are indicated with asterisks. K , overexpression of TMED10/FLAG in A549 cells by adenoviral gene transfer. The experiment was performed as described in G. L , inhibition of TGF-β-induced cell migration by TMED10. After adenoviral infection as described in K , A549 cells were seeded on the upper membrane of the Boyden chamber. Subsequently, 5 ng/ml TGF-β was added to the lower chamber for 18 h. The cells were then stained with hematoxylin/eosin solution, and the number of transmigrated cells was counted. Probability values below 0.05, 0.01, and 0.001 were considered significant: *, p

    Journal: The Journal of Biological Chemistry

    Article Title: TMED10 Protein Interferes with Transforming Growth Factor (TGF)-β Signaling by Disrupting TGF-β Receptor Complex Formation *

    doi: 10.1074/jbc.M116.769109

    Figure Lengend Snippet: Inhibitory action of TMED10 on TGF-β signaling. A , dose-dependent inhibition of TGF-β-driven reporter activity by TMED10. HepG2 cells were transfected with (SBE) 4 ), pCH110, and the indicated plasmids at two different doses. Twenty-four hours later, the cells were stimulated with 5 ng/ml TGF-β for 18 h. Significant differences from the control in the presence of TGF-β are indicated with asterisks. B , effect of TMED10Δ(I 32 -A 80 ) on the activity of TGF-β-driven transcriptional reporter. The experiments were performed as described in A . The combined total amount of TMED10 with TMED10Δ(I 32 -A 80 ) was the same in all columns. Significant differences from the control in the presence of TGF-β are indicated with asterisks. C , inhibitory ability of TMED10 on BMP signaling. The experiments were performed as described in A except for the addition of 25 ng/ml BMP-6. Significant differences from the control in the presence of TGF-β or BMP are indicated with asterisks. D , illustration of C-terminal deletion of TMED10. SP , signal peptide; TM , transmembrane E , dispensation of the C-terminal end of TMED10 for its inhibitory action. The experiments were performed as described in A . Significant differences from the control in the presence of TGF-β are indicated with asterisks. F , inhibition of Smad2 phosphorylation by TMED10 in HaCaT cells. HaCaT cells carrying TMED10/FLAG by the method of lentiviral gene transfer were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-phosphorylated Smad2 ( PS2 ) ( top panel ), anti-Smad2 ( second panel ), anti-FLAG ( third panel ), and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses ( WB ). The expression of phosphorylated Smad2 upon TGF-β stimulation was normalized using the intensity of the band corresponding to Smad2. Relative expression was calculated relative to the value for pLV-CMV-IRES-Puro-infected cells in the absence of TGF-β. G , overexpression of TMED10/FLAG by adenoviral transfer. NMuMG cells were infected with TMED10/FLAG-expressing adenovirus. After preparation of the cell lysates, anti-FLAG ( top panel ) or anti-β-actin antibody ( bottom panel ) was used. H , extension of E-cadherin expression in NMuMG cells expressing TMED10/FLAG upon TGF-β stimulation. TMED10 were introduced into NMuMG cells by an adenoviral gene transfer system as described in G . Forty hours after infection, the cells were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-E-cadherin ( top panel ) and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses. The expression of E-cadherin upon TGF-β stimulation was normalized using the intensity of the band corresponding to β-actin. Relative expression was calculated relative to the value for control cells in the absence of TGF-β. I , inhibition of N-cadherin expression by TMED10 in NMuMG cells. After gene transfer of TMED10/FLAG by adenovirus, the cells were cultured for 40 h. Subsequently, the cells were stimulated with 0.5 ng/ml TGF-β for the indicated times. After preparation of the cell lysates, anti-N-cadherin ( top panel ) and anti-β-actin antibodies ( bottom panel ) were used for Western blotting analyses. The expression of N-cadherin upon TGF-β stimulation was normalized using the intensity of the band corresponding to β-actin. Relative expression was calculated relative to the value for control cells in the absence of TGF-β. J , expression of TMED10 mRNA upon TGF-β stimulation. HepG2 cells were stimulated with 5 ng/ml TGF-β at the different time points. After preparation of total RNA from the cells, PCR was carried out using specific primer sets. As the positive control, the TMEPAI gene, which is well known as a TGF-β target gene, was used. Before qPCR, the amplified PCR product using each primer set could be seen in the agarose gel as a single band. Significant differences from the cells without the treatment of TGF-β are indicated with asterisks. K , overexpression of TMED10/FLAG in A549 cells by adenoviral gene transfer. The experiment was performed as described in G. L , inhibition of TGF-β-induced cell migration by TMED10. After adenoviral infection as described in K , A549 cells were seeded on the upper membrane of the Boyden chamber. Subsequently, 5 ng/ml TGF-β was added to the lower chamber for 18 h. The cells were then stained with hematoxylin/eosin solution, and the number of transmigrated cells was counted. Probability values below 0.05, 0.01, and 0.001 were considered significant: *, p

    Article Snippet: Cell lysates were prepared from COS7 cells transfected with ALK5, TβRII/FLAG, or an empty vector precleared with GST immobilized to GSH-Sepharose 4B (GE Healthcare) for 30 min at 4 °C.

    Techniques: Inhibition, Activity Assay, Transfection, Western Blot, Expressing, Infection, Over Expression, Cell Culture, Polymerase Chain Reaction, Positive Control, Real-time Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Migration, Staining

    IbbHLH4 interacts with IbbHLH3 to repress the transcriptional activation function. (A) Bimolecular fluorescence complementation (BiFC) assay to detect the interactions of IbbHLH3 and IbbHLH4. The NLS-mCherry construct was co-expressed into protoplasts as a nuclei marker. The empty vectors of nYFP and cYFP interacted with IbbHLH3 and IbbHLH4, respectively, as negative controls. GUS protein was used as negative interaction protein of BiFC. (B) BiFC assay to detect the protein interaction between IbbHLH3 and IbbHLH4. (C) Co-IP assay to verify the interaction of IbbHLH3 with IbbHLH4 in plant. The 35S::GST-IbbHLH4 (GST-IbbHLH4) was co-expressed with 35S::GFP-IbbHLH3 (GFP-IbbHLH3) or 35S::GFP (GFP, control) in tobacco leaves. The total protein from the tobacco leaves were immunoprecipitated with the GST resin, and were further analyzed by western blot using anti-GST antibody and anti-GFP antibody. 35S::GST (GST) was used as negative control for IbbHLH3 interaction. (D) Transactivation assay using the GAL4DB-based expression system. The activation of Gal4BS by BD-IbbHLH3 binding was suppressed by IbbHLH4, while IbbHLH4 could not affect the activity of Gal4BS. RLUC driven by the 35S promoter was used as internal control to normalize the transfection efficiency. Error bars mean standard deviations (SDs) (n = 10). Different letters mean statistically significance determined by one-way ANOVA (P

    Journal: PLoS Genetics

    Article Title: The Sweet Potato NAC-Domain Transcription Factor IbNAC1 Is Dynamically Coordinated by the Activator IbbHLH3 and the Repressor IbbHLH4 to Reprogram the Defense Mechanism against Wounding

    doi: 10.1371/journal.pgen.1006397

    Figure Lengend Snippet: IbbHLH4 interacts with IbbHLH3 to repress the transcriptional activation function. (A) Bimolecular fluorescence complementation (BiFC) assay to detect the interactions of IbbHLH3 and IbbHLH4. The NLS-mCherry construct was co-expressed into protoplasts as a nuclei marker. The empty vectors of nYFP and cYFP interacted with IbbHLH3 and IbbHLH4, respectively, as negative controls. GUS protein was used as negative interaction protein of BiFC. (B) BiFC assay to detect the protein interaction between IbbHLH3 and IbbHLH4. (C) Co-IP assay to verify the interaction of IbbHLH3 with IbbHLH4 in plant. The 35S::GST-IbbHLH4 (GST-IbbHLH4) was co-expressed with 35S::GFP-IbbHLH3 (GFP-IbbHLH3) or 35S::GFP (GFP, control) in tobacco leaves. The total protein from the tobacco leaves were immunoprecipitated with the GST resin, and were further analyzed by western blot using anti-GST antibody and anti-GFP antibody. 35S::GST (GST) was used as negative control for IbbHLH3 interaction. (D) Transactivation assay using the GAL4DB-based expression system. The activation of Gal4BS by BD-IbbHLH3 binding was suppressed by IbbHLH4, while IbbHLH4 could not affect the activity of Gal4BS. RLUC driven by the 35S promoter was used as internal control to normalize the transfection efficiency. Error bars mean standard deviations (SDs) (n = 10). Different letters mean statistically significance determined by one-way ANOVA (P

    Article Snippet: Purification of GST-recombinant proteins The open reading flame of IbbHLH3 and IbbHLH4 was cloned behind the GST-tag in the pGEX4T-3 vector (GE Healthcare, Sweden).

    Techniques: Activation Assay, Bimolecular Fluorescence Complementation Assay, Construct, Marker, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Negative Control, Transactivation Assay, Expressing, Binding Assay, Activity Assay, Transfection

    IbbHLH3 and IbbHLH4 physically bind to the NWRE region. (A) Expression levels of bHLH transcription factors in sweet potato leaves upon wounding. The bHLH transcription factors that were selected from the transcriptome dataset of wounded leaves were analyzed by a wounding time-course treatment. IbNAC1 was shown as reference. Error bars indicate standard deviations (SDs) from four independent replicates (B) NWRE binding analysis. Yeast one-hybrid (Y1H) screening was used to analyze the binding ability of IbbHLHs with NWRE DNA cis -elements. NWRE fragments were cloned into the pHIS2.1 vector as bait. Then, the bait and pGADT7-IbbHLHs (AD-IbbHLHs) were co-transformed into yeast strain Y187. The growth of the yeast transformants is shown on both +His medium and -His medium with 100 mM 3-AT. The G-box-mutated NWRE was used as a control to verify the binding abilities of IbbHLH3 and IbbHLH4. (C) NWRE binding analysis of IbbHLH4 by Y1H screening. (D) NWRE binding analysis of IbbHLH3 by EMSA. GST-IbbHLH3 binds to 6FAM-NWRE in the presence of changing protein concentrations. The 50x unlabeled NWRE was included as a competitor (CP), and the GST protein was used as a negative control for NWRE binding. (E) NWRE binding analysis of IbbHLH4 by EMSA. GST-IbbHLH4 binds to 6FAM-NWRE depending on the IbbHLH4 protein concentration. The GST protein was used as a negative control for NWRE binding.

    Journal: PLoS Genetics

    Article Title: The Sweet Potato NAC-Domain Transcription Factor IbNAC1 Is Dynamically Coordinated by the Activator IbbHLH3 and the Repressor IbbHLH4 to Reprogram the Defense Mechanism against Wounding

    doi: 10.1371/journal.pgen.1006397

    Figure Lengend Snippet: IbbHLH3 and IbbHLH4 physically bind to the NWRE region. (A) Expression levels of bHLH transcription factors in sweet potato leaves upon wounding. The bHLH transcription factors that were selected from the transcriptome dataset of wounded leaves were analyzed by a wounding time-course treatment. IbNAC1 was shown as reference. Error bars indicate standard deviations (SDs) from four independent replicates (B) NWRE binding analysis. Yeast one-hybrid (Y1H) screening was used to analyze the binding ability of IbbHLHs with NWRE DNA cis -elements. NWRE fragments were cloned into the pHIS2.1 vector as bait. Then, the bait and pGADT7-IbbHLHs (AD-IbbHLHs) were co-transformed into yeast strain Y187. The growth of the yeast transformants is shown on both +His medium and -His medium with 100 mM 3-AT. The G-box-mutated NWRE was used as a control to verify the binding abilities of IbbHLH3 and IbbHLH4. (C) NWRE binding analysis of IbbHLH4 by Y1H screening. (D) NWRE binding analysis of IbbHLH3 by EMSA. GST-IbbHLH3 binds to 6FAM-NWRE in the presence of changing protein concentrations. The 50x unlabeled NWRE was included as a competitor (CP), and the GST protein was used as a negative control for NWRE binding. (E) NWRE binding analysis of IbbHLH4 by EMSA. GST-IbbHLH4 binds to 6FAM-NWRE depending on the IbbHLH4 protein concentration. The GST protein was used as a negative control for NWRE binding.

    Article Snippet: Purification of GST-recombinant proteins The open reading flame of IbbHLH3 and IbbHLH4 was cloned behind the GST-tag in the pGEX4T-3 vector (GE Healthcare, Sweden).

    Techniques: Expressing, Binding Assay, Clone Assay, Plasmid Preparation, Transformation Assay, Negative Control, Protein Concentration

    RSK1 binds to EBP50 through a PDZ binding motif and PDZ domain interaction which is subjective to signaling regulation A. The C-terminal sequences of all four RSK isoforms contain the putative PDZ binding motif, (S/T)XL, for the tandem repeated PDZ domains of EBP50. B. Cell lysates from HEK293 cells transiently expressing Myc-tagged full-length RSK1 (RSK1) or a PDZ binding motif deletion mutant (RSK1deSTTL) were subjected for in vitro pull-down assay with GST-fused full-length (FL), first PDZ domain (Z1) or second PDZ domain (Z2) EBP50 construct. The pulled down materials were separated by SDS-PAGE and analyzed by Western blotting using anti-Myc antibody. The coomassie blue-stained gel showed the expression and comparable loading of each recombinant protein used in the assay. Molecular marker ( Mr ): kDa. C. Endogenous EBP50 was immunoprecipitated (IP) from HeLa cells using two independent anti-EBP50 antibodies (K31 and O40), and co-immunoprecipitated RSK1 was detected by Western blotting using anti-RSK1 antibody. K31 and O40 were our in-house anti-EBP50 antibodies. They were generated by immunizing rabbits with a synthetic peptide (MARERAHQKRSSKRC for K31 and LQKLGVPVREELLRAQC for O40) and further purified using affinity chromatography. Immunoprecipitation using the antigenic peptide reabsorbed O40 immune serum was included as a negative control. D. EBP50-knocked down HeLa cells that stably re-expressed EGFP-EBP50 or empty vector were serum starved for 20 h, and stimulated with EGF (50 ng/ml) for the indicated intervals. Then, RSK1 was immunoprecipitated from the harvested lysates followed by Western blotting using anti-GFP antibody.

    Journal: Oncotarget

    Article Title: Ras-activated RSK1 phosphorylates EBP50 to regulate its nuclear localization and promote cell proliferation

    doi: 10.18632/oncotarget.7184

    Figure Lengend Snippet: RSK1 binds to EBP50 through a PDZ binding motif and PDZ domain interaction which is subjective to signaling regulation A. The C-terminal sequences of all four RSK isoforms contain the putative PDZ binding motif, (S/T)XL, for the tandem repeated PDZ domains of EBP50. B. Cell lysates from HEK293 cells transiently expressing Myc-tagged full-length RSK1 (RSK1) or a PDZ binding motif deletion mutant (RSK1deSTTL) were subjected for in vitro pull-down assay with GST-fused full-length (FL), first PDZ domain (Z1) or second PDZ domain (Z2) EBP50 construct. The pulled down materials were separated by SDS-PAGE and analyzed by Western blotting using anti-Myc antibody. The coomassie blue-stained gel showed the expression and comparable loading of each recombinant protein used in the assay. Molecular marker ( Mr ): kDa. C. Endogenous EBP50 was immunoprecipitated (IP) from HeLa cells using two independent anti-EBP50 antibodies (K31 and O40), and co-immunoprecipitated RSK1 was detected by Western blotting using anti-RSK1 antibody. K31 and O40 were our in-house anti-EBP50 antibodies. They were generated by immunizing rabbits with a synthetic peptide (MARERAHQKRSSKRC for K31 and LQKLGVPVREELLRAQC for O40) and further purified using affinity chromatography. Immunoprecipitation using the antigenic peptide reabsorbed O40 immune serum was included as a negative control. D. EBP50-knocked down HeLa cells that stably re-expressed EGFP-EBP50 or empty vector were serum starved for 20 h, and stimulated with EGF (50 ng/ml) for the indicated intervals. Then, RSK1 was immunoprecipitated from the harvested lysates followed by Western blotting using anti-GFP antibody.

    Article Snippet: GST pull-down assay The recombinant proteins of GST-fused full-length EBP50 and its individual PDZ domains were produced in Escherichia coli BL21 strain and conventionally purified on glutathione-Sepharose 4B beads (GE Healthcare) in PBS containing 4 mM 2-Mercaptoethanol (Sigma-Aldrich), 1% TritonX-100 and protease inhibitor cocktail.

    Techniques: Binding Assay, Expressing, Mutagenesis, In Vitro, Pull Down Assay, Construct, SDS Page, Western Blot, Staining, Recombinant, Marker, Immunoprecipitation, Generated, Purification, Affinity Chromatography, Negative Control, Stable Transfection, Plasmid Preparation

    RSK1 phosphorylates EBP50 at T156 within a consensus RXRXXpS/T motif A. Mammalian EBP50 conserves a consensus phosphorylation motif of RSK1 substrates, RXRXXpS/T. B. RSK1-knocked down HeLa cells (shRSK1) or mock-transduced cells (shVC) were grown in complete media (untreated), serum-starved, or re-stimulated with 10% (v/v) serum for 7 h (FBS). EBP50 phosphorylation was analyzed by immunoblotting using phospho-T156 antibody. Knock down efficiency and activation of RSK1 was analyzed using anti-RSK1 antibody and phospho-S380 antibody, respectively. Phosphorylation of histone H3 at S10 served as the mitotic marker. Molecular marker ( Mr ): kDa. C. HEK293 cells were transfected with HA-RasV12 or empty vector, serum starved, and stimulated with EGF before EBP50 phosphorylation was analyzed. D. HEK293 cells were transfected with HA-tagged RSK1 (RSK1), its kinase dead mutant (RSK1KD), or an empty vector, serum starved, and stimulated with EGF. The immunoprecipitated RSK1 was incubated with 5 μl purified GST-EBP50, and phosphorylation of EBP50 was analyzed by immunoblotting using an antibody recognizing the RXRXXpS/T motif. Molecular marker ( Mr ): kDa. E. HEK293 cells were transfected with HA-RSK1 or empty vector, serum starved, and stimulated with EGF. The immunoprecipitated RSK1 was incubated with synthetic peptide of EBP50 (T156NP). The samples were then processed for dot blot analysis using phospho-T156 antibody. Synthetic phosphopeptide of EBP50 (T156P) was included as a positive control. Ezrin or α-tubulin was examined as a loading control.

    Journal: Oncotarget

    Article Title: Ras-activated RSK1 phosphorylates EBP50 to regulate its nuclear localization and promote cell proliferation

    doi: 10.18632/oncotarget.7184

    Figure Lengend Snippet: RSK1 phosphorylates EBP50 at T156 within a consensus RXRXXpS/T motif A. Mammalian EBP50 conserves a consensus phosphorylation motif of RSK1 substrates, RXRXXpS/T. B. RSK1-knocked down HeLa cells (shRSK1) or mock-transduced cells (shVC) were grown in complete media (untreated), serum-starved, or re-stimulated with 10% (v/v) serum for 7 h (FBS). EBP50 phosphorylation was analyzed by immunoblotting using phospho-T156 antibody. Knock down efficiency and activation of RSK1 was analyzed using anti-RSK1 antibody and phospho-S380 antibody, respectively. Phosphorylation of histone H3 at S10 served as the mitotic marker. Molecular marker ( Mr ): kDa. C. HEK293 cells were transfected with HA-RasV12 or empty vector, serum starved, and stimulated with EGF before EBP50 phosphorylation was analyzed. D. HEK293 cells were transfected with HA-tagged RSK1 (RSK1), its kinase dead mutant (RSK1KD), or an empty vector, serum starved, and stimulated with EGF. The immunoprecipitated RSK1 was incubated with 5 μl purified GST-EBP50, and phosphorylation of EBP50 was analyzed by immunoblotting using an antibody recognizing the RXRXXpS/T motif. Molecular marker ( Mr ): kDa. E. HEK293 cells were transfected with HA-RSK1 or empty vector, serum starved, and stimulated with EGF. The immunoprecipitated RSK1 was incubated with synthetic peptide of EBP50 (T156NP). The samples were then processed for dot blot analysis using phospho-T156 antibody. Synthetic phosphopeptide of EBP50 (T156P) was included as a positive control. Ezrin or α-tubulin was examined as a loading control.

    Article Snippet: GST pull-down assay The recombinant proteins of GST-fused full-length EBP50 and its individual PDZ domains were produced in Escherichia coli BL21 strain and conventionally purified on glutathione-Sepharose 4B beads (GE Healthcare) in PBS containing 4 mM 2-Mercaptoethanol (Sigma-Aldrich), 1% TritonX-100 and protease inhibitor cocktail.

    Techniques: Activation Assay, Marker, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Incubation, Purification, Dot Blot, Positive Control