Structured Review

GE Healthcare glutathione sepharose 4b beads
Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to <t>glutathione-Sepharose</t> 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
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Images

1) Product Images from "Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking"

Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms15022929

Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
Figure Legend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

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

2) Product Images from "TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction"

Article Title: TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction

Journal: Autophagy

doi: 10.1080/15548627.2019.1580510

TP53INP2 forms a complex with LC3B and ATG7. (a) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-TP53INP2, GFP-TP53INP2[NLSΔ] or GFP-TP53INP2[LIRΔ] from HEK293 cells. TP53INP2 proteins were immunoprecipitated by anti-GFP. The coprecipitated ATG7, ATG3 or ATG12–ATG5 was detected by western blot using anti-ATG3, anti-ATG7 or anti-ATG5 respectively. (b) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ] or TP53INP2[LIRΔ]. GFP-tagged TP53INP2 mutants were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7, anti-ATG3 or anti-ATG5. (c) In vitro TP53INP2-ATG7 binding assay. Purified GST-TP53INP2 or GST-TP53INP2 W35,I38A was incubated with purified LC3B[G120] and ATG7. After affinity-isolating GST-TP53INP2 or GST-TP53INP2 W35,I38A with glutathione-sepharose 4B beads, the bound LC3B[G120] and ATG7 were analyzed by western blot. (d) HEK293T cells were cotransfected with Flag-LC3B, TP53INP2-MYC and HA-ATG7. The cells were lysed 48 h after transfection and Flag-LC3B was immunoprecipitated with anti-Flag. After incubation of the Flag-LC3B precipitates with Flag peptide, the eluate was used for immunoprecipitation with either anti-MYC or anti-HA. The immunoprecipitates were then analyzed by western blot by anti-Flag, anti-MYC and anti-HA respectively. (e) Coimmunoprecipitation of ATG7 with each of the indicated GFP-tagged truncated TP53INP2 mutants in HEK293 cells. TP53INP2 proteins were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7. (f) Purified GST-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ], TP53INP2 W35,I38A [Δ1-28],[NLSΔ] or SQSTM1 was incubated with purified ATG7, then the GST-tagged proteins were affinity-isolated by glutathione-sepharose 4B beads and bound ATG7 was detected by western blot using anti-ATG7.
Figure Legend Snippet: TP53INP2 forms a complex with LC3B and ATG7. (a) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-TP53INP2, GFP-TP53INP2[NLSΔ] or GFP-TP53INP2[LIRΔ] from HEK293 cells. TP53INP2 proteins were immunoprecipitated by anti-GFP. The coprecipitated ATG7, ATG3 or ATG12–ATG5 was detected by western blot using anti-ATG3, anti-ATG7 or anti-ATG5 respectively. (b) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ] or TP53INP2[LIRΔ]. GFP-tagged TP53INP2 mutants were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7, anti-ATG3 or anti-ATG5. (c) In vitro TP53INP2-ATG7 binding assay. Purified GST-TP53INP2 or GST-TP53INP2 W35,I38A was incubated with purified LC3B[G120] and ATG7. After affinity-isolating GST-TP53INP2 or GST-TP53INP2 W35,I38A with glutathione-sepharose 4B beads, the bound LC3B[G120] and ATG7 were analyzed by western blot. (d) HEK293T cells were cotransfected with Flag-LC3B, TP53INP2-MYC and HA-ATG7. The cells were lysed 48 h after transfection and Flag-LC3B was immunoprecipitated with anti-Flag. After incubation of the Flag-LC3B precipitates with Flag peptide, the eluate was used for immunoprecipitation with either anti-MYC or anti-HA. The immunoprecipitates were then analyzed by western blot by anti-Flag, anti-MYC and anti-HA respectively. (e) Coimmunoprecipitation of ATG7 with each of the indicated GFP-tagged truncated TP53INP2 mutants in HEK293 cells. TP53INP2 proteins were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7. (f) Purified GST-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ], TP53INP2 W35,I38A [Δ1-28],[NLSΔ] or SQSTM1 was incubated with purified ATG7, then the GST-tagged proteins were affinity-isolated by glutathione-sepharose 4B beads and bound ATG7 was detected by western blot using anti-ATG7.

Techniques Used: Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Purification, Incubation, Transfection, Isolation

TP53INP2 facilitates LC3B-ATG7 interaction. (a) Coprecipitation of endogenous ATG7 with exogenous Flag-LC3B in TP53INP2-MYC cotransfected HEK293 cells with or without cell starvation. Flag-LC3B was immunoprecipitated using anti-Flag, then ATG7 and TP53INP2-MYC were detected by anti-ATG7 and anti-MYC respectively. (b) Coprecipitation of ATG7 with Flag-LC3B from HEK293 cells transiently expressing RFP-tagged TP53INP2 or each of the indicated TP53INP2 mutants. Flag-LC3B was immunoprecipitated using anti-Flag. (c) HEK293 cells stably expressing non-silencing shRNA or TP53INP2 shRNA were transfected with Flag-LC3B K49,51R and starved. The cells were then fractionated by differential centrifugation. Flag-LC3B K49,51R was immunoprecipitated from the cell cytosol using anti-Flag and the coprecipitated ATG7 was detected by western blot. (d) In vitro affinity-isolation assay of LC3B[G120]-ATG7 interaction. Purified GST-LC3B[G120] was incubated with cell lysate from HEK293 cells expressing the indicated RFP-tagged TP53INP2 mutants. After affinity-isolating GST-LC3B[G120] using glutathione-sepharose 4B beads, GST-LC3B[G120]-bound ATG7 was analyzed by western blot. (e) Confocal images of HEK293 cells stably expressing GFP-LC3B and transfected with plasmids expressing each of the indicated RFP-tagged TP53INP2 truncated mutants. (f) Quantification of GFP-LC3B puncta in (e). The data are presented as mean ± SEM, n = 30 cells. ***, P
Figure Legend Snippet: TP53INP2 facilitates LC3B-ATG7 interaction. (a) Coprecipitation of endogenous ATG7 with exogenous Flag-LC3B in TP53INP2-MYC cotransfected HEK293 cells with or without cell starvation. Flag-LC3B was immunoprecipitated using anti-Flag, then ATG7 and TP53INP2-MYC were detected by anti-ATG7 and anti-MYC respectively. (b) Coprecipitation of ATG7 with Flag-LC3B from HEK293 cells transiently expressing RFP-tagged TP53INP2 or each of the indicated TP53INP2 mutants. Flag-LC3B was immunoprecipitated using anti-Flag. (c) HEK293 cells stably expressing non-silencing shRNA or TP53INP2 shRNA were transfected with Flag-LC3B K49,51R and starved. The cells were then fractionated by differential centrifugation. Flag-LC3B K49,51R was immunoprecipitated from the cell cytosol using anti-Flag and the coprecipitated ATG7 was detected by western blot. (d) In vitro affinity-isolation assay of LC3B[G120]-ATG7 interaction. Purified GST-LC3B[G120] was incubated with cell lysate from HEK293 cells expressing the indicated RFP-tagged TP53INP2 mutants. After affinity-isolating GST-LC3B[G120] using glutathione-sepharose 4B beads, GST-LC3B[G120]-bound ATG7 was analyzed by western blot. (e) Confocal images of HEK293 cells stably expressing GFP-LC3B and transfected with plasmids expressing each of the indicated RFP-tagged TP53INP2 truncated mutants. (f) Quantification of GFP-LC3B puncta in (e). The data are presented as mean ± SEM, n = 30 cells. ***, P

Techniques Used: Immunoprecipitation, Expressing, Stable Transfection, shRNA, Transfection, Centrifugation, Western Blot, In Vitro, Isolation, Purification, Incubation

3) Product Images from "MCD1 Associates with FtsZ Filaments via the Membrane-Tethering Protein ARC6 to Guide Chloroplast Division"

Article Title: MCD1 Associates with FtsZ Filaments via the Membrane-Tethering Protein ARC6 to Guide Chloroplast Division

Journal: The Plant Cell

doi: 10.1105/tpc.18.00189

In Vitro Pull-Down Analysis of the Stromal Regions of MCD1 and ARC6. (A) Recombinant His-ARC6 N binds to GST-MCD1 C or GST-MCD1 C(∆277-314) . Glutathione-Sepharose 4B beads were treated with buffer only (lane 2) or coated with GST (lane 3), GST-tagged MCD1 C (lane 4), MCD1C (∆277-314) (lane 5), or MCD1 N (lane 6). The beads were then incubated with crude extracts of E. coli cells expressing His-ARC6 N . Proteins were eluted and analyzed by immunoblotting with anti-His and anti-GST antibodies. (B) Recombinant MBP-PARC6 N -His or His-ARC6 C was not precipitated from crude E. coli extracts by Glutathione-Sepharose 4B beads coated with GST-MCD1 C . All assays were performed more than three times.
Figure Legend Snippet: In Vitro Pull-Down Analysis of the Stromal Regions of MCD1 and ARC6. (A) Recombinant His-ARC6 N binds to GST-MCD1 C or GST-MCD1 C(∆277-314) . Glutathione-Sepharose 4B beads were treated with buffer only (lane 2) or coated with GST (lane 3), GST-tagged MCD1 C (lane 4), MCD1C (∆277-314) (lane 5), or MCD1 N (lane 6). The beads were then incubated with crude extracts of E. coli cells expressing His-ARC6 N . Proteins were eluted and analyzed by immunoblotting with anti-His and anti-GST antibodies. (B) Recombinant MBP-PARC6 N -His or His-ARC6 C was not precipitated from crude E. coli extracts by Glutathione-Sepharose 4B beads coated with GST-MCD1 C . All assays were performed more than three times.

Techniques Used: In Vitro, Recombinant, Incubation, Expressing

4) Product Images from "Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions"

Article Title: Retromer Regulates HIV-1 Envelope Glycoprotein Trafficking and Incorporation into Virions

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004518

The gp41 cytoplasmic tail binds directly to retromer. A) Native coimmunoprecipitation with anti-CD8 identifies retromer components Vps26 and Vps35 as interacting with the HIV-1 gp41CT. Cell lysates prepared from untransfected (UT), CD8-CIMPR or CD8-gp41CT expressing HeLa cells were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and western blotting for Vps35 and Vps26. Untransfected HeLa cells were used as a negative control and CD8-CIMPR as a positive control. B) GST-pulldown confirms direct binding of the gp41CT to retromer. Purified recombinant FLAG-tagged retromer complex (3xFLAG-Vps26-Vps29-Vps35-His 6 ) was incubated with purified GST or GST fusion proteins containing the CIMPR (GST-CIMPR) or Envgp41 (GST-gp41CT) cytoplasmic tail and proteins were pulled down with glutathione-Sepharose 4B beads. Bound retromer components Vps26 and Vps35 were detected by immunoblotting.
Figure Legend Snippet: The gp41 cytoplasmic tail binds directly to retromer. A) Native coimmunoprecipitation with anti-CD8 identifies retromer components Vps26 and Vps35 as interacting with the HIV-1 gp41CT. Cell lysates prepared from untransfected (UT), CD8-CIMPR or CD8-gp41CT expressing HeLa cells were incubated with anti-CD8 coated beads and co-IP proteins were subjected to SDS-PAGE and western blotting for Vps35 and Vps26. Untransfected HeLa cells were used as a negative control and CD8-CIMPR as a positive control. B) GST-pulldown confirms direct binding of the gp41CT to retromer. Purified recombinant FLAG-tagged retromer complex (3xFLAG-Vps26-Vps29-Vps35-His 6 ) was incubated with purified GST or GST fusion proteins containing the CIMPR (GST-CIMPR) or Envgp41 (GST-gp41CT) cytoplasmic tail and proteins were pulled down with glutathione-Sepharose 4B beads. Bound retromer components Vps26 and Vps35 were detected by immunoblotting.

Techniques Used: Expressing, Incubation, Co-Immunoprecipitation Assay, SDS Page, Western Blot, Negative Control, Positive Control, Binding Assay, Purification, Recombinant

5) Product Images from "Phactr3/Scapinin, a Member of Protein Phosphatase 1 and Actin Regulator (Phactr) Family, Interacts with the Plasma Membrane via Basic and Hydrophobic Residues in the N-Terminus"

Article Title: Phactr3/Scapinin, a Member of Protein Phosphatase 1 and Actin Regulator (Phactr) Family, Interacts with the Plasma Membrane via Basic and Hydrophobic Residues in the N-Terminus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0113289

The interaction between the Nt and lipid bilayers. (A) Liposome co-sedimentation assay. Nt was produced as a fusion protein with glutathione transferase (GST-Nt) in the BL21 strain of Escherichia coli and purified with glutathione-Sepharose 4B beads. GST was used as a control. Diagram showing the liposome sedimentation assay. GST and and GST-Nt were centrifuged before use to sediment insoluble proteins and mixed with liposomes (input). After centrifugation, the liposome-bound (ppt) and liposome-unbound (sup) fractions were recovered. Aliquots of each fraction were separated on an SDS-PAGE and stained with Coomassie brilliant blue. (B) Lipid binding assay using the spot array method. Lipid-spotted membrane strips were incubated with GST or GST-Nt and the lipid-bound proteins were detected using anti-GST antibody. LPA, lysophosphatidic acid; PI, phosphatidylinositol; PI(3)P, phosphatidylinositol-(3)-phosphate; PI(4)P, phosphatidylinositol-(4)-phosphate; PI(5), phosphatidylinositol-(5)-phosphate; PE, phosphatidylethanolamine, PC, phosphatidylcholine; SIP, sphingosine-1-phosphate; PI(3,4)P 2 ; phosphatidylinositol-(3,4)-bisphosphate; PI(3,5)P 2 , phosphatidylinositol-(3,5)-bisphosphate; PI(3,4,5)P 3 , phosphatidylinositol-(3,4,5)-trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine.
Figure Legend Snippet: The interaction between the Nt and lipid bilayers. (A) Liposome co-sedimentation assay. Nt was produced as a fusion protein with glutathione transferase (GST-Nt) in the BL21 strain of Escherichia coli and purified with glutathione-Sepharose 4B beads. GST was used as a control. Diagram showing the liposome sedimentation assay. GST and and GST-Nt were centrifuged before use to sediment insoluble proteins and mixed with liposomes (input). After centrifugation, the liposome-bound (ppt) and liposome-unbound (sup) fractions were recovered. Aliquots of each fraction were separated on an SDS-PAGE and stained with Coomassie brilliant blue. (B) Lipid binding assay using the spot array method. Lipid-spotted membrane strips were incubated with GST or GST-Nt and the lipid-bound proteins were detected using anti-GST antibody. LPA, lysophosphatidic acid; PI, phosphatidylinositol; PI(3)P, phosphatidylinositol-(3)-phosphate; PI(4)P, phosphatidylinositol-(4)-phosphate; PI(5), phosphatidylinositol-(5)-phosphate; PE, phosphatidylethanolamine, PC, phosphatidylcholine; SIP, sphingosine-1-phosphate; PI(3,4)P 2 ; phosphatidylinositol-(3,4)-bisphosphate; PI(3,5)P 2 , phosphatidylinositol-(3,5)-bisphosphate; PI(3,4,5)P 3 , phosphatidylinositol-(3,4,5)-trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine.

Techniques Used: Sedimentation, Produced, Purification, Centrifugation, SDS Page, Staining, Binding Assay, Incubation

6) Product Images from "GABAA Receptor Phospho-Dependent Modulation Is Regulated by Phospholipase C-Related Inactive Protein Type 1, a Novel Protein Phosphatase 1 Anchoring Protein"

Article Title: GABAA Receptor Phospho-Dependent Modulation Is Regulated by Phospholipase C-Related Inactive Protein Type 1, a Novel Protein Phosphatase 1 Anchoring Protein

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.1323-04.2004

Phosphatase activities in hippocampal extracts and dephosphorylation of β3 subunit. A , GST-tagged β3 subunit (200 pmol) immobilized on glutathione Sepharose 4B beads was phosphorylated with [γ- 32 P]ATP using the catalytic subunit of PKA. The beads were subjected to dephosphorylation by 1 U of PP1α, and released 32 ). Results are expressed as mean ± SE from four separate experiments. The top panel shows an autoradiogram of GST-β3 subunit after the treatment with PP1α alone or together with various versions of PRIP-1 molecule. B , GST-β3 subunit phosphorylated as described above was subjected to dephosphorylation by PP1α, PP2A, or PP2B, and released 32 P was counted by a scintillation counter. Results are expressed as mean ± SE from four separate experiments. 1, 2, and 3 represent the presence of 0, 0.5, and 1 U of each phosphatase, respectively. The top panel shows an autoradiogram of GST-β3 subunit after the treatment with each phosphatase. C , Hippocampal extract (∼200 μg of protein) from the control mice was assayed for the phosphatase activities, which dephosphorylate phosphorylated GST-β3 subunit in the presence of various concentrations of okadaic acid. The absence of okadaic acid caused the release of 18,900 dpm (mean of 3 experiments), which was given a value of 100%. The relative radioactivities released in the presence of okadaic acid at concentrations indicated are shown. Results are expressed as mean ± SE from three separate experiments. D , The same experiments described in C were also performed using hippocampal extracts from the PRIP-1 -/- mice, and the results were compared with those seen in the control mice. Results are expressed as mean ± SE from three separate experiments. * p
Figure Legend Snippet: Phosphatase activities in hippocampal extracts and dephosphorylation of β3 subunit. A , GST-tagged β3 subunit (200 pmol) immobilized on glutathione Sepharose 4B beads was phosphorylated with [γ- 32 P]ATP using the catalytic subunit of PKA. The beads were subjected to dephosphorylation by 1 U of PP1α, and released 32 ). Results are expressed as mean ± SE from four separate experiments. The top panel shows an autoradiogram of GST-β3 subunit after the treatment with PP1α alone or together with various versions of PRIP-1 molecule. B , GST-β3 subunit phosphorylated as described above was subjected to dephosphorylation by PP1α, PP2A, or PP2B, and released 32 P was counted by a scintillation counter. Results are expressed as mean ± SE from four separate experiments. 1, 2, and 3 represent the presence of 0, 0.5, and 1 U of each phosphatase, respectively. The top panel shows an autoradiogram of GST-β3 subunit after the treatment with each phosphatase. C , Hippocampal extract (∼200 μg of protein) from the control mice was assayed for the phosphatase activities, which dephosphorylate phosphorylated GST-β3 subunit in the presence of various concentrations of okadaic acid. The absence of okadaic acid caused the release of 18,900 dpm (mean of 3 experiments), which was given a value of 100%. The relative radioactivities released in the presence of okadaic acid at concentrations indicated are shown. Results are expressed as mean ± SE from three separate experiments. D , The same experiments described in C were also performed using hippocampal extracts from the PRIP-1 -/- mice, and the results were compared with those seen in the control mice. Results are expressed as mean ± SE from three separate experiments. * p

Techniques Used: De-Phosphorylation Assay, Mouse Assay

7) Product Images from "Identification of Novel Amelogenin-Binding Proteins by Proteomics Analysis"

Article Title: Identification of Novel Amelogenin-Binding Proteins by Proteomics Analysis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0078129

Proteomic analysis of amelogenin-interacting proteins in osteoblastic cells. Purified GST-rM180 immobilized on glutathione-Sepharose 4B beads was incubated with no extract (GST-rM180), fractionated soluble protein extract (GST-rM180 + cytoplasm) ( A ) or membrane-associated protein extract (GST-rM180 + membrane) ( B ) prepared from SaOS-2 cells. GST control gels for the both extracts ware also shown to exclude the possibility to non-specific bindings (GST + cytoplasm, GST + membrane). To minimize binding of nonspecific proteins, the cell extracts were pre-cleaned with glutathione beads. The proteins bound to affinity matrices were eluted and separated by isoelectric focusing and SDS-PAGE was performed on a 7.5–15% gradient gel. A typical two-dimensional gel is illustrated. The pH gradient of the separation in the first dimension is shown on the top of the gels, and the molecular weight markers are shown in kDa on the left of the gels. Proteins were visualized with Coomassie brilliant blue staining, excised, trypsinized, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis as described in Table 2 , 3. Magnified views of indicated areas were shown to demonstrate the analyzed spots of amelogenin-interacting proteins (Protein spots).
Figure Legend Snippet: Proteomic analysis of amelogenin-interacting proteins in osteoblastic cells. Purified GST-rM180 immobilized on glutathione-Sepharose 4B beads was incubated with no extract (GST-rM180), fractionated soluble protein extract (GST-rM180 + cytoplasm) ( A ) or membrane-associated protein extract (GST-rM180 + membrane) ( B ) prepared from SaOS-2 cells. GST control gels for the both extracts ware also shown to exclude the possibility to non-specific bindings (GST + cytoplasm, GST + membrane). To minimize binding of nonspecific proteins, the cell extracts were pre-cleaned with glutathione beads. The proteins bound to affinity matrices were eluted and separated by isoelectric focusing and SDS-PAGE was performed on a 7.5–15% gradient gel. A typical two-dimensional gel is illustrated. The pH gradient of the separation in the first dimension is shown on the top of the gels, and the molecular weight markers are shown in kDa on the left of the gels. Proteins were visualized with Coomassie brilliant blue staining, excised, trypsinized, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis as described in Table 2 , 3. Magnified views of indicated areas were shown to demonstrate the analyzed spots of amelogenin-interacting proteins (Protein spots).

Techniques Used: Purification, Incubation, Binding Assay, SDS Page, Two-Dimensional Gel Electrophoresis, Molecular Weight, Staining, Mass Spectrometry

8) Product Images from "An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex *"

Article Title: An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.751867

APLF interacts with the Ku·DNA-PKcs·DNA complex. A, His-APLF was immobilized on nitrilotriacetic acid beads and incubated with HeLa whole cell extracts. Beads were washed either in the absence (−) or presence (+) of ethidium bromide (EtBr, 50 μg/ml), then boiled in SDS sample buffer, loaded onto SDS-PAGE gels, and immunoblotted with antibodies to His (for His-APLF), DNA-PKcs, and Ku80 as indicated. B, GST ( lane 2 ) or GST-APLF ( lanes 3–6 ) were immobilized on glutathione-Sepharose 4B beads and incubated with whole cell extracts from HeLa cells that had been either unirradiated (−) or irradiated (10 gray IR) and allowed to recover for 1 h. Beads were washed either in the absence (−) or presence (+) of EtBr (50 μg/ml), then boiled in SDS sample buffer, loaded onto SDS-PAGE gels, and immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs, and Ku80 as indicated. The lower panel represents a longer exposure of the Ku80 blot to show a signal in the input lanes. Lane 1 contained 50 μg of extract from unirradiated cells as a positive control. C, HeLa cells were transiently transfected with FLAG-tagged APLF ( lanes 3 and 4 ) or empty vector ( lane 2 ), then extracts were immunoprecipitated with anti-FLAG antibody, run on SDS-PAGE, and immunoblotted with antibodies to FLAG (for FLAG-APLF), DNA-PKcs and Ku as indicated. Where indicated, ethidium bromide (50 μg/ml) was added to immunoprecipitation wash buffers. Note: a duplicated sample lane has been removed between lanes 2 and 3 . All blots were from the same exposure of the same gels. D, purified DNA-PKcs and/or Ku were incubated with GST-APLF immobilized on glutathione-Sepharose 4B beads in either the absence (−) or presence (+) of CT-DNA (10 μg/ml). Samples were run on SDS-PAGE and immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs and Ku as indicated. E, purified DNA-PKcs and Ku were incubated with GST-APLF ( lanes 3–8 ) or GST ( lane 2 ) immobilized on glutathione-Sepharose 4B beads in the presence of different lengths of DNA (10 μg/ml) and then immunoblotted with antibodies as indicated. In lanes 2 and 4 , proteins were incubated in the presence of 10 μg/ml of CT-DNA, lane 5 contained 40 base ssDNA; lane 6 , 40-bp dsDNA; lane 7 , 100 base ssDNA; and lane 8 , 100-bp dsDNA. Lane 1 contained 100 ng each DNA-PKcs and Ku. Lane 3 contained no DNA. F, purified DNA-PKcs and Ku were incubated with either GST alone ( lanes 2 and 3 ), GST-APLF ( lanes 4 and 5 ), or GST-APLF residues 1–120 ( lanes 6 and 7 ), 110–360 ( lanes 8 and 9 ), or 360–511 ( lanes 9 and 10 ) that had been bound to glutathione-Sepharose 4B beads either in the absence (−) or presence (+) of CT-DNA (80 μg/ml). Samples were washed, run on SDS-PAGE, and immunoblotted. Lane 1 contains 100 ng each DNA-PKcs and Ku. The upper panel is a Ponceau Red-stained membrane, whereas the lower panels show immunoblots for DNA-PKcs and Ku80, respectively. Positions of molecular mass markers (in kDa) are shown on the left-hand side on the Ponceau-stained blot. G, GST alone, GST-APLF, or GST-APLF with mutations of R182E/K183E/R184E or W189G were bound to glutathione-Sepharose 4B beads and incubated with purified DNA-PKcs and Ku in the absence (−) or presence (+) of CT-DNA as above then immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs, and Ku80 as indicated.
Figure Legend Snippet: APLF interacts with the Ku·DNA-PKcs·DNA complex. A, His-APLF was immobilized on nitrilotriacetic acid beads and incubated with HeLa whole cell extracts. Beads were washed either in the absence (−) or presence (+) of ethidium bromide (EtBr, 50 μg/ml), then boiled in SDS sample buffer, loaded onto SDS-PAGE gels, and immunoblotted with antibodies to His (for His-APLF), DNA-PKcs, and Ku80 as indicated. B, GST ( lane 2 ) or GST-APLF ( lanes 3–6 ) were immobilized on glutathione-Sepharose 4B beads and incubated with whole cell extracts from HeLa cells that had been either unirradiated (−) or irradiated (10 gray IR) and allowed to recover for 1 h. Beads were washed either in the absence (−) or presence (+) of EtBr (50 μg/ml), then boiled in SDS sample buffer, loaded onto SDS-PAGE gels, and immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs, and Ku80 as indicated. The lower panel represents a longer exposure of the Ku80 blot to show a signal in the input lanes. Lane 1 contained 50 μg of extract from unirradiated cells as a positive control. C, HeLa cells were transiently transfected with FLAG-tagged APLF ( lanes 3 and 4 ) or empty vector ( lane 2 ), then extracts were immunoprecipitated with anti-FLAG antibody, run on SDS-PAGE, and immunoblotted with antibodies to FLAG (for FLAG-APLF), DNA-PKcs and Ku as indicated. Where indicated, ethidium bromide (50 μg/ml) was added to immunoprecipitation wash buffers. Note: a duplicated sample lane has been removed between lanes 2 and 3 . All blots were from the same exposure of the same gels. D, purified DNA-PKcs and/or Ku were incubated with GST-APLF immobilized on glutathione-Sepharose 4B beads in either the absence (−) or presence (+) of CT-DNA (10 μg/ml). Samples were run on SDS-PAGE and immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs and Ku as indicated. E, purified DNA-PKcs and Ku were incubated with GST-APLF ( lanes 3–8 ) or GST ( lane 2 ) immobilized on glutathione-Sepharose 4B beads in the presence of different lengths of DNA (10 μg/ml) and then immunoblotted with antibodies as indicated. In lanes 2 and 4 , proteins were incubated in the presence of 10 μg/ml of CT-DNA, lane 5 contained 40 base ssDNA; lane 6 , 40-bp dsDNA; lane 7 , 100 base ssDNA; and lane 8 , 100-bp dsDNA. Lane 1 contained 100 ng each DNA-PKcs and Ku. Lane 3 contained no DNA. F, purified DNA-PKcs and Ku were incubated with either GST alone ( lanes 2 and 3 ), GST-APLF ( lanes 4 and 5 ), or GST-APLF residues 1–120 ( lanes 6 and 7 ), 110–360 ( lanes 8 and 9 ), or 360–511 ( lanes 9 and 10 ) that had been bound to glutathione-Sepharose 4B beads either in the absence (−) or presence (+) of CT-DNA (80 μg/ml). Samples were washed, run on SDS-PAGE, and immunoblotted. Lane 1 contains 100 ng each DNA-PKcs and Ku. The upper panel is a Ponceau Red-stained membrane, whereas the lower panels show immunoblots for DNA-PKcs and Ku80, respectively. Positions of molecular mass markers (in kDa) are shown on the left-hand side on the Ponceau-stained blot. G, GST alone, GST-APLF, or GST-APLF with mutations of R182E/K183E/R184E or W189G were bound to glutathione-Sepharose 4B beads and incubated with purified DNA-PKcs and Ku in the absence (−) or presence (+) of CT-DNA as above then immunoblotted with antibodies to GST (for GST-APLF), DNA-PKcs, and Ku80 as indicated.

Techniques Used: Incubation, SDS Page, Irradiation, Positive Control, Transfection, Plasmid Preparation, Immunoprecipitation, Purification, Staining, Western Blot

9) Product Images from "B-Cell Translocation Gene 2 (Btg2) Regulates Vertebral Patterning by Modulating Bone Morphogenetic Protein/Smad Signaling"

Article Title: B-Cell Translocation Gene 2 (Btg2) Regulates Vertebral Patterning by Modulating Bone Morphogenetic Protein/Smad Signaling

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.23.10256-10262.2004

BTG2 activates BMP-dependent transcription and associates with Smad1 or Smad8 in vivo. (a and b) Effect of Btg2 on TGF-β1-induced transcription of SBE 4 -Luc (a) and ARE-Luc reporters (b). C2C12 cells were transiently transfected with a control vector (pCMV-β-gal) or Btg2 (1 μg/well) along with either SBE 4 -Luc (a) or ARE reporter plus FAST1 (b). (c and d) Effect of Btg2 on BMP-induced transcription of BRE-Luc reporter. (c) C2C12 cells were transiently transfected with the control vector or Btg2 (1 μg/well) along with BRE-Luc. The cells were treated with 50 ng of BMP-2/ml (black bars) or left untreated (white bars). (d) C2C12 cells were transiently transfected with the control vector or increasing amounts of Btg2 along with BRE-Luc. Luciferase activity was normalized to β-galactosidase activity and plotted as the mean and standard deviation for triplicates from a representative experiment. (e) GST-BTG2 was transfected into 293T cells with the Flag-tagged Smad1 or Smad5 or Myc-tagged Smad8 construct. Cell extracts were subjected to GST pull-down assay using glutathione-Sepharose 4B beads, followed by immunoblotting with anti-Flag or anti-Myc antibody. Expression of GST, GST-BTG2, and Smads was monitored as indicated.
Figure Legend Snippet: BTG2 activates BMP-dependent transcription and associates with Smad1 or Smad8 in vivo. (a and b) Effect of Btg2 on TGF-β1-induced transcription of SBE 4 -Luc (a) and ARE-Luc reporters (b). C2C12 cells were transiently transfected with a control vector (pCMV-β-gal) or Btg2 (1 μg/well) along with either SBE 4 -Luc (a) or ARE reporter plus FAST1 (b). (c and d) Effect of Btg2 on BMP-induced transcription of BRE-Luc reporter. (c) C2C12 cells were transiently transfected with the control vector or Btg2 (1 μg/well) along with BRE-Luc. The cells were treated with 50 ng of BMP-2/ml (black bars) or left untreated (white bars). (d) C2C12 cells were transiently transfected with the control vector or increasing amounts of Btg2 along with BRE-Luc. Luciferase activity was normalized to β-galactosidase activity and plotted as the mean and standard deviation for triplicates from a representative experiment. (e) GST-BTG2 was transfected into 293T cells with the Flag-tagged Smad1 or Smad5 or Myc-tagged Smad8 construct. Cell extracts were subjected to GST pull-down assay using glutathione-Sepharose 4B beads, followed by immunoblotting with anti-Flag or anti-Myc antibody. Expression of GST, GST-BTG2, and Smads was monitored as indicated.

Techniques Used: In Vivo, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Construct, Pull Down Assay, Expressing

10) Product Images from "Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor"

Article Title: Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0065266

CaM binding sites in the i3 loop and carboxyl tail of the AT 1A receptor. A, Schematic representation of constructs of GST-fusion proteins. Four GST-fusion proteins containing truncated peptides in the i3 loop and carboxyl tail of the receptor were constructed, including GST-N-terminus of the i3 loop (GST-ATi3N), GST-C-terminus of the i3 loop (GST-ATi3C), GST-N-terminus of carboxyl tail (GST-ATctN) and GST-C-terminus of carboxyl tail (GST-ATctC). The numbers under the first or the last residues represent amino acid positions in the receptor. B, Interaction of GST-fusion proteins with CaM. The GST-fusion proteins (50 pmol) were incubated with purified bovine CaM (50 pmol) in 250 µl of buffer containing 100 mM Tris-HCl (pH 7.5) with 0.1 mM CaCl 2 . Proteins were pulled down by gluthathione-sepharose 4B beads, following which immunoblots were probed with a specific anti-CaM antibody. These experiments were repeated five times with similar results.
Figure Legend Snippet: CaM binding sites in the i3 loop and carboxyl tail of the AT 1A receptor. A, Schematic representation of constructs of GST-fusion proteins. Four GST-fusion proteins containing truncated peptides in the i3 loop and carboxyl tail of the receptor were constructed, including GST-N-terminus of the i3 loop (GST-ATi3N), GST-C-terminus of the i3 loop (GST-ATi3C), GST-N-terminus of carboxyl tail (GST-ATctN) and GST-C-terminus of carboxyl tail (GST-ATctC). The numbers under the first or the last residues represent amino acid positions in the receptor. B, Interaction of GST-fusion proteins with CaM. The GST-fusion proteins (50 pmol) were incubated with purified bovine CaM (50 pmol) in 250 µl of buffer containing 100 mM Tris-HCl (pH 7.5) with 0.1 mM CaCl 2 . Proteins were pulled down by gluthathione-sepharose 4B beads, following which immunoblots were probed with a specific anti-CaM antibody. These experiments were repeated five times with similar results.

Techniques Used: Chick Chorioallantoic Membrane Assay, Binding Assay, Construct, Incubation, Purification, Western Blot

Effect of CaM on G protein βγ subunit interaction with wild ATi3 or ATct. A. Interaction of G protein βγ subunits and ATi3 or ATct. 50 pmol of GST-ATi3(213–242) or GST-ATct(297–359) were incubated with different amounts of G protein Gβγ subunits in a buffer containing 20 mM Tris-HCl and 70 mM NaCl (pH 7.5). Interactions were assessed by GST-fusion protein pull-down assay, and blots were probed with a specific antibody against Gβ subunits (upper panel). 1 pmol of Gβγ subunit was interacted with different amounts of GST-ATi3(213–242) and GST-ATct(297–359). Interaction was assessed by immunoblot against Gβ subunits (lower panel). B. CaM inhibits the interaction between G protein Gβγ subunits and ATi3 or ATct. 2 pmol of GST-ATi3(213–242) or GST-ATct(297–359) were incubated with different concentrations of pure bovine brain CaM for 30 minutes in a buffer (20 mM Tris-HCl and 70 mM NaCl with 0.1 mM CaCl 2 , pH 7.5) following which 2 pmol of Gβγ subunits were added and incubated for 1 hour. GST-fusion proteins and their interacting proteins were pulled down by gluthathione-sepharose 4B beads, and subjected to immunoblot. Blots were probed with a specific antibody against Gβ subunits. The summary graph represents means ± S.E. from four independent experiments. C. Effects of CaM on the interactions between G protein βγ subunits and mutated ATi3 and ATct. The method is same as the described in the Figure 7B , except that we used mutated GST-fusion proteins, GST-ATi3(213–242)W219A, ATct(297–359)F309A, and ATct(297–359)F313A. The summary graph represents mean ± S.E. from 4 or 5 independent experiments.
Figure Legend Snippet: Effect of CaM on G protein βγ subunit interaction with wild ATi3 or ATct. A. Interaction of G protein βγ subunits and ATi3 or ATct. 50 pmol of GST-ATi3(213–242) or GST-ATct(297–359) were incubated with different amounts of G protein Gβγ subunits in a buffer containing 20 mM Tris-HCl and 70 mM NaCl (pH 7.5). Interactions were assessed by GST-fusion protein pull-down assay, and blots were probed with a specific antibody against Gβ subunits (upper panel). 1 pmol of Gβγ subunit was interacted with different amounts of GST-ATi3(213–242) and GST-ATct(297–359). Interaction was assessed by immunoblot against Gβ subunits (lower panel). B. CaM inhibits the interaction between G protein Gβγ subunits and ATi3 or ATct. 2 pmol of GST-ATi3(213–242) or GST-ATct(297–359) were incubated with different concentrations of pure bovine brain CaM for 30 minutes in a buffer (20 mM Tris-HCl and 70 mM NaCl with 0.1 mM CaCl 2 , pH 7.5) following which 2 pmol of Gβγ subunits were added and incubated for 1 hour. GST-fusion proteins and their interacting proteins were pulled down by gluthathione-sepharose 4B beads, and subjected to immunoblot. Blots were probed with a specific antibody against Gβ subunits. The summary graph represents means ± S.E. from four independent experiments. C. Effects of CaM on the interactions between G protein βγ subunits and mutated ATi3 and ATct. The method is same as the described in the Figure 7B , except that we used mutated GST-fusion proteins, GST-ATi3(213–242)W219A, ATct(297–359)F309A, and ATct(297–359)F313A. The summary graph represents mean ± S.E. from 4 or 5 independent experiments.

Techniques Used: Chick Chorioallantoic Membrane Assay, Incubation, Pull Down Assay

Interactions of the i3 loop and carboxyl tail of the AT 1A receptor with CaM. A, Construction of GST-fusion proteins. Upper panel illustrates a schematic structure of the AT 1A receptor and constructs of GST-fusion proteins of the i3 loop and carboxyl tail of the receptor. The numbers represent positions of amino acids in the receptor. The GST-fusion proteins were constructed, expressed in E. coli , purified by using gluthathione-sepharose 4B beads, separated by SDS-PAGE, and stained by Coomassie blue (lower panel). B. Interactions of the i3 loop or carboxyl tail of AT 1A receptor with CaM in rat brain lysates (upper panel) and with purified bovine brain CaM (lower panel). 50 pmol of GST-fusion proteins were incubated with 500 µg of rat brain lysates in 250 µl of buffer containing 20 mM Tris-HCl, 70 mM NaCl, pH 7.5 with 1 mM EGTA or 0.1 mM CaCl 2 . Interacting protein complexes were pulled down by gluthathione-sepharose 4B beads, and visualized by immunoblot with a specific anti-CaM antibody. The same methods were applied to interactions with pure bovine CaM (50 pmol), except that the buffer contained 100 mM Tris-HCl (pH 7.5). These experiments were repeated five times with similar results.
Figure Legend Snippet: Interactions of the i3 loop and carboxyl tail of the AT 1A receptor with CaM. A, Construction of GST-fusion proteins. Upper panel illustrates a schematic structure of the AT 1A receptor and constructs of GST-fusion proteins of the i3 loop and carboxyl tail of the receptor. The numbers represent positions of amino acids in the receptor. The GST-fusion proteins were constructed, expressed in E. coli , purified by using gluthathione-sepharose 4B beads, separated by SDS-PAGE, and stained by Coomassie blue (lower panel). B. Interactions of the i3 loop or carboxyl tail of AT 1A receptor with CaM in rat brain lysates (upper panel) and with purified bovine brain CaM (lower panel). 50 pmol of GST-fusion proteins were incubated with 500 µg of rat brain lysates in 250 µl of buffer containing 20 mM Tris-HCl, 70 mM NaCl, pH 7.5 with 1 mM EGTA or 0.1 mM CaCl 2 . Interacting protein complexes were pulled down by gluthathione-sepharose 4B beads, and visualized by immunoblot with a specific anti-CaM antibody. The same methods were applied to interactions with pure bovine CaM (50 pmol), except that the buffer contained 100 mM Tris-HCl (pH 7.5). These experiments were repeated five times with similar results.

Techniques Used: Chick Chorioallantoic Membrane Assay, Construct, Purification, SDS Page, Staining, Incubation

Effect of point mutations in ATi3N or ATctN on their interactions with CaM. A. Modeled structures of CaM-ATi3(214–231) and CaM-ATct(302–317). The complexes of CaM and CaM binding motif in the i3 loop ATi3(214–231) or the carboxyl terminal tail ATct(302–317) of the receptor were modeled as described in Experimental Procedures. The target peptides are colored in red. Residues W219 in ATi3(214–231)−SYTLIWKALKKAYEIQKN, and F309 and F313 in ATct(302–317)−YGFLGKKFKKYFLQLL are displayed with sticks and are colored in blue. Calcium atoms are shown as orange spheres. The N- and C- termini of CaM are also labeled. Helices and sheets in CaM are colored in green and yellow, respectively. B. Effect of point mutations at ATi3N or ATctN on their interaction with CaM. 50 pmol of wild type GST-fusion proteins including GST-ATi3N(213–234) and GST-ATctN (297–324), and 50 pmol of mutated GST-fusion proteins including GST-ATi3N(W219A), GST-ATctN(F309A) and GST-ATctN(F313A), were incubated with purified bovine brain CaM in a buffer containing 100 mM Tris-HCl (pH 7.5) with 0.1 mM CaCl 2 . The protein complexes were pulled down by gluthathione-sepharose 4B beads, and subjected to immunoblot with a specific anti-CaM antibody. GST-fusion proteins were visualized in the gels by Coomassie blue staining (the lower gel panel). The summary graph represents relative densities of the ratio of the CaM in the immunoblots and the loaded GST-fusion proteins as determined by Coomassie blue staining. The bars represent mean ± S.E. from 5 independent experiments. * or # stand for P
Figure Legend Snippet: Effect of point mutations in ATi3N or ATctN on their interactions with CaM. A. Modeled structures of CaM-ATi3(214–231) and CaM-ATct(302–317). The complexes of CaM and CaM binding motif in the i3 loop ATi3(214–231) or the carboxyl terminal tail ATct(302–317) of the receptor were modeled as described in Experimental Procedures. The target peptides are colored in red. Residues W219 in ATi3(214–231)−SYTLIWKALKKAYEIQKN, and F309 and F313 in ATct(302–317)−YGFLGKKFKKYFLQLL are displayed with sticks and are colored in blue. Calcium atoms are shown as orange spheres. The N- and C- termini of CaM are also labeled. Helices and sheets in CaM are colored in green and yellow, respectively. B. Effect of point mutations at ATi3N or ATctN on their interaction with CaM. 50 pmol of wild type GST-fusion proteins including GST-ATi3N(213–234) and GST-ATctN (297–324), and 50 pmol of mutated GST-fusion proteins including GST-ATi3N(W219A), GST-ATctN(F309A) and GST-ATctN(F313A), were incubated with purified bovine brain CaM in a buffer containing 100 mM Tris-HCl (pH 7.5) with 0.1 mM CaCl 2 . The protein complexes were pulled down by gluthathione-sepharose 4B beads, and subjected to immunoblot with a specific anti-CaM antibody. GST-fusion proteins were visualized in the gels by Coomassie blue staining (the lower gel panel). The summary graph represents relative densities of the ratio of the CaM in the immunoblots and the loaded GST-fusion proteins as determined by Coomassie blue staining. The bars represent mean ± S.E. from 5 independent experiments. * or # stand for P

Techniques Used: Chick Chorioallantoic Membrane Assay, Binding Assay, Labeling, Incubation, Purification, Staining, Western Blot

11) Product Images from "CFTR-associated ligand is a negative regulator of Mrp2 expression"

Article Title: CFTR-associated ligand is a negative regulator of Mrp2 expression

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00100.2016

Mrp2 binds to CAL via the COOH-terminal PDZ-binding motif in GST pull-down assays. A : diagram of GST-rat Mrp2 COOH-terminus constructs for GST pull-down assays. a : Full-length rat Mrp2 consists of three membrane-spanning domains (in green) and an intracellular COOH-terminal tail (amino acid 1255–1541, in yellow). b : Construct 1 encodes GST (in orange) and amino acid 1255–1541 of rMrp2, including the COOH-terminal PDZ-binding motif that is composed of 4 amino acids (red asterisks). c : Construct 2 encodes GST and amino acid 1255–1538 of rMrp2 with deletion of the last 3 amino acids. d : Construct 3 encodes GST and amino acid 1255–1534 of rMrp2 with deletion of the last 7 amino acids. B : GST pull-down assays. HEK-293 cells were transfected with Lipofectamine 2000, and lysates of cells transfected with HA-CAL were incubated with GST control or GST-rMrp2 fusion protein encoded by the constructs shown in A . The samples were then supplemented with glutathione Sepharose 4B beads. Both the pull-down complex ( top ) and the unbound fraction ( bottom ) were immunoblotted with anti-HA antibody. Note the presence of HA-CAL in the pull-down complex when the COOH-terminus PDZ-binding motif of Mrp2 was included in the GST-Mrp2 bait, but was not detected in the pull-down complex when the COOH-terminus PDZ-binding motif was deleted in ( Construct 1 vs. Construct 2 and Construct 3 ). The bottom panel confirms the presence of HA-CAL in the unbound fraction of all of the pull-down assays. Data are representative of three independent experiments.
Figure Legend Snippet: Mrp2 binds to CAL via the COOH-terminal PDZ-binding motif in GST pull-down assays. A : diagram of GST-rat Mrp2 COOH-terminus constructs for GST pull-down assays. a : Full-length rat Mrp2 consists of three membrane-spanning domains (in green) and an intracellular COOH-terminal tail (amino acid 1255–1541, in yellow). b : Construct 1 encodes GST (in orange) and amino acid 1255–1541 of rMrp2, including the COOH-terminal PDZ-binding motif that is composed of 4 amino acids (red asterisks). c : Construct 2 encodes GST and amino acid 1255–1538 of rMrp2 with deletion of the last 3 amino acids. d : Construct 3 encodes GST and amino acid 1255–1534 of rMrp2 with deletion of the last 7 amino acids. B : GST pull-down assays. HEK-293 cells were transfected with Lipofectamine 2000, and lysates of cells transfected with HA-CAL were incubated with GST control or GST-rMrp2 fusion protein encoded by the constructs shown in A . The samples were then supplemented with glutathione Sepharose 4B beads. Both the pull-down complex ( top ) and the unbound fraction ( bottom ) were immunoblotted with anti-HA antibody. Note the presence of HA-CAL in the pull-down complex when the COOH-terminus PDZ-binding motif of Mrp2 was included in the GST-Mrp2 bait, but was not detected in the pull-down complex when the COOH-terminus PDZ-binding motif was deleted in ( Construct 1 vs. Construct 2 and Construct 3 ). The bottom panel confirms the presence of HA-CAL in the unbound fraction of all of the pull-down assays. Data are representative of three independent experiments.

Techniques Used: Binding Assay, Construct, Transfection, Incubation

12) Product Images from "Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA *"

Article Title: Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.154831

TDP-43 interacts with PABP2 and FUS/TLS. A , identification of TDP-43 interacting proteins by mass spectrometry. HeLa cells transfected with HA-TDP-43 were immunoprecipitated with α-HA-conjugated agarose, and the immunoprecipitated proteins were separated by SDS-PAGE. The gel was stained with Colloidal Blue. Candidate TDP-43-associated proteins were analyzed by mass spectrometry. B , co-IP of HA-TDP-43 with endogenous FUS/TLS and PABP2. HA-TDP-43 was immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA, α-FUS/TLS, and α-PABP2 antibodies. C , co-IP of HA-FUS/TLS and endogenous TDP-43. HeLa cells transfected with HA-FUS/TLS were immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA and α-TDP-43 antibodies. D , interaction of endogenous TDP-43 and FUS/TLS. Exponentially growing HeLa cells were immunoprecipitated with α-FUS/TLS antibodies, and the immunoprecipitated fraction was analyzed by immunoblotting with α-FUS/TLS and α-TDP-43 antibodies. E , HA-TDP-43 interacts with GST-FUS/TLS in vitro . GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with HEK 293T cell extract containing HA-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HA antibodies. F , interaction of purified proteins. Purified GST and GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with purified HIS-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HIS antibodies.
Figure Legend Snippet: TDP-43 interacts with PABP2 and FUS/TLS. A , identification of TDP-43 interacting proteins by mass spectrometry. HeLa cells transfected with HA-TDP-43 were immunoprecipitated with α-HA-conjugated agarose, and the immunoprecipitated proteins were separated by SDS-PAGE. The gel was stained with Colloidal Blue. Candidate TDP-43-associated proteins were analyzed by mass spectrometry. B , co-IP of HA-TDP-43 with endogenous FUS/TLS and PABP2. HA-TDP-43 was immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA, α-FUS/TLS, and α-PABP2 antibodies. C , co-IP of HA-FUS/TLS and endogenous TDP-43. HeLa cells transfected with HA-FUS/TLS were immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA and α-TDP-43 antibodies. D , interaction of endogenous TDP-43 and FUS/TLS. Exponentially growing HeLa cells were immunoprecipitated with α-FUS/TLS antibodies, and the immunoprecipitated fraction was analyzed by immunoblotting with α-FUS/TLS and α-TDP-43 antibodies. E , HA-TDP-43 interacts with GST-FUS/TLS in vitro . GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with HEK 293T cell extract containing HA-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HA antibodies. F , interaction of purified proteins. Purified GST and GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with purified HIS-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HIS antibodies.

Techniques Used: Mass Spectrometry, Transfection, Immunoprecipitation, SDS Page, Staining, Co-Immunoprecipitation Assay, In Vitro, Incubation, Purification

The Gly-rich and RRM2 domains of TDP-43 contribute to FUS/TLS binding. A , stick diagrams of TDP-43 deletion mutants used in the GST-FUS/TLS pulldown assays. B , GST-FUS/TLS pulldown assay using C-terminal deletion mutants of TDP-43. HA-tagged wild-type TDP-43 or deletion mutants of TDP-43 were expressed in HEK 293T cells, and the cell lysates were incubated with GST or GST-FUS/TLS proteins conjugated to glutathione-Sepharose 4B beads. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with α-GST and α-HA antibodies. C , interaction of FUS/TLS with N-terminal TDP-43 truncation mutants. The indicated TDP-43 N-terminal truncation mutants were expressed in HEK 293T cell and tested for interaction with GST-FUS/TLS in GST pulldown assays. These findings demonstrate that a region spanning amino acids 170–414 of TDP-43 is sufficient for binding to GST-FUS/TLS in vitro. vec , vector; wt , wild type.
Figure Legend Snippet: The Gly-rich and RRM2 domains of TDP-43 contribute to FUS/TLS binding. A , stick diagrams of TDP-43 deletion mutants used in the GST-FUS/TLS pulldown assays. B , GST-FUS/TLS pulldown assay using C-terminal deletion mutants of TDP-43. HA-tagged wild-type TDP-43 or deletion mutants of TDP-43 were expressed in HEK 293T cells, and the cell lysates were incubated with GST or GST-FUS/TLS proteins conjugated to glutathione-Sepharose 4B beads. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with α-GST and α-HA antibodies. C , interaction of FUS/TLS with N-terminal TDP-43 truncation mutants. The indicated TDP-43 N-terminal truncation mutants were expressed in HEK 293T cell and tested for interaction with GST-FUS/TLS in GST pulldown assays. These findings demonstrate that a region spanning amino acids 170–414 of TDP-43 is sufficient for binding to GST-FUS/TLS in vitro. vec , vector; wt , wild type.

Techniques Used: Binding Assay, Incubation, SDS Page, In Vitro, Plasmid Preparation

Wild-type and ALS-associated TDP-43 mutants interact with FUS/TLS comparably. A , co-immunoprecipitation assay. HeLa cells were transfected with HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants. Wild-type and mutant HA-TDP-43 proteins were immunoprecipitated with α-HA. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. B , reciprocal co-immunoprecipitation assay. HeLa cells were transfected with plasmids encoding HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants, and cell extracts were immunoprecipitated with α-FUS/TLS antibodies. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. C , GST-FUS/TLS pulldown of ALS-associated TDP-43 mutants. GST and GST-FUS/TLS were induced in BL 21 cells and purified using glutathione-Sepharose 4B beads. HA-tagged wild-type TDP-43 or ALS-associated mutants of TDP-43 were expressed in HEK 293T cells, and corresponding cell extracts were incubated with GST or GST-FUS/TLS. The affinity-purified proteins were separated by 10% SDS-PAGE and analyzed by Western blotting with α-GST and α-HA antibodies. vec , vector.
Figure Legend Snippet: Wild-type and ALS-associated TDP-43 mutants interact with FUS/TLS comparably. A , co-immunoprecipitation assay. HeLa cells were transfected with HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants. Wild-type and mutant HA-TDP-43 proteins were immunoprecipitated with α-HA. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. B , reciprocal co-immunoprecipitation assay. HeLa cells were transfected with plasmids encoding HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants, and cell extracts were immunoprecipitated with α-FUS/TLS antibodies. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. C , GST-FUS/TLS pulldown of ALS-associated TDP-43 mutants. GST and GST-FUS/TLS were induced in BL 21 cells and purified using glutathione-Sepharose 4B beads. HA-tagged wild-type TDP-43 or ALS-associated mutants of TDP-43 were expressed in HEK 293T cells, and corresponding cell extracts were incubated with GST or GST-FUS/TLS. The affinity-purified proteins were separated by 10% SDS-PAGE and analyzed by Western blotting with α-GST and α-HA antibodies. vec , vector.

Techniques Used: Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Immunoprecipitation, Purification, Incubation, Affinity Purification, SDS Page, Western Blot, Plasmid Preparation

13) Product Images from "Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking"

Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms15022929

Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
Figure Legend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

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

14) Product Images from "Interaction of the Putative Human Cytomegalovirus Portal Protein pUL104 with the Large Terminase Subunit pUL56 and Its Inhibition by Benzimidazole-d-Ribonucleosides"

Article Title: Interaction of the Putative Human Cytomegalovirus Portal Protein pUL104 with the Large Terminase Subunit pUL56 and Its Inhibition by Benzimidazole-d-Ribonucleosides

Journal: Journal of Virology

doi: 10.1128/JVI.79.23.14660-14667.2005

Binding of pUL104 to an immobilized carboxy-terminal portion of pUL56 (aa 404 through 850). Glutathione-Sepharose 4B beads loaded with GST, GST-UL56C or GST-UL89 were incubated with in vitro translated UL104 (in vitro UL104) or luciferase (in vitro luciferase).
Figure Legend Snippet: Binding of pUL104 to an immobilized carboxy-terminal portion of pUL56 (aa 404 through 850). Glutathione-Sepharose 4B beads loaded with GST, GST-UL56C or GST-UL89 were incubated with in vitro translated UL104 (in vitro UL104) or luciferase (in vitro luciferase).

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

15) Product Images from "Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA *"

Article Title: Amyotrophic Lateral Sclerosis-associated Proteins TDP-43 and FUS/TLS Function in a Common Biochemical Complex to Co-regulate HDAC6 mRNA *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.154831

TDP-43 interacts with PABP2 and FUS/TLS. A , identification of TDP-43 interacting proteins by mass spectrometry. HeLa cells transfected with HA-TDP-43 were immunoprecipitated with α-HA-conjugated agarose, and the immunoprecipitated proteins were separated by SDS-PAGE. The gel was stained with Colloidal Blue. Candidate TDP-43-associated proteins were analyzed by mass spectrometry. B , co-IP of HA-TDP-43 with endogenous FUS/TLS and PABP2. HA-TDP-43 was immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA, α-FUS/TLS, and α-PABP2 antibodies. C , co-IP of HA-FUS/TLS and endogenous TDP-43. HeLa cells transfected with HA-FUS/TLS were immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA and α-TDP-43 antibodies. D , interaction of endogenous TDP-43 and FUS/TLS. Exponentially growing HeLa cells were immunoprecipitated with α-FUS/TLS antibodies, and the immunoprecipitated fraction was analyzed by immunoblotting with α-FUS/TLS and α-TDP-43 antibodies. E , HA-TDP-43 interacts with GST-FUS/TLS in vitro . GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with HEK 293T cell extract containing HA-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HA antibodies. F , interaction of purified proteins. Purified GST and GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with purified HIS-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HIS antibodies.
Figure Legend Snippet: TDP-43 interacts with PABP2 and FUS/TLS. A , identification of TDP-43 interacting proteins by mass spectrometry. HeLa cells transfected with HA-TDP-43 were immunoprecipitated with α-HA-conjugated agarose, and the immunoprecipitated proteins were separated by SDS-PAGE. The gel was stained with Colloidal Blue. Candidate TDP-43-associated proteins were analyzed by mass spectrometry. B , co-IP of HA-TDP-43 with endogenous FUS/TLS and PABP2. HA-TDP-43 was immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA, α-FUS/TLS, and α-PABP2 antibodies. C , co-IP of HA-FUS/TLS and endogenous TDP-43. HeLa cells transfected with HA-FUS/TLS were immunoprecipitated with α-HA, and the immunoprecipitated fractions were analyzed by immunoblotting with α-HA and α-TDP-43 antibodies. D , interaction of endogenous TDP-43 and FUS/TLS. Exponentially growing HeLa cells were immunoprecipitated with α-FUS/TLS antibodies, and the immunoprecipitated fraction was analyzed by immunoblotting with α-FUS/TLS and α-TDP-43 antibodies. E , HA-TDP-43 interacts with GST-FUS/TLS in vitro . GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with HEK 293T cell extract containing HA-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HA antibodies. F , interaction of purified proteins. Purified GST and GST-FUS/TLS fusion proteins conjugated to glutathione-Sepharose 4B beads were incubated with purified HIS-TDP-43, and bound proteins were analyzed by immunoblotting with α-GST and α-HIS antibodies.

Techniques Used: Mass Spectrometry, Transfection, Immunoprecipitation, SDS Page, Staining, Co-Immunoprecipitation Assay, In Vitro, Incubation, Purification

The Gly-rich and RRM2 domains of TDP-43 contribute to FUS/TLS binding. A , stick diagrams of TDP-43 deletion mutants used in the GST-FUS/TLS pulldown assays. B , GST-FUS/TLS pulldown assay using C-terminal deletion mutants of TDP-43. HA-tagged wild-type TDP-43 or deletion mutants of TDP-43 were expressed in HEK 293T cells, and the cell lysates were incubated with GST or GST-FUS/TLS proteins conjugated to glutathione-Sepharose 4B beads. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with α-GST and α-HA antibodies. C , interaction of FUS/TLS with N-terminal TDP-43 truncation mutants. The indicated TDP-43 N-terminal truncation mutants were expressed in HEK 293T cell and tested for interaction with GST-FUS/TLS in GST pulldown assays. These findings demonstrate that a region spanning amino acids 170–414 of TDP-43 is sufficient for binding to GST-FUS/TLS in vitro. vec , vector; wt , wild type.
Figure Legend Snippet: The Gly-rich and RRM2 domains of TDP-43 contribute to FUS/TLS binding. A , stick diagrams of TDP-43 deletion mutants used in the GST-FUS/TLS pulldown assays. B , GST-FUS/TLS pulldown assay using C-terminal deletion mutants of TDP-43. HA-tagged wild-type TDP-43 or deletion mutants of TDP-43 were expressed in HEK 293T cells, and the cell lysates were incubated with GST or GST-FUS/TLS proteins conjugated to glutathione-Sepharose 4B beads. Bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with α-GST and α-HA antibodies. C , interaction of FUS/TLS with N-terminal TDP-43 truncation mutants. The indicated TDP-43 N-terminal truncation mutants were expressed in HEK 293T cell and tested for interaction with GST-FUS/TLS in GST pulldown assays. These findings demonstrate that a region spanning amino acids 170–414 of TDP-43 is sufficient for binding to GST-FUS/TLS in vitro. vec , vector; wt , wild type.

Techniques Used: Binding Assay, Incubation, SDS Page, In Vitro, Plasmid Preparation

Wild-type and ALS-associated TDP-43 mutants interact with FUS/TLS comparably. A , co-immunoprecipitation assay. HeLa cells were transfected with HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants. Wild-type and mutant HA-TDP-43 proteins were immunoprecipitated with α-HA. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. B , reciprocal co-immunoprecipitation assay. HeLa cells were transfected with plasmids encoding HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants, and cell extracts were immunoprecipitated with α-FUS/TLS antibodies. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. C , GST-FUS/TLS pulldown of ALS-associated TDP-43 mutants. GST and GST-FUS/TLS were induced in BL 21 cells and purified using glutathione-Sepharose 4B beads. HA-tagged wild-type TDP-43 or ALS-associated mutants of TDP-43 were expressed in HEK 293T cells, and corresponding cell extracts were incubated with GST or GST-FUS/TLS. The affinity-purified proteins were separated by 10% SDS-PAGE and analyzed by Western blotting with α-GST and α-HA antibodies. vec , vector.
Figure Legend Snippet: Wild-type and ALS-associated TDP-43 mutants interact with FUS/TLS comparably. A , co-immunoprecipitation assay. HeLa cells were transfected with HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants. Wild-type and mutant HA-TDP-43 proteins were immunoprecipitated with α-HA. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. B , reciprocal co-immunoprecipitation assay. HeLa cells were transfected with plasmids encoding HA-tagged wild-type TDP-43 or ALS-associated TDP-43 mutants, and cell extracts were immunoprecipitated with α-FUS/TLS antibodies. The IP fractions were analyzed by immunoblotting with α-HA and α-FUS/TLS antibodies. C , GST-FUS/TLS pulldown of ALS-associated TDP-43 mutants. GST and GST-FUS/TLS were induced in BL 21 cells and purified using glutathione-Sepharose 4B beads. HA-tagged wild-type TDP-43 or ALS-associated mutants of TDP-43 were expressed in HEK 293T cells, and corresponding cell extracts were incubated with GST or GST-FUS/TLS. The affinity-purified proteins were separated by 10% SDS-PAGE and analyzed by Western blotting with α-GST and α-HA antibodies. vec , vector.

Techniques Used: Co-Immunoprecipitation Assay, Transfection, Mutagenesis, Immunoprecipitation, Purification, Incubation, Affinity Purification, SDS Page, Western Blot, Plasmid Preparation

16) Product Images from "OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation 1"

Article Title: OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation 1

Journal: Plant Physiology

doi: 10.1104/pp.15.01668

The TPR domain of BSK3 prevents it from interacting with AtBSU1. A, Yeast two-hybrid assays of the interaction between full-length OsBSK3, the kinase domain of OsBSK3 (N390), and the TPR domain of OsBSK3 (TPR). B, Yeast two-hybrid assays of the interaction between full-length AtBSK3, full-length OsBSK3, the kinase domain of AtBSK3 (N334), and N390 with AtBSU1, AtBIN2, and the TPR domain from OsBSK3. C, AtBSU1 showed increased binding with the kinase domains of OsBSK3 and AtBSK3. The recombinant 6His-tagged kinase domain and full-length versions of OsBSK3 (N390 and OsBSK3) or AtBSK3 (N334 and AtBSK3) were dot blotted onto nitrocellulose membranes and then incubated with MBP-AtBSU1 and horseradish peroxidase-labeled anti-MBP antibodies. Total protein was visualized by Ponceau S staining. D, OsBRI1 phosphorylation prevents the TPR and kinase domains of OsBSK3 from binding. GST-TPR on Glutathione Sepharose 4B beads was used to pull down 6His-tagged N390, which had been incubated with MBP-tagged OsBRI1 kinase domain in the presence (pN390) or absence (N390) of ATP. The proteins were blotted onto nitrocellulose membranes and detected using anti-His or -GST antibodies. E, Ser-215 phosphorylation reduced the binding of the kinase and TPR domains of OsBSK3. Shown are quantitative β-glycosidase activity assays of the interaction between the TPR domain and wild type or Ser-215-substituted mutant form of N390. A one-way ANOVA was performed. Statistically significant differences are indicated by different lowercase letters ( P
Figure Legend Snippet: The TPR domain of BSK3 prevents it from interacting with AtBSU1. A, Yeast two-hybrid assays of the interaction between full-length OsBSK3, the kinase domain of OsBSK3 (N390), and the TPR domain of OsBSK3 (TPR). B, Yeast two-hybrid assays of the interaction between full-length AtBSK3, full-length OsBSK3, the kinase domain of AtBSK3 (N334), and N390 with AtBSU1, AtBIN2, and the TPR domain from OsBSK3. C, AtBSU1 showed increased binding with the kinase domains of OsBSK3 and AtBSK3. The recombinant 6His-tagged kinase domain and full-length versions of OsBSK3 (N390 and OsBSK3) or AtBSK3 (N334 and AtBSK3) were dot blotted onto nitrocellulose membranes and then incubated with MBP-AtBSU1 and horseradish peroxidase-labeled anti-MBP antibodies. Total protein was visualized by Ponceau S staining. D, OsBRI1 phosphorylation prevents the TPR and kinase domains of OsBSK3 from binding. GST-TPR on Glutathione Sepharose 4B beads was used to pull down 6His-tagged N390, which had been incubated with MBP-tagged OsBRI1 kinase domain in the presence (pN390) or absence (N390) of ATP. The proteins were blotted onto nitrocellulose membranes and detected using anti-His or -GST antibodies. E, Ser-215 phosphorylation reduced the binding of the kinase and TPR domains of OsBSK3. Shown are quantitative β-glycosidase activity assays of the interaction between the TPR domain and wild type or Ser-215-substituted mutant form of N390. A one-way ANOVA was performed. Statistically significant differences are indicated by different lowercase letters ( P

Techniques Used: Binding Assay, Recombinant, Incubation, Labeling, Staining, Activity Assay, Mutagenesis

17) Product Images from "A Dictyostelium nuclear phosphatidylinositol phosphate kinase required for developmental gene expression"

Article Title: A Dictyostelium nuclear phosphatidylinositol phosphate kinase required for developmental gene expression

Journal: The EMBO Journal

doi: 10.1093/emboj/20.21.6017

Fig. 5. PIP kinase activity of the C-terminal domain of PIPkinA.GST alone or a fusion protein of GST and the C-terminal domain of PIPkinA was purified by elution from glutathione–Sepharose 4B beads, and incubated with PIP in the presence of [γ- 32 P]ATP for 30 min at 22°C. Recombinant human type IIα PIP kinase was used as a control. Lipids were extracted into chloroform/methanol and the generation of radiolabelled PIP 2 was assessed by TLC.
Figure Legend Snippet: Fig. 5. PIP kinase activity of the C-terminal domain of PIPkinA.GST alone or a fusion protein of GST and the C-terminal domain of PIPkinA was purified by elution from glutathione–Sepharose 4B beads, and incubated with PIP in the presence of [γ- 32 P]ATP for 30 min at 22°C. Recombinant human type IIα PIP kinase was used as a control. Lipids were extracted into chloroform/methanol and the generation of radiolabelled PIP 2 was assessed by TLC.

Techniques Used: Activity Assay, Purification, Incubation, Recombinant, Thin Layer Chromatography

18) Product Images from "SASPase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing"

Article Title: SASPase regulates stratum corneum hydration through profilaggrin-to-filaggrin processing

Journal: EMBO Molecular Medicine

doi: 10.1002/emmm.201100140

Recombinant hSASP14 directly cleaves recombinant filaggrin in vitro Production and purification of hSASP14 by autoprocessing of GST-hSASP28. Purified GST-hSASP28 (arrow) was incubated for the indicated times (0′, 0 min; 60′, 60 min) with 700 mM NaCl at pH 6.0. GST-hSASP28 underwent autoprocessing and produced hSASP14 (arrowhead). Cleaved GST-fusion proteins were removed by passing through Glutathione Sepharose 4B beads to purify hSASP14 (arrowhead). Asterisk indicates a dimer of hSASP14. Cleavage of profilaggrin linker peptide by hSASP14 in vitro . The purified MBP-hFilaggrin/MBP-hFilaggrin-ΔC (arrow) was incubated with or without the purified hSASP14 (arrowhead) with 700 mM NaCl at pH 6.0 for 60 min at 37°C. The linker peptide of profilaggrin between MBP and hFilaggrin in MBP-hFilaggrin/MBP-hFilaggrin-ΔC was cleaved by hSASP14, resulting in the production of MBP (42 kDa), hFilaggrin (37 kDa), and hFilaggrin-ΔC (23 kDa). The N-terminal amino acid sequencing of hFilaggrin (37 kDa) and hFilaggrin-ΔC (23 kDa) protein identified QVSTH amino acids, which corresponded to the linker peptide of profilaggrin. Schematic representation of the mode of processing of MBP-hFilaggrin by hSASP14 as described in B. Schematic representation of the possible cleavage site of profilaggrin by hSASP14. Homodimerized hSASP14 proteins were suggested to primarily cleave between GSFLY-QVSTH in the profilaggrin linker sequence (arrow).
Figure Legend Snippet: Recombinant hSASP14 directly cleaves recombinant filaggrin in vitro Production and purification of hSASP14 by autoprocessing of GST-hSASP28. Purified GST-hSASP28 (arrow) was incubated for the indicated times (0′, 0 min; 60′, 60 min) with 700 mM NaCl at pH 6.0. GST-hSASP28 underwent autoprocessing and produced hSASP14 (arrowhead). Cleaved GST-fusion proteins were removed by passing through Glutathione Sepharose 4B beads to purify hSASP14 (arrowhead). Asterisk indicates a dimer of hSASP14. Cleavage of profilaggrin linker peptide by hSASP14 in vitro . The purified MBP-hFilaggrin/MBP-hFilaggrin-ΔC (arrow) was incubated with or without the purified hSASP14 (arrowhead) with 700 mM NaCl at pH 6.0 for 60 min at 37°C. The linker peptide of profilaggrin between MBP and hFilaggrin in MBP-hFilaggrin/MBP-hFilaggrin-ΔC was cleaved by hSASP14, resulting in the production of MBP (42 kDa), hFilaggrin (37 kDa), and hFilaggrin-ΔC (23 kDa). The N-terminal amino acid sequencing of hFilaggrin (37 kDa) and hFilaggrin-ΔC (23 kDa) protein identified QVSTH amino acids, which corresponded to the linker peptide of profilaggrin. Schematic representation of the mode of processing of MBP-hFilaggrin by hSASP14 as described in B. Schematic representation of the possible cleavage site of profilaggrin by hSASP14. Homodimerized hSASP14 proteins were suggested to primarily cleave between GSFLY-QVSTH in the profilaggrin linker sequence (arrow).

Techniques Used: Recombinant, In Vitro, Purification, Incubation, Produced, Sequencing

19) Product Images from "Cloning and Characterization of a 2-Cys Peroxiredoxin from Babesiagibsoni"

Article Title: Cloning and Characterization of a 2-Cys Peroxiredoxin from Babesiagibsoni

Journal: The Journal of Veterinary Medical Science

doi: 10.1292/jvms.13-0274

Amino acid sequence alignment and SDS-PAGE analysis of BgTPx-1 protein. (A) Multiple sequence alignment of B. gibsoni BgTPx-1 protein (deduced sequence) with the sequences of other 2-Cys Prxs of apicomplexan parasites. Sequences are from B. bovis (BbTPx-1; XP_001610019), T. gondii (TgPrx; AAG25678), C. parvum (CpTPx; ACV31867) and P. falciparum (BAA97121). Black boxes with white letters show identical residues, and gray boxes with black letters show chemically similar residues. The dashes indicate gaps introduced between the sequences. Two conserved cysteine residues that correspond to Cys47 and Cys170 of the yeast Prx [ 5 ] are marked with asterisks. (B) Expression of BgTPx-1 protein by using the E. coli. expression system and SDS-PAGE analysis. A recombinant plasmid containing the sequence of BgTPx-1 in pGEX-6P1 was transformed in E.coli strain BL21 (DE3), and the transformed colony was cultured in 1 l of LB broth with ampicillin sodium (100 µ g/m l ) at 37°C. When the optical density at 600 n m reached 0.6, expression of the recombinant fusion protein was induced by adding 1 mM isopropyl thio-β-D-galactoside (IPTG) and incubating for another 5 hr at 24°C. The bacterial cultures were lysed with PBS containing 100 µ g/m l lysozyme and 1.5% Triton X-100 with sonication. The supernatant was subjected to protein purification using Glutathione-Sepharose 4B beads and PreScission protease. An SDS-PAGE image of rBgTPx-1 protein is shown. M, protein marker.
Figure Legend Snippet: Amino acid sequence alignment and SDS-PAGE analysis of BgTPx-1 protein. (A) Multiple sequence alignment of B. gibsoni BgTPx-1 protein (deduced sequence) with the sequences of other 2-Cys Prxs of apicomplexan parasites. Sequences are from B. bovis (BbTPx-1; XP_001610019), T. gondii (TgPrx; AAG25678), C. parvum (CpTPx; ACV31867) and P. falciparum (BAA97121). Black boxes with white letters show identical residues, and gray boxes with black letters show chemically similar residues. The dashes indicate gaps introduced between the sequences. Two conserved cysteine residues that correspond to Cys47 and Cys170 of the yeast Prx [ 5 ] are marked with asterisks. (B) Expression of BgTPx-1 protein by using the E. coli. expression system and SDS-PAGE analysis. A recombinant plasmid containing the sequence of BgTPx-1 in pGEX-6P1 was transformed in E.coli strain BL21 (DE3), and the transformed colony was cultured in 1 l of LB broth with ampicillin sodium (100 µ g/m l ) at 37°C. When the optical density at 600 n m reached 0.6, expression of the recombinant fusion protein was induced by adding 1 mM isopropyl thio-β-D-galactoside (IPTG) and incubating for another 5 hr at 24°C. The bacterial cultures were lysed with PBS containing 100 µ g/m l lysozyme and 1.5% Triton X-100 with sonication. The supernatant was subjected to protein purification using Glutathione-Sepharose 4B beads and PreScission protease. An SDS-PAGE image of rBgTPx-1 protein is shown. M, protein marker.

Techniques Used: Sequencing, SDS Page, Expressing, Recombinant, Plasmid Preparation, Transformation Assay, Cell Culture, Sonication, Protein Purification, Marker

20) Product Images from "Helicase SUV3, Polynucleotide Phosphorylase, and Mitochondrial Polyadenylation Polymerase Form a Transient Complex to Modulate Mitochondrial mRNA Polyadenylated Tail Lengths in Response to Energetic Changes *"

Article Title: Helicase SUV3, Polynucleotide Phosphorylase, and Mitochondrial Polyadenylation Polymerase Form a Transient Complex to Modulate Mitochondrial mRNA Polyadenylated Tail Lengths in Response to Energetic Changes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.536540

SUV3 serves as a bridge for PNPase and mtPAP binding. A, in vitro binding assay. GST-mtPAP full-length ( FL ) or GST proteins were bound to glutathione-Sepharose 4B beads. Group C was preincubated with increasing amounts of His-SUV3 for 1 h. Subsequently, Groups A, B, and C were incubated with increasing amounts of His-PNPase, whereas Group D was incubated with increasing amounts of preformed SUV3·PNPase complex for 1 h. Post washing, the bound proteins were resolved by SDS-PAGE and visualized by immunoblotting. B , in vitro binding assay. Increasing amounts of un-tagged SUV3 was preincubated with His-PNPase on Ni + resin for 1 h. Subsequently, equal amounts of GST-mtPAP were added to the mixture and further incubated for 1 h. Post washing, the bound proteins were resolved by SDS-PAGE and visualized by immunoblotting. C, size exclusion chromatography (Superdex 200) elution profiles of the purified recombinant SUV3·PNPase·mtPAP complex and the individual proteins. The molecular masses ( M.M .) of the individual proteins were determined by analytical ultracentrifuge. The molecular mass of the SUV3·PNPase·mtPAP complex was approximated from its peak elution volume. D, SDS-PAGE followed by silver staining of the complex. E, schematic representation of the SUV3·PNPase·mtPAP complex.
Figure Legend Snippet: SUV3 serves as a bridge for PNPase and mtPAP binding. A, in vitro binding assay. GST-mtPAP full-length ( FL ) or GST proteins were bound to glutathione-Sepharose 4B beads. Group C was preincubated with increasing amounts of His-SUV3 for 1 h. Subsequently, Groups A, B, and C were incubated with increasing amounts of His-PNPase, whereas Group D was incubated with increasing amounts of preformed SUV3·PNPase complex for 1 h. Post washing, the bound proteins were resolved by SDS-PAGE and visualized by immunoblotting. B , in vitro binding assay. Increasing amounts of un-tagged SUV3 was preincubated with His-PNPase on Ni + resin for 1 h. Subsequently, equal amounts of GST-mtPAP were added to the mixture and further incubated for 1 h. Post washing, the bound proteins were resolved by SDS-PAGE and visualized by immunoblotting. C, size exclusion chromatography (Superdex 200) elution profiles of the purified recombinant SUV3·PNPase·mtPAP complex and the individual proteins. The molecular masses ( M.M .) of the individual proteins were determined by analytical ultracentrifuge. The molecular mass of the SUV3·PNPase·mtPAP complex was approximated from its peak elution volume. D, SDS-PAGE followed by silver staining of the complex. E, schematic representation of the SUV3·PNPase·mtPAP complex.

Techniques Used: Binding Assay, In Vitro, Incubation, SDS Page, Size-exclusion Chromatography, Purification, Recombinant, Silver Staining

N-terminal region of SUV3 binds to the C-terminal region of mtPAP. A and C, schematics of SUV3 constructs used in the in vitro binding assays. B and D, in vitro binding assay. Top panels, immunoblotting of His-tagged mtPAP remained bound to GST-SUV3 fusion proteins on glutathione-Sepharose 4B beads after binding and washing. Bottom panels , protein quantifications of GST-SUV3 fusion proteins by Coomassie Blue staining. *, desired protein species. E and G, schematics of mtPAP constructs used for the in vitro binding assays. F and H, in vitro binding assay. Top panels : immunoblotting of His-tagged SUV3 remained bound to GST-mtPAP fusion proteins on-Sepharose 4B beads after binding and washing. Bottom panels , protein quantifications of GST-mtPAP fusion proteins by Coomassie Blue staining. FL, full-length.
Figure Legend Snippet: N-terminal region of SUV3 binds to the C-terminal region of mtPAP. A and C, schematics of SUV3 constructs used in the in vitro binding assays. B and D, in vitro binding assay. Top panels, immunoblotting of His-tagged mtPAP remained bound to GST-SUV3 fusion proteins on glutathione-Sepharose 4B beads after binding and washing. Bottom panels , protein quantifications of GST-SUV3 fusion proteins by Coomassie Blue staining. *, desired protein species. E and G, schematics of mtPAP constructs used for the in vitro binding assays. F and H, in vitro binding assay. Top panels : immunoblotting of His-tagged SUV3 remained bound to GST-mtPAP fusion proteins on-Sepharose 4B beads after binding and washing. Bottom panels , protein quantifications of GST-mtPAP fusion proteins by Coomassie Blue staining. FL, full-length.

Techniques Used: Construct, In Vitro, Binding Assay, Staining

21) Product Images from "Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq"

Article Title: Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq

Journal: Cell Research

doi: 10.1038/cr.2017.112

Osr1 interacts directly with H2A.Z in vivo and in vitro . (A) Osr1 co-immunoprecipitates with H2A.Z but not H2A from cell extracts. Whole-cell extracts were prepared from NIH3T3 cells co-transfected with Osr1-FLAG-GFPC or Tinf2-FLAG-GFPC and GFPN-HA-H2A or GFPN-HA-H2A.Z for 48 h, followed by immunoprecipitation using anti-FLAG or anti-HA antibodies. The immunoprecipitates were washed with buffers containing 300 or 500 mM NaCl and detected by western blotting with anti-HA antibody. (B) Cartoons showing the full length and truncated H2A.Z proteins expressed in bacteria as MBP fusion proteins, which were purified using Amylose beads. The helices and M6 acid patch domains are indicated. The full length and different truncated Osr1 proteins were expressed in bacteria as GST fusion proteins and purified using glutathione-Sepharose 4B beads. (C) GST or GST-Osr1 fusion proteins bound to beads were incubated with different MBP-H2A.Z fusion proteins. The H2A.Z proteins captured by the beads were detected by western blotting using anti-HA antibody. (D) MBP or MBP-H2A.Z fusion proteins bound to beads were incubated with different GST-Osr1 fusion proteins. The Osr1 proteins captured by the beads were detected by western blotting using anti-FLAG antibody.
Figure Legend Snippet: Osr1 interacts directly with H2A.Z in vivo and in vitro . (A) Osr1 co-immunoprecipitates with H2A.Z but not H2A from cell extracts. Whole-cell extracts were prepared from NIH3T3 cells co-transfected with Osr1-FLAG-GFPC or Tinf2-FLAG-GFPC and GFPN-HA-H2A or GFPN-HA-H2A.Z for 48 h, followed by immunoprecipitation using anti-FLAG or anti-HA antibodies. The immunoprecipitates were washed with buffers containing 300 or 500 mM NaCl and detected by western blotting with anti-HA antibody. (B) Cartoons showing the full length and truncated H2A.Z proteins expressed in bacteria as MBP fusion proteins, which were purified using Amylose beads. The helices and M6 acid patch domains are indicated. The full length and different truncated Osr1 proteins were expressed in bacteria as GST fusion proteins and purified using glutathione-Sepharose 4B beads. (C) GST or GST-Osr1 fusion proteins bound to beads were incubated with different MBP-H2A.Z fusion proteins. The H2A.Z proteins captured by the beads were detected by western blotting using anti-HA antibody. (D) MBP or MBP-H2A.Z fusion proteins bound to beads were incubated with different GST-Osr1 fusion proteins. The Osr1 proteins captured by the beads were detected by western blotting using anti-FLAG antibody.

Techniques Used: In Vivo, In Vitro, Transfection, Immunoprecipitation, Western Blot, Purification, Incubation

22) Product Images from "Physical and functional interaction between DDB and XPA in nucleotide excision repair"

Article Title: Physical and functional interaction between DDB and XPA in nucleotide excision repair

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn964

DDB directly binds to XPA through DDB2 subunit. Purified DDB heterodimer ( A ) or each DDB subunit ( B ) was incubated with GST alone (lane 2) or GST-XPA (lane 3) coupled to glutathione–sepharose 4B beads. The bound proteins were separated on a SDS–polyacrylamide gel and analyzed by western blotting with anti-Flag antibody for detecting DDB1 and DDB2. ( C ) Purified (His) 6 -XPA protein was incubated with MBP alone (lane 2) or MBP-DDB2 (lane 3) coupled to amylose beads. The bound proteins were analyzed by western blotting with anti-His antibody.
Figure Legend Snippet: DDB directly binds to XPA through DDB2 subunit. Purified DDB heterodimer ( A ) or each DDB subunit ( B ) was incubated with GST alone (lane 2) or GST-XPA (lane 3) coupled to glutathione–sepharose 4B beads. The bound proteins were separated on a SDS–polyacrylamide gel and analyzed by western blotting with anti-Flag antibody for detecting DDB1 and DDB2. ( C ) Purified (His) 6 -XPA protein was incubated with MBP alone (lane 2) or MBP-DDB2 (lane 3) coupled to amylose beads. The bound proteins were analyzed by western blotting with anti-His antibody.

Techniques Used: Purification, Incubation, Western Blot

R207G mutation reduces XPA binding to DDB. ( A ) Purified DDB heterodimer or in vitro translated ERCC1 was incubated with GST alone (lane 2), GST-XPA (lane 3) or GST-XPA(R207G) (lane 4) coupled to glutathione–sepharose 4B beads. The bound proteins were analyzed by western blotting with either anti-Flag followed by anti-mouse IgG conjugated with alkaline phosphatase (for DDB1 and DDB2) or streptavidin conjugated with alkaline phosphatase (for ERCC1). ( B) and ( C ) myc-tagged XPA protein, wild-type or R207G mutant, was expressed in Tet-on U2OS/3xF-DDB2 cells in the presence of doxycycline and cell lysates were prepared after 40-h incubation. One or 0.3 mg of the lysates were incubated for 1.5 h with anti-FLAG M2 agarose (B) or anti-myc antibody followed by protein A/G plus agarose (C), respectively. Proteins retained on the beads were analyzed by western blotting using anti-Flag and anti-myc antibodies.
Figure Legend Snippet: R207G mutation reduces XPA binding to DDB. ( A ) Purified DDB heterodimer or in vitro translated ERCC1 was incubated with GST alone (lane 2), GST-XPA (lane 3) or GST-XPA(R207G) (lane 4) coupled to glutathione–sepharose 4B beads. The bound proteins were analyzed by western blotting with either anti-Flag followed by anti-mouse IgG conjugated with alkaline phosphatase (for DDB1 and DDB2) or streptavidin conjugated with alkaline phosphatase (for ERCC1). ( B) and ( C ) myc-tagged XPA protein, wild-type or R207G mutant, was expressed in Tet-on U2OS/3xF-DDB2 cells in the presence of doxycycline and cell lysates were prepared after 40-h incubation. One or 0.3 mg of the lysates were incubated for 1.5 h with anti-FLAG M2 agarose (B) or anti-myc antibody followed by protein A/G plus agarose (C), respectively. Proteins retained on the beads were analyzed by western blotting using anti-Flag and anti-myc antibodies.

Techniques Used: Mutagenesis, Binding Assay, Purification, In Vitro, Incubation, Western Blot

Domain mapping of XPA responsible for the binding to DDB2. ( A ) Schematic diagram of various XPA deletion mutants and summary of pull-down experiments with these mutants. ( B ) The lysates from insect cells overproducing DDB2 were incubated with GST alone (lane 1) or various GST-XPA derivatives (lanes 2–7) coupled to glutathione–sepharose 4B beads. The bound proteins were analyzed by western blotting with anti-Flag antibody.
Figure Legend Snippet: Domain mapping of XPA responsible for the binding to DDB2. ( A ) Schematic diagram of various XPA deletion mutants and summary of pull-down experiments with these mutants. ( B ) The lysates from insect cells overproducing DDB2 were incubated with GST alone (lane 1) or various GST-XPA derivatives (lanes 2–7) coupled to glutathione–sepharose 4B beads. The bound proteins were analyzed by western blotting with anti-Flag antibody.

Techniques Used: Binding Assay, Incubation, Western Blot

23) Product Images from "AP2/ERF Family Transcription Factors ORA59 and RAP2.3 Interact in the Nucleus and Function Together in Ethylene Responses"

Article Title: AP2/ERF Family Transcription Factors ORA59 and RAP2.3 Interact in the Nucleus and Function Together in Ethylene Responses

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.01675

Physical interaction of ORA59 with RAP2.3. (A) Yeast two-hybrid assay. ORA59 with N-terminal 60 amino acids deleted (ORA59Δ1-60) and full-length RAP2.3 were fused with GAL4 AD and BD, respectively. Their interactions were tested on selective media SD/-AHLT and in the presence of X-α-Gal. (B) In vitro GST pull-down assay. GST or ORA59-GST was incubated with RAP2.3-His and precipitated with glutathione sepharose 4B beads. Proteins were detected by immunoblotting with anti-GST and anti-His antibodies. Input shows 1% of the amount used in binding reactions. WB, western blotting. (C) BiFC assay. YFP NE , YFP CE , and their fusion proteins bZIP63 NE , bZIP63 CE , ORA59 NE , and RAP2.3 CE were expressed in Arabidopsis protoplasts as indicated. YFP fluorescence signals were visualized under a confocal microscope. Bars, 10 μm. Experiments were repeated three times with similar results.
Figure Legend Snippet: Physical interaction of ORA59 with RAP2.3. (A) Yeast two-hybrid assay. ORA59 with N-terminal 60 amino acids deleted (ORA59Δ1-60) and full-length RAP2.3 were fused with GAL4 AD and BD, respectively. Their interactions were tested on selective media SD/-AHLT and in the presence of X-α-Gal. (B) In vitro GST pull-down assay. GST or ORA59-GST was incubated with RAP2.3-His and precipitated with glutathione sepharose 4B beads. Proteins were detected by immunoblotting with anti-GST and anti-His antibodies. Input shows 1% of the amount used in binding reactions. WB, western blotting. (C) BiFC assay. YFP NE , YFP CE , and their fusion proteins bZIP63 NE , bZIP63 CE , ORA59 NE , and RAP2.3 CE were expressed in Arabidopsis protoplasts as indicated. YFP fluorescence signals were visualized under a confocal microscope. Bars, 10 μm. Experiments were repeated three times with similar results.

Techniques Used: Y2H Assay, In Vitro, Pull Down Assay, Incubation, Binding Assay, Western Blot, Bimolecular Fluorescence Complementation Assay, Fluorescence, Microscopy

24) Product Images from "Cloning of a Truncated Babesia equi Gene Encoding an 82-Kilodalton Protein and Its Potential Use in an Enzyme-Linked Immunosorbent Assay"

Article Title: Cloning of a Truncated Babesia equi Gene Encoding an 82-Kilodalton Protein and Its Potential Use in an Enzyme-Linked Immunosorbent Assay

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.40.4.1470-1474.2002

Purification of GST-Be82 with glutathione-Sepharose 4B beads. Shown are results with whole GST-Be82 (lane a), the supernatant of GST-Be82 (lane b), and purified GST-Be82 and GST proteins (lanes c and d, respectively).
Figure Legend Snippet: Purification of GST-Be82 with glutathione-Sepharose 4B beads. Shown are results with whole GST-Be82 (lane a), the supernatant of GST-Be82 (lane b), and purified GST-Be82 and GST proteins (lanes c and d, respectively).

Techniques Used: Purification

25) Product Images from "Molecular mechanism by which acyclic retinoid induces nuclear localization of transglutaminase 2 in human hepatocellular carcinoma cells"

Article Title: Molecular mechanism by which acyclic retinoid induces nuclear localization of transglutaminase 2 in human hepatocellular carcinoma cells

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.339

Co-factor(s) for nuclear import of TG2. Recombinant human TG2 (about 1.5 pmol) was incubated for 1 h at room temperature with glutathione sepharose 4B beads conjugated with six times molar excess of ( a ) GST (lane 1), GST-tagged importin- β (lane 2), - α 5 (lane 3), - α 1 (lane 4) and - α 3 (lane 7) or with GST-tagged importin- α 5, α 1, α 3 in presence of HA-tagged importin- β in (lane 4, 6 and 8 respectively). ( b ) GST (lane 1) or GST-importins- α 3/HA-importin- β complex (lane 2) or in presence of peptide SV40 NLS, TG2 NES and TG2 NLS (lane 3, 4 and 5, respectively) ( c ) GST (lane 1) or GST-tagged exportin-1 (Exp-1) in the presence or absence of 0.1% EtOH, 1 mM ACR and 10 μ g/ml LMB as indicated. After spin-down, proteins were eluted with SDS-PAGE sample buffer and TG2 level in each pull down obtained under each condition were determined by western blotting using an antibody against TG2. A representative image, showing intensity value of each blot relative to lane 2 is presented as mean for three independent experiments ( a ) or as mean for two independent experiments ( b and c ). Specificity in the immunoprecipitation experiment of the TG2-exportin-1 complex was ensured in the control experiment ( Supplementary Figure 7 ). ( d ) JHH-7 cells were seeded at 1 × 10 3 cells per well in 96-well plate and incubated at 37 °C overnight. PLA was performed according to the manufacturer's instruction. In control (row 1), the cells were treated with media for 3 h and cells were fixed, permeabilized and no antibodies were used against TG2 and importins for PLA assay. While in others, cells were treated with 0.1% EtOH for 3 h, fixed, permeabilized and treated with combinations of mouse anti-TG2 (CUB7402) and rabbit anti-KPNA4 (left panel, row 2), mouse anti-TG2 (CUB7402) and rabbit anti-SRP-1 (central panel, row 2) or mouse anti-TG2 (CUB7402) and rabbit anti-importin- β (right panel, row 2). After amplification and staining with H33258, the cells were observed under a confocal microscope. Red fluorescence dots derived from amplification of detected protein interaction was monitored with blue fluorescence from H33258. A scale bar=20 μ m. A representative image from three independent experiments with similar results is presented
Figure Legend Snippet: Co-factor(s) for nuclear import of TG2. Recombinant human TG2 (about 1.5 pmol) was incubated for 1 h at room temperature with glutathione sepharose 4B beads conjugated with six times molar excess of ( a ) GST (lane 1), GST-tagged importin- β (lane 2), - α 5 (lane 3), - α 1 (lane 4) and - α 3 (lane 7) or with GST-tagged importin- α 5, α 1, α 3 in presence of HA-tagged importin- β in (lane 4, 6 and 8 respectively). ( b ) GST (lane 1) or GST-importins- α 3/HA-importin- β complex (lane 2) or in presence of peptide SV40 NLS, TG2 NES and TG2 NLS (lane 3, 4 and 5, respectively) ( c ) GST (lane 1) or GST-tagged exportin-1 (Exp-1) in the presence or absence of 0.1% EtOH, 1 mM ACR and 10 μ g/ml LMB as indicated. After spin-down, proteins were eluted with SDS-PAGE sample buffer and TG2 level in each pull down obtained under each condition were determined by western blotting using an antibody against TG2. A representative image, showing intensity value of each blot relative to lane 2 is presented as mean for three independent experiments ( a ) or as mean for two independent experiments ( b and c ). Specificity in the immunoprecipitation experiment of the TG2-exportin-1 complex was ensured in the control experiment ( Supplementary Figure 7 ). ( d ) JHH-7 cells were seeded at 1 × 10 3 cells per well in 96-well plate and incubated at 37 °C overnight. PLA was performed according to the manufacturer's instruction. In control (row 1), the cells were treated with media for 3 h and cells were fixed, permeabilized and no antibodies were used against TG2 and importins for PLA assay. While in others, cells were treated with 0.1% EtOH for 3 h, fixed, permeabilized and treated with combinations of mouse anti-TG2 (CUB7402) and rabbit anti-KPNA4 (left panel, row 2), mouse anti-TG2 (CUB7402) and rabbit anti-SRP-1 (central panel, row 2) or mouse anti-TG2 (CUB7402) and rabbit anti-importin- β (right panel, row 2). After amplification and staining with H33258, the cells were observed under a confocal microscope. Red fluorescence dots derived from amplification of detected protein interaction was monitored with blue fluorescence from H33258. A scale bar=20 μ m. A representative image from three independent experiments with similar results is presented

Techniques Used: Recombinant, Incubation, SDS Page, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Amplification, Staining, Microscopy, Fluorescence, Derivative Assay

Effect of ACR in trimeric complex formation. ( a ) Recombinant human TG2 (1.5 pmol) was incubated for 1 h at room temperature with glutathione sepharose 4B beads conjugated with six times molar excess of GST-importins- α 3/HA-tagged importin- β complex in the presence or absence of ATP, EtOH or ACR as indicated. After spin-down, proteins were eluted with SDS-PAGE sample buffer and TG2 levels in each co-precipitate obtained under each condition were determined by western blotting using an antibody against TG2. A representative result from two independent experiments with similar results is presented. ( b ) JHH-7 cells were seeded at 1 × 10 6 cells per 10-cm dish overnight. The cells were then treated with 0.1% EtOH (column 1 and 3) or 10 μ M ACR (column 2 and 4) for next 5 h. The cells were lysed using Tris buffer (pH 7.4) with 1% Triton X-100, 0.1 mg/ml PMSF and the protease inhibitor cocktail. Importin- β (row 1) and importin- α 3 (row 2) were co-immunoprecipitated using TG2 antibody (CUB7402) from samples containing equal amount of total protein determined by bicinchoninic acid (BCA) protein assay method. After precipitation, proteins were eluted with SDS-PAGE sample buffer and levels of importin- β and - α 3 in each co-precipitation obtained under each condition were determined by western blotting using an antibody indicated. ( c ) JHH-7 cells were seeded at 1 × 10 3 cells per well in 96-well plate and incubated at 37 °C overnight. The cells were then treated with 0.1% EtOH (columns 1–3) or 10 μ M ACR (columns 4–6) for the next 3, 5 and 7 h (rows 1–3). The cells were then fixed and PLA was performed as per manufacturer's instruction. The cells were treated with mouse anti-TG2 (CUB 7402) and rabbit anti-importin- β . After amplification and staining with H33258, the cells were observed under a confocal microscope. Red fluorescence dots derived from amplification of detected protein interaction were monitored with blue fluorescence from H33258. A scale bar, 20 μ m. A representative image from two independent experiments is presented. White arrow heads signify the nuclear TG2-importin β complex.
Figure Legend Snippet: Effect of ACR in trimeric complex formation. ( a ) Recombinant human TG2 (1.5 pmol) was incubated for 1 h at room temperature with glutathione sepharose 4B beads conjugated with six times molar excess of GST-importins- α 3/HA-tagged importin- β complex in the presence or absence of ATP, EtOH or ACR as indicated. After spin-down, proteins were eluted with SDS-PAGE sample buffer and TG2 levels in each co-precipitate obtained under each condition were determined by western blotting using an antibody against TG2. A representative result from two independent experiments with similar results is presented. ( b ) JHH-7 cells were seeded at 1 × 10 6 cells per 10-cm dish overnight. The cells were then treated with 0.1% EtOH (column 1 and 3) or 10 μ M ACR (column 2 and 4) for next 5 h. The cells were lysed using Tris buffer (pH 7.4) with 1% Triton X-100, 0.1 mg/ml PMSF and the protease inhibitor cocktail. Importin- β (row 1) and importin- α 3 (row 2) were co-immunoprecipitated using TG2 antibody (CUB7402) from samples containing equal amount of total protein determined by bicinchoninic acid (BCA) protein assay method. After precipitation, proteins were eluted with SDS-PAGE sample buffer and levels of importin- β and - α 3 in each co-precipitation obtained under each condition were determined by western blotting using an antibody indicated. ( c ) JHH-7 cells were seeded at 1 × 10 3 cells per well in 96-well plate and incubated at 37 °C overnight. The cells were then treated with 0.1% EtOH (columns 1–3) or 10 μ M ACR (columns 4–6) for the next 3, 5 and 7 h (rows 1–3). The cells were then fixed and PLA was performed as per manufacturer's instruction. The cells were treated with mouse anti-TG2 (CUB 7402) and rabbit anti-importin- β . After amplification and staining with H33258, the cells were observed under a confocal microscope. Red fluorescence dots derived from amplification of detected protein interaction were monitored with blue fluorescence from H33258. A scale bar, 20 μ m. A representative image from two independent experiments is presented. White arrow heads signify the nuclear TG2-importin β complex.

Techniques Used: Recombinant, Incubation, SDS Page, Western Blot, Protease Inhibitor, Immunoprecipitation, BIA-KA, Proximity Ligation Assay, Amplification, Staining, Microscopy, Fluorescence, Derivative Assay

26) Product Images from "The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment"

Article Title: The genome-linked protein VPg of the Norwalk virus binds eIF3, suggesting its role in translation initiation complex recruitment

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg251

Fig. 3. VPg interactions with eIF4GI and other initiation factors. Pull-down assays were conducted with GST, GST–VPg or GST–VPg mutant constructs immobilized on glutathione–Sepharose 4B beads and incubated with CaCo-2 cell extracts. Western blots were probed with ( A ) anti-eIF4GI, or ( B ) anti-eIF2α, anti-eIF4E, anti-S6 or anti-eIF4B.
Figure Legend Snippet: Fig. 3. VPg interactions with eIF4GI and other initiation factors. Pull-down assays were conducted with GST, GST–VPg or GST–VPg mutant constructs immobilized on glutathione–Sepharose 4B beads and incubated with CaCo-2 cell extracts. Western blots were probed with ( A ) anti-eIF4GI, or ( B ) anti-eIF2α, anti-eIF4E, anti-S6 or anti-eIF4B.

Techniques Used: Mutagenesis, Construct, Incubation, Western Blot

Fig. 2. VPg binds eIF3 present in cell lysates. Pull-down assays were conducted with GST, GST–VPg or GST–VPg mutant constructs immobilized on glutathione–Sepharose 4B beads and incubated with CaCo-2 cell extracts. ( A ) CaCo-2 cell lysates. ( B ) S7-treated CaCo-2 lysates. ( C ) GST–SMV-VPg immobilized to glutathione–Sepharose beads. ( D ) VPg deletion mutants. Western blots were probed with anti-eIF3 polyclonal antibody. Asterisks indicate a protein likely to be eIF4GI.
Figure Legend Snippet: Fig. 2. VPg binds eIF3 present in cell lysates. Pull-down assays were conducted with GST, GST–VPg or GST–VPg mutant constructs immobilized on glutathione–Sepharose 4B beads and incubated with CaCo-2 cell extracts. ( A ) CaCo-2 cell lysates. ( B ) S7-treated CaCo-2 lysates. ( C ) GST–SMV-VPg immobilized to glutathione–Sepharose beads. ( D ) VPg deletion mutants. Western blots were probed with anti-eIF3 polyclonal antibody. Asterisks indicate a protein likely to be eIF4GI.

Techniques Used: Mutagenesis, Construct, Incubation, Western Blot

Fig. 1. VPg binds initiation factor eIF3. Pull-down assays were conducted with GST or GST–VPg immobilized on glutathione–Sepharose 4B beads. ( A ) Pull-down with purified eIF3. The input eIF3 lane represents 10% of the amount of protein used in the pull-down reaction, and the western blot was probed with anti-eIF3 polyclonal antibody. ( B ) Pull-down with 35 S-labeled in vitro translated subunit eIF3d. The input eIF3d lane represents 25% of the amount used in the GST and GST–VPg pull-downs. Pull-down eluates were separated by 10% SDS–PAGE and exposed to film for autoradiography.
Figure Legend Snippet: Fig. 1. VPg binds initiation factor eIF3. Pull-down assays were conducted with GST or GST–VPg immobilized on glutathione–Sepharose 4B beads. ( A ) Pull-down with purified eIF3. The input eIF3 lane represents 10% of the amount of protein used in the pull-down reaction, and the western blot was probed with anti-eIF3 polyclonal antibody. ( B ) Pull-down with 35 S-labeled in vitro translated subunit eIF3d. The input eIF3d lane represents 25% of the amount used in the GST and GST–VPg pull-downs. Pull-down eluates were separated by 10% SDS–PAGE and exposed to film for autoradiography.

Techniques Used: Purification, Western Blot, Labeling, In Vitro, SDS Page, Autoradiography

27) Product Images from "Fructose Uptake in Bifidobacterium longum NCC2705 Is Mediated by an ATP-binding Cassette Transporter *"

Article Title: Fructose Uptake in Bifidobacterium longum NCC2705 Is Mediated by an ATP-binding Cassette Transporter *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.266213

SDS-PAGE and GST pulldown assays to analyze protein-protein interactions of the FruEKFG ABC transporter subunits. A , Coomassie-stained SDS-PAGE of crude extracts ( lanes 1 , 3 , 5 , 7 , and 9 ; 30 μg of protein were loaded per sample) and purified proteins ( lanes 2 , 4 , 6 , 8 , and 10 ; 5–10 μg of purified protein was loaded per sample) of IPTG-induced E. coli BL21(DE3) containing pET32a-FruE ( lanes 1 and 2 ), pET32a-FruK ( lanes 3 and 4 ), pGEX-4T-1-FruF ( lanes 5 and 6 ), pGEX-4T-1-FruG ( lanes 7 and 8 ), and pGEX-4T-1-FruE ( lanes 9 and 10 ). Lane M , molecular weight marker. The proteins were purified by Ni + affinity column (His-FruE and His-FruK) or GST beads (GST-FruF, GST-FruG, and GST-FruE). B , GST pulldown assays probing interactions between FruE or FruK with the membrane permeases FruF and FruG. For pulldown, 25 μg of GST fusion protein was incubated with 5 μl of glutathione-Sepharose 4B beads for 2 h in PBS at 4 °C. Then 200 μl of lysate containing a total of 25 μg of protein of an E. coli BL21 strain expressing the respective His 6 -tagged protein were added, and the bound proteins were precipitated by centrifugation. Lane 1 , GST + His-FruE (negative control); lane 2 , GST-FruF + His-FruE; lane 3 , His-FruE (positive control); lane 4 , GST + His-FruK (negative control); lane 5 , GST-FruF + His-FruK; lane 6 , His-FruK (positive control); lane 7 , GST + His-FruE (negative control); lane 8 , GST-FruG + His-FruE; lane 9 , His-FruE (positive control); lane 10 , GST + His-FruK (negative control); lane 11 , GST-FruF + His-FruK; Lane 12 , His-FruK (positive control). IB , immunoblot.
Figure Legend Snippet: SDS-PAGE and GST pulldown assays to analyze protein-protein interactions of the FruEKFG ABC transporter subunits. A , Coomassie-stained SDS-PAGE of crude extracts ( lanes 1 , 3 , 5 , 7 , and 9 ; 30 μg of protein were loaded per sample) and purified proteins ( lanes 2 , 4 , 6 , 8 , and 10 ; 5–10 μg of purified protein was loaded per sample) of IPTG-induced E. coli BL21(DE3) containing pET32a-FruE ( lanes 1 and 2 ), pET32a-FruK ( lanes 3 and 4 ), pGEX-4T-1-FruF ( lanes 5 and 6 ), pGEX-4T-1-FruG ( lanes 7 and 8 ), and pGEX-4T-1-FruE ( lanes 9 and 10 ). Lane M , molecular weight marker. The proteins were purified by Ni + affinity column (His-FruE and His-FruK) or GST beads (GST-FruF, GST-FruG, and GST-FruE). B , GST pulldown assays probing interactions between FruE or FruK with the membrane permeases FruF and FruG. For pulldown, 25 μg of GST fusion protein was incubated with 5 μl of glutathione-Sepharose 4B beads for 2 h in PBS at 4 °C. Then 200 μl of lysate containing a total of 25 μg of protein of an E. coli BL21 strain expressing the respective His 6 -tagged protein were added, and the bound proteins were precipitated by centrifugation. Lane 1 , GST + His-FruE (negative control); lane 2 , GST-FruF + His-FruE; lane 3 , His-FruE (positive control); lane 4 , GST + His-FruK (negative control); lane 5 , GST-FruF + His-FruK; lane 6 , His-FruK (positive control); lane 7 , GST + His-FruE (negative control); lane 8 , GST-FruG + His-FruE; lane 9 , His-FruE (positive control); lane 10 , GST + His-FruK (negative control); lane 11 , GST-FruF + His-FruK; Lane 12 , His-FruK (positive control). IB , immunoblot.

Techniques Used: SDS Page, Staining, Purification, Molecular Weight, Marker, Affinity Column, Incubation, Expressing, Centrifugation, Negative Control, Positive Control

28) Product Images from "Inhibition of CRM1-mediated Nuclear Export of Transcription Factors by Leukemogenic NUP98 Fusion Proteins *"

Article Title: Inhibition of CRM1-mediated Nuclear Export of Transcription Factors by Leukemogenic NUP98 Fusion Proteins *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.048785

NUP98-HOXA9 binds CRM1 through the FG motif in a Ran-GTP-dependent manner. A , schematic of NUP98-HOXA9 and its variants used in the binding assays. GLEBS , Gle2p-binding motif; HD , homeodomain. B , 35 S-labeled NUP98-HOXA9 and its variants were incubated with GST ( Control ) or GST-CRM1 ( CRM1 . C , recombinant GFP-NUP98-HOXA9 protein was incubated with GST ( Control ) or GST-CRM1 ( CRM1 ) immobilized on glutathione-Sepharose 4B beads in the presence of RanGTP. Approximately 0.67% of the total bound material and 0.13% of total unbound material for each reaction were analyzed by immunoblotting against GFP. D , 35 .
Figure Legend Snippet: NUP98-HOXA9 binds CRM1 through the FG motif in a Ran-GTP-dependent manner. A , schematic of NUP98-HOXA9 and its variants used in the binding assays. GLEBS , Gle2p-binding motif; HD , homeodomain. B , 35 S-labeled NUP98-HOXA9 and its variants were incubated with GST ( Control ) or GST-CRM1 ( CRM1 . C , recombinant GFP-NUP98-HOXA9 protein was incubated with GST ( Control ) or GST-CRM1 ( CRM1 ) immobilized on glutathione-Sepharose 4B beads in the presence of RanGTP. Approximately 0.67% of the total bound material and 0.13% of total unbound material for each reaction were analyzed by immunoblotting against GFP. D , 35 .

Techniques Used: Binding Assay, Labeling, Incubation, Recombinant

29) Product Images from "Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking"

Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms15022929

Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
Figure Legend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

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

30) Product Images from "The Small C-terminal Domain Phosphatase 1 Inhibits Cancer Cell Migration and Invasion by Dephosphorylating Ser(P)68-Twist1 to Accelerate Twist1 Protein Degradation *"

Article Title: The Small C-terminal Domain Phosphatase 1 Inhibits Cancer Cell Migration and Invasion by Dephosphorylating Ser(P)68-Twist1 to Accelerate Twist1 Protein Degradation *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.721795

The amino acid 43–63 region of SCP1 is responsible for interacting with the N terminus of Twist1. A , GST-tagged full-length SCP1 and nine SCP1 fragments were purified from bacteria using glutathione-Sepharose 4B beads. B , Twist1-F protein was
Figure Legend Snippet: The amino acid 43–63 region of SCP1 is responsible for interacting with the N terminus of Twist1. A , GST-tagged full-length SCP1 and nine SCP1 fragments were purified from bacteria using glutathione-Sepharose 4B beads. B , Twist1-F protein was

Techniques Used: Purification

31) Product Images from "TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction"

Article Title: TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction

Journal: Autophagy

doi: 10.1080/15548627.2019.1580510

TP53INP2 forms a complex with LC3B and ATG7. (a) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-TP53INP2, GFP-TP53INP2[NLSΔ] or GFP-TP53INP2[LIRΔ] from HEK293 cells. TP53INP2 proteins were immunoprecipitated by anti-GFP. The coprecipitated ATG7, ATG3 or ATG12–ATG5 was detected by western blot using anti-ATG3, anti-ATG7 or anti-ATG5 respectively. (b) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ] or TP53INP2[LIRΔ]. GFP-tagged TP53INP2 mutants were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7, anti-ATG3 or anti-ATG5. (c) In vitro TP53INP2-ATG7 binding assay. Purified GST-TP53INP2 or GST-TP53INP2 W35,I38A was incubated with purified LC3B[G120] and ATG7. After affinity-isolating GST-TP53INP2 or GST-TP53INP2 W35,I38A with glutathione-sepharose 4B beads, the bound LC3B[G120] and ATG7 were analyzed by western blot. (d) HEK293T cells were cotransfected with Flag-LC3B, TP53INP2-MYC and HA-ATG7. The cells were lysed 48 h after transfection and Flag-LC3B was immunoprecipitated with anti-Flag. After incubation of the Flag-LC3B precipitates with Flag peptide, the eluate was used for immunoprecipitation with either anti-MYC or anti-HA. The immunoprecipitates were then analyzed by western blot by anti-Flag, anti-MYC and anti-HA respectively. (e) Coimmunoprecipitation of ATG7 with each of the indicated GFP-tagged truncated TP53INP2 mutants in HEK293 cells. TP53INP2 proteins were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7. (f) Purified GST-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ], TP53INP2 W35,I38A [Δ1-28],[NLSΔ] or SQSTM1 was incubated with purified ATG7, then the GST-tagged proteins were affinity-isolated by glutathione-sepharose 4B beads and bound ATG7 was detected by western blot using anti-ATG7.
Figure Legend Snippet: TP53INP2 forms a complex with LC3B and ATG7. (a) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-TP53INP2, GFP-TP53INP2[NLSΔ] or GFP-TP53INP2[LIRΔ] from HEK293 cells. TP53INP2 proteins were immunoprecipitated by anti-GFP. The coprecipitated ATG7, ATG3 or ATG12–ATG5 was detected by western blot using anti-ATG3, anti-ATG7 or anti-ATG5 respectively. (b) Coimmunoprecipitation of ATG7, ATG3 or ATG12–ATG5 with GFP-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ] or TP53INP2[LIRΔ]. GFP-tagged TP53INP2 mutants were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7, anti-ATG3 or anti-ATG5. (c) In vitro TP53INP2-ATG7 binding assay. Purified GST-TP53INP2 or GST-TP53INP2 W35,I38A was incubated with purified LC3B[G120] and ATG7. After affinity-isolating GST-TP53INP2 or GST-TP53INP2 W35,I38A with glutathione-sepharose 4B beads, the bound LC3B[G120] and ATG7 were analyzed by western blot. (d) HEK293T cells were cotransfected with Flag-LC3B, TP53INP2-MYC and HA-ATG7. The cells were lysed 48 h after transfection and Flag-LC3B was immunoprecipitated with anti-Flag. After incubation of the Flag-LC3B precipitates with Flag peptide, the eluate was used for immunoprecipitation with either anti-MYC or anti-HA. The immunoprecipitates were then analyzed by western blot by anti-Flag, anti-MYC and anti-HA respectively. (e) Coimmunoprecipitation of ATG7 with each of the indicated GFP-tagged truncated TP53INP2 mutants in HEK293 cells. TP53INP2 proteins were immunoprecipitated using anti-GFP and the precipitates were analyzed using anti-ATG7. (f) Purified GST-tagged TP53INP2[NLSΔ], TP53INP2 W35,I38A [NLSΔ], TP53INP2 W35,I38A [Δ1-28],[NLSΔ] or SQSTM1 was incubated with purified ATG7, then the GST-tagged proteins were affinity-isolated by glutathione-sepharose 4B beads and bound ATG7 was detected by western blot using anti-ATG7.

Techniques Used: Immunoprecipitation, Western Blot, In Vitro, Binding Assay, Purification, Incubation, Transfection, Isolation

TP53INP2 facilitates LC3B-ATG7 interaction. (a) Coprecipitation of endogenous ATG7 with exogenous Flag-LC3B in TP53INP2-MYC cotransfected HEK293 cells with or without cell starvation. Flag-LC3B was immunoprecipitated using anti-Flag, then ATG7 and TP53INP2-MYC were detected by anti-ATG7 and anti-MYC respectively. (b) Coprecipitation of ATG7 with Flag-LC3B from HEK293 cells transiently expressing RFP-tagged TP53INP2 or each of the indicated TP53INP2 mutants. Flag-LC3B was immunoprecipitated using anti-Flag. (c) HEK293 cells stably expressing non-silencing shRNA or TP53INP2 shRNA were transfected with Flag-LC3B K49,51R and starved. The cells were then fractionated by differential centrifugation. Flag-LC3B K49,51R was immunoprecipitated from the cell cytosol using anti-Flag and the coprecipitated ATG7 was detected by western blot. (d) In vitro affinity-isolation assay of LC3B[G120]-ATG7 interaction. Purified GST-LC3B[G120] was incubated with cell lysate from HEK293 cells expressing the indicated RFP-tagged TP53INP2 mutants. After affinity-isolating GST-LC3B[G120] using glutathione-sepharose 4B beads, GST-LC3B[G120]-bound ATG7 was analyzed by western blot. (e) Confocal images of HEK293 cells stably expressing GFP-LC3B and transfected with plasmids expressing each of the indicated RFP-tagged TP53INP2 truncated mutants. (f) Quantification of GFP-LC3B puncta in (e). The data are presented as mean ± SEM, n = 30 cells. ***, P
Figure Legend Snippet: TP53INP2 facilitates LC3B-ATG7 interaction. (a) Coprecipitation of endogenous ATG7 with exogenous Flag-LC3B in TP53INP2-MYC cotransfected HEK293 cells with or without cell starvation. Flag-LC3B was immunoprecipitated using anti-Flag, then ATG7 and TP53INP2-MYC were detected by anti-ATG7 and anti-MYC respectively. (b) Coprecipitation of ATG7 with Flag-LC3B from HEK293 cells transiently expressing RFP-tagged TP53INP2 or each of the indicated TP53INP2 mutants. Flag-LC3B was immunoprecipitated using anti-Flag. (c) HEK293 cells stably expressing non-silencing shRNA or TP53INP2 shRNA were transfected with Flag-LC3B K49,51R and starved. The cells were then fractionated by differential centrifugation. Flag-LC3B K49,51R was immunoprecipitated from the cell cytosol using anti-Flag and the coprecipitated ATG7 was detected by western blot. (d) In vitro affinity-isolation assay of LC3B[G120]-ATG7 interaction. Purified GST-LC3B[G120] was incubated with cell lysate from HEK293 cells expressing the indicated RFP-tagged TP53INP2 mutants. After affinity-isolating GST-LC3B[G120] using glutathione-sepharose 4B beads, GST-LC3B[G120]-bound ATG7 was analyzed by western blot. (e) Confocal images of HEK293 cells stably expressing GFP-LC3B and transfected with plasmids expressing each of the indicated RFP-tagged TP53INP2 truncated mutants. (f) Quantification of GFP-LC3B puncta in (e). The data are presented as mean ± SEM, n = 30 cells. ***, P

Techniques Used: Immunoprecipitation, Expressing, Stable Transfection, shRNA, Transfection, Centrifugation, Western Blot, In Vitro, Isolation, Purification, Incubation

32) Product Images from "Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1"

Article Title: Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200108089

MURF family members interact with SUMO modifying enzymes ISOT-3 and Ubc9, but only MURF-1 interacts with the transcriptional regulator GMEB-1. (A) Y2H screens using full-length cDNAs of individual MURF family members as baits identified ISOT-3 (light gray) and Ubc9 (black) as MURF-binding proteins. However, GMEB-1 (dark gray) was found to interact only with MURF-1. β-Galactosidase assays were performed to confirm positive clones, and the levels were compared with colonies transformed with each prey construct and the empty bait vector (white). Data are presented as mean levels of β-galactosidase from triplicate experiments ± SD. ***, P > 0.001. (B) RT-PCR analysis of human heart total RNA revealed that GMEB-1 mRNA transcripts are detectable in heart (H) and skeletal (Sk) tissues. Lane 1, no reverse transcriptase control in human heart RNA (−); lane 2, 511-bp GMEB-1 PCR product amplified from human heart RNA (+); lane 3, 511-bp GMEB-1 PCR product amplified from human skeletal RNA (+); lane 4, no reverse transcriptase control in human skeletal RNA (−). (C) GMEB-1 specifically binds to MURF-1 in GST pull-down assays. GMEB-1 was translated in vitro (lane 3). When incubated with bacterially expressed GST–MURF-1 fusion peptides, GMEB-1 and MURF-1 binding to glutathione–sepharose 4B beads was detectable (lane 2). Lane 1 contains no detectable binding of GMEB-1 to the beads alone. IVT, in vitro translated. (D) GMEB-1–GFP targets to the nuclei of cardiac myocytes (a and c). MURF-1 staining also was present in some of the nuclei that contained GMEB-1–GFP (b). Note, MURF-1 is also detected at the M-line region in the same myocytes (b, double arrows). Expression of GMEB-1–GFP in cardiac myocytes does not appear to affect the integrity of the COOH-terminal region of titin (d, staining with anti-titin A168–170 antibodies). Double arrows mark regular, striated titin staining. N, nuclei. Bars, 10 μm.
Figure Legend Snippet: MURF family members interact with SUMO modifying enzymes ISOT-3 and Ubc9, but only MURF-1 interacts with the transcriptional regulator GMEB-1. (A) Y2H screens using full-length cDNAs of individual MURF family members as baits identified ISOT-3 (light gray) and Ubc9 (black) as MURF-binding proteins. However, GMEB-1 (dark gray) was found to interact only with MURF-1. β-Galactosidase assays were performed to confirm positive clones, and the levels were compared with colonies transformed with each prey construct and the empty bait vector (white). Data are presented as mean levels of β-galactosidase from triplicate experiments ± SD. ***, P > 0.001. (B) RT-PCR analysis of human heart total RNA revealed that GMEB-1 mRNA transcripts are detectable in heart (H) and skeletal (Sk) tissues. Lane 1, no reverse transcriptase control in human heart RNA (−); lane 2, 511-bp GMEB-1 PCR product amplified from human heart RNA (+); lane 3, 511-bp GMEB-1 PCR product amplified from human skeletal RNA (+); lane 4, no reverse transcriptase control in human skeletal RNA (−). (C) GMEB-1 specifically binds to MURF-1 in GST pull-down assays. GMEB-1 was translated in vitro (lane 3). When incubated with bacterially expressed GST–MURF-1 fusion peptides, GMEB-1 and MURF-1 binding to glutathione–sepharose 4B beads was detectable (lane 2). Lane 1 contains no detectable binding of GMEB-1 to the beads alone. IVT, in vitro translated. (D) GMEB-1–GFP targets to the nuclei of cardiac myocytes (a and c). MURF-1 staining also was present in some of the nuclei that contained GMEB-1–GFP (b). Note, MURF-1 is also detected at the M-line region in the same myocytes (b, double arrows). Expression of GMEB-1–GFP in cardiac myocytes does not appear to affect the integrity of the COOH-terminal region of titin (d, staining with anti-titin A168–170 antibodies). Double arrows mark regular, striated titin staining. N, nuclei. Bars, 10 μm.

Techniques Used: Binding Assay, Clone Assay, Transformation Assay, Construct, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, In Vitro, Incubation, Staining, Expressing

33) Product Images from "Dynamic ubiquitylation of Sox2 regulates proteostasis and governs neural progenitor cell differentiation"

Article Title: Dynamic ubiquitylation of Sox2 regulates proteostasis and governs neural progenitor cell differentiation

Journal: Nature Communications

doi: 10.1038/s41467-018-07025-z

CUL4A DET1-COP1 interacts with Sox2 and regulates its stability. a Network view of E3–Sox2 interactions (left panel) and the E3 hierarchical tree for Sox2 (right panel). UbiBrowser was employed to explore the E3 ligases for Sox2. The representative predicted E3 ligases surround Sox2. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. The predicted E3s and their position in the E3 family hierarchical tree was presented. In this tree, texts in each circle (just like “U”, “D” and “SO”) represent the E3 family. The number in the bracket following each E3 family represents the number of corresponding predicted E3–Sox2 interaction. b NPCs cell lysates were subjected to immunoprecipitation with control IgG or anti-Sox2 antibodies and detected CUL4A, COP1, DET1, DDB1, Roc1, and Sox2 protein levels. c The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Myc or Histidine tag-specific affinity resin (agarose beads). The immunoprecipitates or the eluates were then blotted. d Overview of the structures of COP1 wild type and different truncates. HEK293T cells were co-transfected with Myc-Sox2 and the indicated COP1 truncates. The lysates were collected and subjected to immunoprecipitation with anti-Flag. The immunoprecipitates were then blotted. e Overview of the structure of Sox2 wild type and different VP mutants. Recombinant proteins (His-COP1, GST-Sox2, GST-Sox2-A1, GST-Sox2-A2, and GST-Sox2-AA) were expressed and purified. GST-Sox2 bound to glutathione-Sepharose 4B beads was incubated with His- COP1 for 24 h at 4 °C. Then the beads were washed and proteins were eluted, followed by western blotting. f HEK293T cells were transfected with indicated constructs. The lysates were collected and blotted with anti-Flag and anti-Myc antibody. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9
Figure Legend Snippet: CUL4A DET1-COP1 interacts with Sox2 and regulates its stability. a Network view of E3–Sox2 interactions (left panel) and the E3 hierarchical tree for Sox2 (right panel). UbiBrowser was employed to explore the E3 ligases for Sox2. The representative predicted E3 ligases surround Sox2. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. The predicted E3s and their position in the E3 family hierarchical tree was presented. In this tree, texts in each circle (just like “U”, “D” and “SO”) represent the E3 family. The number in the bracket following each E3 family represents the number of corresponding predicted E3–Sox2 interaction. b NPCs cell lysates were subjected to immunoprecipitation with control IgG or anti-Sox2 antibodies and detected CUL4A, COP1, DET1, DDB1, Roc1, and Sox2 protein levels. c The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Myc or Histidine tag-specific affinity resin (agarose beads). The immunoprecipitates or the eluates were then blotted. d Overview of the structures of COP1 wild type and different truncates. HEK293T cells were co-transfected with Myc-Sox2 and the indicated COP1 truncates. The lysates were collected and subjected to immunoprecipitation with anti-Flag. The immunoprecipitates were then blotted. e Overview of the structure of Sox2 wild type and different VP mutants. Recombinant proteins (His-COP1, GST-Sox2, GST-Sox2-A1, GST-Sox2-A2, and GST-Sox2-AA) were expressed and purified. GST-Sox2 bound to glutathione-Sepharose 4B beads was incubated with His- COP1 for 24 h at 4 °C. Then the beads were washed and proteins were eluted, followed by western blotting. f HEK293T cells were transfected with indicated constructs. The lysates were collected and blotted with anti-Flag and anti-Myc antibody. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9

Techniques Used: Immunoprecipitation, Transfection, Construct, Recombinant, Purification, Incubation, Western Blot

34) Product Images from "Cep57 is a Mis12-interacting kinetochore protein involved in kinetochore targeting of Mad1–Mad2"

Article Title: Cep57 is a Mis12-interacting kinetochore protein involved in kinetochore targeting of Mad1–Mad2

Journal: Nature Communications

doi: 10.1038/ncomms10151

Cep57 interacts with Mad1. ( a ) Mitotic HeLa cells arrested by nocodazole were used for immunoprecipitation (IP) with anti-Cep57 antibody and western blotting with anti-Cep57 and anti-Mad1 antibodies. IgG served as the negative control. ( b ) HEK293T cells were co-transfected with the indicated plasmids, and were used to for IP and western blotting. ( c ) Binding assays of Mad1 and Cep57. Flag-Cep57 and Mad1-GFP (expressed in HEK293T cells and purified) were incubated for IP with anti-GFP antibody. IP samples were analysed by western blotting with anti-Flag and anti-GFP antibodies. ( d ) In vitro pull-down assays of Mad1 and Cep57. GST-Mad1 (176–718 amino acids) and MBP-Cep57 (151–500 amino acids) (expressed in E. coli and purified) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by western blotting with anti-GST antibody and Coomassie blue staining. ( e ) GST pull-down assays of Cep57 and Mad1. Lysates of HEK293T cells overexpressing Mad1-GFP were incubated with Glutathione Sepharose 4B beads coated with GST, GST-Cep57 (1–242 amino acids) or GST-Cep57 (195–500 amino acids). The samples were analysed by western blotting with anti-GFP antibody. GST and GST-tagged proteins were stained with Coomassie blue. ( f ) Binding assays of Mad1 and Cep57 (195–500 amino acids). GST-Cep57 (195–500 amino acids; expressed in E. coli ) and Flag-Mad1 (expressed in HEK293T cells) were purified and incubated with anti-Flag antibody. The IP samples with anti-Flag antibody were analysed by western blotting with anti-GST and anti-Flag antibodies. ( g ) Schematic of truncated mutants of Mad1. ( h , i ) IP using lysates of HEK293T cells co-overexpressing GFP-Cep57 and Flag-Mad1 (FL, 1–530 and 531–718 amino acids) ( h ) or Flag-Mad1 (1–530, 1–175, 176–350 and 351–530 amino acids) ( i ) with anti-Flag antibody. The IP samples were analysed by western blotting with anti-GFP and anti-Flag antibodies. FL, full length. ( j ) Quantification and normalization of the kinetochore signal of Flag-Mad1 in HeLa cells that were transfected with FL and truncated mutants of Flag-Mad1 and treated with nocodazole for 1 h. Greater than 50 kinetochores from 5 cells were measured. The experiment was repeated three times. Data are mean±s.e.m.
Figure Legend Snippet: Cep57 interacts with Mad1. ( a ) Mitotic HeLa cells arrested by nocodazole were used for immunoprecipitation (IP) with anti-Cep57 antibody and western blotting with anti-Cep57 and anti-Mad1 antibodies. IgG served as the negative control. ( b ) HEK293T cells were co-transfected with the indicated plasmids, and were used to for IP and western blotting. ( c ) Binding assays of Mad1 and Cep57. Flag-Cep57 and Mad1-GFP (expressed in HEK293T cells and purified) were incubated for IP with anti-GFP antibody. IP samples were analysed by western blotting with anti-Flag and anti-GFP antibodies. ( d ) In vitro pull-down assays of Mad1 and Cep57. GST-Mad1 (176–718 amino acids) and MBP-Cep57 (151–500 amino acids) (expressed in E. coli and purified) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by western blotting with anti-GST antibody and Coomassie blue staining. ( e ) GST pull-down assays of Cep57 and Mad1. Lysates of HEK293T cells overexpressing Mad1-GFP were incubated with Glutathione Sepharose 4B beads coated with GST, GST-Cep57 (1–242 amino acids) or GST-Cep57 (195–500 amino acids). The samples were analysed by western blotting with anti-GFP antibody. GST and GST-tagged proteins were stained with Coomassie blue. ( f ) Binding assays of Mad1 and Cep57 (195–500 amino acids). GST-Cep57 (195–500 amino acids; expressed in E. coli ) and Flag-Mad1 (expressed in HEK293T cells) were purified and incubated with anti-Flag antibody. The IP samples with anti-Flag antibody were analysed by western blotting with anti-GST and anti-Flag antibodies. ( g ) Schematic of truncated mutants of Mad1. ( h , i ) IP using lysates of HEK293T cells co-overexpressing GFP-Cep57 and Flag-Mad1 (FL, 1–530 and 531–718 amino acids) ( h ) or Flag-Mad1 (1–530, 1–175, 176–350 and 351–530 amino acids) ( i ) with anti-Flag antibody. The IP samples were analysed by western blotting with anti-GFP and anti-Flag antibodies. FL, full length. ( j ) Quantification and normalization of the kinetochore signal of Flag-Mad1 in HeLa cells that were transfected with FL and truncated mutants of Flag-Mad1 and treated with nocodazole for 1 h. Greater than 50 kinetochores from 5 cells were measured. The experiment was repeated three times. Data are mean±s.e.m.

Techniques Used: Immunoprecipitation, Western Blot, Negative Control, Transfection, Binding Assay, Purification, Incubation, In Vitro, Magnetic Beads, Staining

Cep57 is a novel kinetochore component in human cells. ( a ) Three-dimensional structured illumination microscopy (SIM) images of HeLa cells double-immunostained with antibodies against Cep57 (green) and CREST (red). ( b ) Immunofluorescence of Cep57 (green) and CENP-A (red) in HeLa cells. ( c ) Immunofluorescence of Cep57 (green) and Mis12 (red) in HeLa cells at metaphase after treatment with MG132 for 1 h. A linescan through the kinetochore pair indicates the co-localization of Cep57 with Mis12. ( d ) Stimulated emission depletion (STED) images of immunofluorescence of Cep57 (green) and Zwint-1 (red) in RPE1 cells. ( e ) Immunofluorescence of Cep57 (green) and CREST (red) in HeLa cells at different stages during mitosis. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). ( f ) HEK293T cells were co-transfected with Cep57-GFP and Mis12-HA. The cell lysates were immunoprecipitated (IP) and analysed by western blotting (WB) with the indicated antibodies. ( g ) Binding assays of Mis12 and Cep57. GST-Mis12 (expressed in E. coli and purified) was incubated with Flag-Cep57 (expressed in HEK293T cells and purified). The IP samples with anti-Flag antibody were analysed by WB with anti-Flag and anti-Mis12 antibodies. ( h ) Schematic of Cep57 truncated mutants. ( i ) GST pull-down assays of Cep57 and Mis12. Lysates of HEK293T cells overexpressing Mis12-HA were incubated with Glutathione Sepharose 4B beads coated with GST or GST-Cep57N/C. The samples were analysed by WB with anti-HA antibody. GST-tagged proteins were stained with Coomassie blue. ( j ) In vitro pull-down assays of Mis12 and Cep57 (1–242 amino acids). GST-Mis12 (expressed in E. coli and purified) and MBP-Cep57 (1–242 amino acids) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by WB with anti-GST antibody and Coomassie blue staining. ( k ) Quantification of kinetochore signals of Cep57 and Mis12 in HeLa cells depleted of Cep57, Mis12 or negative control (NC) by siRNAs. The signal from control siRNA-treated cells was normalized to 1.0. More than 200 kinetochores from 20 cells were measured. The experiment was repeated three times. Data are mean±s.e.m. **** P
Figure Legend Snippet: Cep57 is a novel kinetochore component in human cells. ( a ) Three-dimensional structured illumination microscopy (SIM) images of HeLa cells double-immunostained with antibodies against Cep57 (green) and CREST (red). ( b ) Immunofluorescence of Cep57 (green) and CENP-A (red) in HeLa cells. ( c ) Immunofluorescence of Cep57 (green) and Mis12 (red) in HeLa cells at metaphase after treatment with MG132 for 1 h. A linescan through the kinetochore pair indicates the co-localization of Cep57 with Mis12. ( d ) Stimulated emission depletion (STED) images of immunofluorescence of Cep57 (green) and Zwint-1 (red) in RPE1 cells. ( e ) Immunofluorescence of Cep57 (green) and CREST (red) in HeLa cells at different stages during mitosis. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). ( f ) HEK293T cells were co-transfected with Cep57-GFP and Mis12-HA. The cell lysates were immunoprecipitated (IP) and analysed by western blotting (WB) with the indicated antibodies. ( g ) Binding assays of Mis12 and Cep57. GST-Mis12 (expressed in E. coli and purified) was incubated with Flag-Cep57 (expressed in HEK293T cells and purified). The IP samples with anti-Flag antibody were analysed by WB with anti-Flag and anti-Mis12 antibodies. ( h ) Schematic of Cep57 truncated mutants. ( i ) GST pull-down assays of Cep57 and Mis12. Lysates of HEK293T cells overexpressing Mis12-HA were incubated with Glutathione Sepharose 4B beads coated with GST or GST-Cep57N/C. The samples were analysed by WB with anti-HA antibody. GST-tagged proteins were stained with Coomassie blue. ( j ) In vitro pull-down assays of Mis12 and Cep57 (1–242 amino acids). GST-Mis12 (expressed in E. coli and purified) and MBP-Cep57 (1–242 amino acids) were incubated with Amylose Magnetic beads. The precipitated samples were analysed by WB with anti-GST antibody and Coomassie blue staining. ( k ) Quantification of kinetochore signals of Cep57 and Mis12 in HeLa cells depleted of Cep57, Mis12 or negative control (NC) by siRNAs. The signal from control siRNA-treated cells was normalized to 1.0. More than 200 kinetochores from 20 cells were measured. The experiment was repeated three times. Data are mean±s.e.m. **** P

Techniques Used: Microscopy, Immunofluorescence, Staining, Transfection, Immunoprecipitation, Western Blot, Binding Assay, Purification, Incubation, In Vitro, Magnetic Beads, Negative Control

Microtubule-binding activity of Cep57 contributes to checkpoint silencing. ( a ) Microtubule-binding assays in vitro . GST-Cep57 (195–500 amino acids; 0.1 μM) expressed in E. coli and Flag-Mad1 (0.05 μM) expressed in HEK293T cells were purified and incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. After centrifugation, supernatant (S) and pellet (P) were separated and used for Coomassie blue staining (top), and western blotting with anti-Flag antibody (bottom). ( b ) GST-Cep57 (195–500 amino acids; 0.1 μM)-coupled Glutathione Sepharose 4B beads were incubated with taxol-stabilized microtubules and purified Flag-Mad1 (0.05 μM) in BRB80 buffer at room temperature. The bead-bound proteins were analysed by western blotting with anti-Flag and anti-tubulin antibodies. GST-Cep57 (195–500 amino acids) was detected by Coomassie blue staining. ( c ) Microtubule-binding assays in vitro . Flag-Cep57 (0.05 μM) and Flag-Cep57-12A (0.05 μM) expressed in HEK293T cells and purified, and were incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. Samples were separated by centrifugation, and analysed by western blotting with anti-Flag antibody (top) and Coomassie blue staining (bottom). 12A: K432A, K434A, K435A, K438A, K441A, K442A, K467A, R469A, K473A, R474A, R475A and K476A. ( d ) Immunostaining of α-tubulin (red) in HeLa cells expressing Cep57-GFP or Cep57-12A-GFP. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 5 μm. ( e ) Immunostaining of Flag-Cep57 (green) and Mad1 (red) in metaphase HeLa cells expressing RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A after transfection with Cep57-siRNA. DNA was stained with DAPI (blue). Scale bars, 5 μm. ( f ) Quantification of the percentage of metaphase cells with Mad1 signals at kinetochores from ( e ). Fifty cells were measured. ( g ) Quantification of the percentage of kinetochores with Mad1 signals in metaphase cells from ( e ). Greater than 100 kinetochores from 10 cells were measured. ( h ) Quantification of the percentage of metaphase cells in negative control (NC) or Cep57-depleted prometaphase and metaphase HeLa cells that expressed RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A. Mitotic stages were quantified by the morphology of DNA and spindles. Greater than 100 cells were measured. For f , g and h , the experiment was repeated three times. Data are mean±s.e.m. ** P
Figure Legend Snippet: Microtubule-binding activity of Cep57 contributes to checkpoint silencing. ( a ) Microtubule-binding assays in vitro . GST-Cep57 (195–500 amino acids; 0.1 μM) expressed in E. coli and Flag-Mad1 (0.05 μM) expressed in HEK293T cells were purified and incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. After centrifugation, supernatant (S) and pellet (P) were separated and used for Coomassie blue staining (top), and western blotting with anti-Flag antibody (bottom). ( b ) GST-Cep57 (195–500 amino acids; 0.1 μM)-coupled Glutathione Sepharose 4B beads were incubated with taxol-stabilized microtubules and purified Flag-Mad1 (0.05 μM) in BRB80 buffer at room temperature. The bead-bound proteins were analysed by western blotting with anti-Flag and anti-tubulin antibodies. GST-Cep57 (195–500 amino acids) was detected by Coomassie blue staining. ( c ) Microtubule-binding assays in vitro . Flag-Cep57 (0.05 μM) and Flag-Cep57-12A (0.05 μM) expressed in HEK293T cells and purified, and were incubated with or without taxol-stabilized microtubules (1.0 μM) in BRB80 buffer. Samples were separated by centrifugation, and analysed by western blotting with anti-Flag antibody (top) and Coomassie blue staining (bottom). 12A: K432A, K434A, K435A, K438A, K441A, K442A, K467A, R469A, K473A, R474A, R475A and K476A. ( d ) Immunostaining of α-tubulin (red) in HeLa cells expressing Cep57-GFP or Cep57-12A-GFP. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 5 μm. ( e ) Immunostaining of Flag-Cep57 (green) and Mad1 (red) in metaphase HeLa cells expressing RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A after transfection with Cep57-siRNA. DNA was stained with DAPI (blue). Scale bars, 5 μm. ( f ) Quantification of the percentage of metaphase cells with Mad1 signals at kinetochores from ( e ). Fifty cells were measured. ( g ) Quantification of the percentage of kinetochores with Mad1 signals in metaphase cells from ( e ). Greater than 100 kinetochores from 10 cells were measured. ( h ) Quantification of the percentage of metaphase cells in negative control (NC) or Cep57-depleted prometaphase and metaphase HeLa cells that expressed RNAi-resistant wild-type Flag-Cep57 or Flag-Cep57-12A. Mitotic stages were quantified by the morphology of DNA and spindles. Greater than 100 cells were measured. For f , g and h , the experiment was repeated three times. Data are mean±s.e.m. ** P

Techniques Used: Binding Assay, Activity Assay, In Vitro, Purification, Incubation, Centrifugation, Staining, Western Blot, Immunostaining, Expressing, Transfection, Negative Control

35) Product Images from "Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain"

Article Title: Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain

Journal: Open Biology

doi: 10.1098/rsob.160089

Details of the molecular recognition in the Aurora-A/vNAR-D01 complex. ( a ) Key interactions are shown in the three panels. Aurora-A is coloured teal and vNAR-D01 is coloured orange. ( b ) Co-precipitation assay between GST-Aurora-A KD DN and WT, and mutant vNAR-D01 constructs. GST-Aurora-A KD DN was immobilized on Glutathione Sepharose 4B beads and then incubated with vNAR-D01 proteins. GST was used as a binding control.
Figure Legend Snippet: Details of the molecular recognition in the Aurora-A/vNAR-D01 complex. ( a ) Key interactions are shown in the three panels. Aurora-A is coloured teal and vNAR-D01 is coloured orange. ( b ) Co-precipitation assay between GST-Aurora-A KD DN and WT, and mutant vNAR-D01 constructs. GST-Aurora-A KD DN was immobilized on Glutathione Sepharose 4B beads and then incubated with vNAR-D01 proteins. GST was used as a binding control.

Techniques Used: Mutagenesis, Construct, Incubation, Binding Assay

vNAR-D01 is an Aurora-A inhibitor that competes with TPX2. ( a ) Surface plasmon resonance binding assays between Aurora-A KD CA-Avi and vNAR-D01. The kinase was immobilized on Biacore Sensor SA chips at 550, 350 and 250 RU and interacted with 0.01–50 µM vNAR-D01. Maximum responses were plotted against vNAR-D01 concentration and fitted to a one-site specific binding equation (solid lines) in P rism 6 (GraphPad) to calculate binding affinities. ( b ) Co-precipitation assay between the Aurora-A KD CA/vNAR-D01 complex or His 6 -Aurora-A KD CA and GST-TPX2 1–43 . The complex and Aurora-A were immobilized on Nickel Sepharose beads using the His 6 -tag on the vNAR domain and kinase, respectively. GST was used as a binding control. ( c ) Co-precipitation assay between GST-Aurora-A KD DN and vNAR-D01 and His 6 -TPX2 1–43 . In total, 2 µM GST-Aurora-A KD DN was immobilized on Glutathione Sepharose 4B beads and incubated with 5 µM vNAR-D01 and 0, 1, 2, 5, 10, 20 and 50 µM His 6 -TPX2 (black triangle). GST was used as a binding control. ( d ) In vitro kinase activity assay of Aurora-A KD in the presence of vNAR-D01. MBP was used as a generic kinase substrate. Reactions were analysed by SDS-PAGE (top left panel) and incorporation of radioisotope resolved by autoradiography (bottom left panel). Incorporation of radioisotope was measured by scintillation counting (right). Error bars represent the standard error for two independent reactions. ** = p
Figure Legend Snippet: vNAR-D01 is an Aurora-A inhibitor that competes with TPX2. ( a ) Surface plasmon resonance binding assays between Aurora-A KD CA-Avi and vNAR-D01. The kinase was immobilized on Biacore Sensor SA chips at 550, 350 and 250 RU and interacted with 0.01–50 µM vNAR-D01. Maximum responses were plotted against vNAR-D01 concentration and fitted to a one-site specific binding equation (solid lines) in P rism 6 (GraphPad) to calculate binding affinities. ( b ) Co-precipitation assay between the Aurora-A KD CA/vNAR-D01 complex or His 6 -Aurora-A KD CA and GST-TPX2 1–43 . The complex and Aurora-A were immobilized on Nickel Sepharose beads using the His 6 -tag on the vNAR domain and kinase, respectively. GST was used as a binding control. ( c ) Co-precipitation assay between GST-Aurora-A KD DN and vNAR-D01 and His 6 -TPX2 1–43 . In total, 2 µM GST-Aurora-A KD DN was immobilized on Glutathione Sepharose 4B beads and incubated with 5 µM vNAR-D01 and 0, 1, 2, 5, 10, 20 and 50 µM His 6 -TPX2 (black triangle). GST was used as a binding control. ( d ) In vitro kinase activity assay of Aurora-A KD in the presence of vNAR-D01. MBP was used as a generic kinase substrate. Reactions were analysed by SDS-PAGE (top left panel) and incorporation of radioisotope resolved by autoradiography (bottom left panel). Incorporation of radioisotope was measured by scintillation counting (right). Error bars represent the standard error for two independent reactions. ** = p

Techniques Used: SPR Assay, Binding Assay, Concentration Assay, Incubation, In Vitro, Kinase Assay, SDS Page, Autoradiography

36) Product Images from "Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication"

Article Title: Suppression of Rac1 Signaling by Influenza A Virus NS1 Facilitates Viral Replication

Journal: Scientific Reports

doi: 10.1038/srep35041

NS1 interacts with Rac1 in vivo . ( A ) GST-pull down assay showing the interaction between NS1 and Rac1. Whole 293T cell lysates transfected with Myc-tagged Rac1 were incubated with an equal amount of GST or GST-NS1 bound to glutathione-Sepharose 4B beads, followed by IB using the anti-Myc antibody. CBB, Coomassie brilliant blue staining. ( B ) Co-immunoprecipitation of Flag-NS1 and Myc-Rac1 in 293T cells. The 293T cells were co-transfected with Flag-tagged NS1 and pcDNA4.0 or pcDNA4.0-myc-Rac1; Myc-tagged Rac1 and pcDNA3.0 or pcDNA3.0-flag-NS1. Rac1 antibodies (rabbit) were applied to the cell lysates for IP, followed by IB with Flag (mouse); alternatively, the IP was performed with a Flag antibody (mouse) followed by IB with Rac1 (rabbit). ( C ) Co-localization of GFP-NS1 and Myc-Rac1 in A549 cells. A549 cells were co-transfected with pEGFP-NS1 and pcDNA4.0-myc-Rac1 for 28 hours and then fixed, permeabilized, and stained for Myc-Rac1 (red). Yellow indicates overlap. ( D ) Co-immunoprecipitation of virus NS1 and endogenous Rac1 in A549 cells. A549 cells were infected with the A/WSN/33 H1N1 virus and the mutated NS1 − H1N1 virus (MOI = 0.5) for 24 hours, and the cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. ( E ) Co-localization of viral NS1 and endogenous Rac1 in A549 cells. A549 cells were infected with the A/WSN/33 H1N1 virus (MOI = 0.1) for 24 hours and then fixed, permeabilized, and stained with DAPI (blue), anti-NS1 antibody (red), and anti-Rac1 antibody (green). Yellow indicates overlap with red and green. Orange indicates overlap with blue, red and green.
Figure Legend Snippet: NS1 interacts with Rac1 in vivo . ( A ) GST-pull down assay showing the interaction between NS1 and Rac1. Whole 293T cell lysates transfected with Myc-tagged Rac1 were incubated with an equal amount of GST or GST-NS1 bound to glutathione-Sepharose 4B beads, followed by IB using the anti-Myc antibody. CBB, Coomassie brilliant blue staining. ( B ) Co-immunoprecipitation of Flag-NS1 and Myc-Rac1 in 293T cells. The 293T cells were co-transfected with Flag-tagged NS1 and pcDNA4.0 or pcDNA4.0-myc-Rac1; Myc-tagged Rac1 and pcDNA3.0 or pcDNA3.0-flag-NS1. Rac1 antibodies (rabbit) were applied to the cell lysates for IP, followed by IB with Flag (mouse); alternatively, the IP was performed with a Flag antibody (mouse) followed by IB with Rac1 (rabbit). ( C ) Co-localization of GFP-NS1 and Myc-Rac1 in A549 cells. A549 cells were co-transfected with pEGFP-NS1 and pcDNA4.0-myc-Rac1 for 28 hours and then fixed, permeabilized, and stained for Myc-Rac1 (red). Yellow indicates overlap. ( D ) Co-immunoprecipitation of virus NS1 and endogenous Rac1 in A549 cells. A549 cells were infected with the A/WSN/33 H1N1 virus and the mutated NS1 − H1N1 virus (MOI = 0.5) for 24 hours, and the cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. ( E ) Co-localization of viral NS1 and endogenous Rac1 in A549 cells. A549 cells were infected with the A/WSN/33 H1N1 virus (MOI = 0.1) for 24 hours and then fixed, permeabilized, and stained with DAPI (blue), anti-NS1 antibody (red), and anti-Rac1 antibody (green). Yellow indicates overlap with red and green. Orange indicates overlap with blue, red and green.

Techniques Used: In Vivo, Pull Down Assay, Transfection, Incubation, Staining, Immunoprecipitation, Infection

NS1 down-regulates Rac1 activity. ( A ) There was no significant difference in the Rac1 protein expression level between the two different cell lines. The two cell lines were seeded into six-well plates. After 24 hours, the Rac1 protein expression levels were assessed in the whole cell lysates using the indicated antibodies as appropriate. ( B ) GTP-bound Rac1 levels were lower in the NS1 over-expressing cell lines than in the negative control cell lines. Lysates from the two stable cell lines were incubated with equal amounts of GST-PAK bound glutathione Sepharose 4B beads. After washing, the bound proteins were analyzed by western blotting using an anti-Rac1 antibody. The protein band intensity was measured using the ImageJ software (NIH). Data represent the mean fold change ± S.D. of three independent experiments (*p
Figure Legend Snippet: NS1 down-regulates Rac1 activity. ( A ) There was no significant difference in the Rac1 protein expression level between the two different cell lines. The two cell lines were seeded into six-well plates. After 24 hours, the Rac1 protein expression levels were assessed in the whole cell lysates using the indicated antibodies as appropriate. ( B ) GTP-bound Rac1 levels were lower in the NS1 over-expressing cell lines than in the negative control cell lines. Lysates from the two stable cell lines were incubated with equal amounts of GST-PAK bound glutathione Sepharose 4B beads. After washing, the bound proteins were analyzed by western blotting using an anti-Rac1 antibody. The protein band intensity was measured using the ImageJ software (NIH). Data represent the mean fold change ± S.D. of three independent experiments (*p

Techniques Used: Activity Assay, Expressing, Negative Control, Stable Transfection, Incubation, Western Blot, Software

37) Product Images from "14-3-3? Protein Regulates Anterograde Transport of the Human ?-Opioid Receptor (hKOPR) *"

Article Title: 14-3-3? Protein Regulates Anterograde Transport of the Human ?-Opioid Receptor (hKOPR) *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.359679

A , interaction of the hKOPR C-tail with 14-3-3ζ in rat brain extracts by pulldown assay is shown. Rat brains were homogenized, solubilized, and centrifuged at 100,000 × g for 40 min, and the supernatants were filtered through 0.45-μm and then 0.22-μm membranes. The filtrate was used for pulldown experiments by incubating with glutathione-agarose beads preloaded with GST or GST-hKOPR-C-tail overnight at 4 °C. IB , immunoblot. B , direct interaction between the hKOR C-tail and 14-3-3ζ is shown. Purified 14-3-3ζ was incubated with glutathione-Sepharose 4B beads preloaded with GST and GST-hKOPR C-tail overnight at 4 °C. A and B , the beads were washed extensively, and the bound proteins were eluted from the beads, resolved by 8% SDS-PAGE, and transferred onto Immobilon™-P PVDF membranes. A 1/250 supernatant was also loaded as an input control. Upper panel , 14-3-3 was detected by a rabbit anti-14-3-3ζ antibody. Lower panel , the same membrane was stained with Ponceau S, showing the relative sizes and the amounts of the GST and GST-hKOPR C-tail loaded. The figure represents one of the three experiments with similar results.
Figure Legend Snippet: A , interaction of the hKOPR C-tail with 14-3-3ζ in rat brain extracts by pulldown assay is shown. Rat brains were homogenized, solubilized, and centrifuged at 100,000 × g for 40 min, and the supernatants were filtered through 0.45-μm and then 0.22-μm membranes. The filtrate was used for pulldown experiments by incubating with glutathione-agarose beads preloaded with GST or GST-hKOPR-C-tail overnight at 4 °C. IB , immunoblot. B , direct interaction between the hKOR C-tail and 14-3-3ζ is shown. Purified 14-3-3ζ was incubated with glutathione-Sepharose 4B beads preloaded with GST and GST-hKOPR C-tail overnight at 4 °C. A and B , the beads were washed extensively, and the bound proteins were eluted from the beads, resolved by 8% SDS-PAGE, and transferred onto Immobilon™-P PVDF membranes. A 1/250 supernatant was also loaded as an input control. Upper panel , 14-3-3 was detected by a rabbit anti-14-3-3ζ antibody. Lower panel , the same membrane was stained with Ponceau S, showing the relative sizes and the amounts of the GST and GST-hKOPR C-tail loaded. The figure represents one of the three experiments with similar results.

Techniques Used: Purification, Incubation, SDS Page, Staining

38) Product Images from "NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47"

Article Title: NOP132 is required for proper nucleolus localization of DEAD-box RNA helicase DDX47

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl603

Association of NOP132 with DDX18 and DDX47. ( a ) 293 cells were transfected with NOP132 and either FLAG-tagged DDX18 or FLAG-tagged DDX47. Cell lysates were prepared and used for immunoprecipitation with anti-FLAG as described ( 15 ). Total cell protein (2% input) (lanes 1–3). Immunoprecipitates (lanes 4–9). Proteins were detected with anti-NOP132N (upper panels), anti-FLAG (middle panels) or a RAN antibody against the nuclear protein RAN as a loading control (lower panels). Lane 4, control immunoprecipitate (NOP132 transfected); lane 5, control immunoprecipitate (NOP132 transfected) treated with ribonuclease; lane 6, immunoprecipitation with anti-FLAG of FLAG-tagged DDX47 lysate; lane 7, immunoprecipitation with anti-FLAG of FLAG-tagged DDX47 lysate treated with ribonuclease; lane 8, immunoprecipitation with anti-FLAG of FLAG-tagged DDX18 lysate; lane 9, immunoprecipitation with anti-FLAG of FLAG-tagged DDX18 lysate treated with ribonuclease. ( b ) 293 cells were transfected with either FLAG-NOP132, FLAG-DDX18, or FLAG-DDX47. Silver-stained 10% SDS–PAGE gel of FLAG-NOP132-, FLAG-DDX47-, or FLAG-DDX18-associated complexes immunoprecipitated with anti-FLAG. Lane 1, molecular weight marker; lane 2, NOP132-associated proteins treated with ribonuclease; lane 3, NOP132-associated proteins; lane 4, DDX47-associated proteins treated with ribonuclease; lane 5, DDX47-associated proteins; lane 6, DDX18-associated proteins treated with ribonuclease; lane 7, DDX18-associated proteins; lane 8, control FLAG tag-associated proteins treated with ribonuclease; lane 9, control FLAG tag-associated proteins. Proteins which were identified by mass spectrometry are shown at the right of the gel image. ( c ) Baculovirus-produced NOP132 was mixed with GST (lane 1), GST-GRWD1 (lane 2), GST-DDX47 (lane 3), or GST-DDX18 (lane 4) bound to the glutathione-Sepharose-4B beads. Bound NOP132 was detected by western blotting using anti-NOP132N (upper panel). GST-fusion proteins stained with Coomassie brilliant blue are shown in the lower panel. Asterisks indicate the positions of the GST-fusion proteins.
Figure Legend Snippet: Association of NOP132 with DDX18 and DDX47. ( a ) 293 cells were transfected with NOP132 and either FLAG-tagged DDX18 or FLAG-tagged DDX47. Cell lysates were prepared and used for immunoprecipitation with anti-FLAG as described ( 15 ). Total cell protein (2% input) (lanes 1–3). Immunoprecipitates (lanes 4–9). Proteins were detected with anti-NOP132N (upper panels), anti-FLAG (middle panels) or a RAN antibody against the nuclear protein RAN as a loading control (lower panels). Lane 4, control immunoprecipitate (NOP132 transfected); lane 5, control immunoprecipitate (NOP132 transfected) treated with ribonuclease; lane 6, immunoprecipitation with anti-FLAG of FLAG-tagged DDX47 lysate; lane 7, immunoprecipitation with anti-FLAG of FLAG-tagged DDX47 lysate treated with ribonuclease; lane 8, immunoprecipitation with anti-FLAG of FLAG-tagged DDX18 lysate; lane 9, immunoprecipitation with anti-FLAG of FLAG-tagged DDX18 lysate treated with ribonuclease. ( b ) 293 cells were transfected with either FLAG-NOP132, FLAG-DDX18, or FLAG-DDX47. Silver-stained 10% SDS–PAGE gel of FLAG-NOP132-, FLAG-DDX47-, or FLAG-DDX18-associated complexes immunoprecipitated with anti-FLAG. Lane 1, molecular weight marker; lane 2, NOP132-associated proteins treated with ribonuclease; lane 3, NOP132-associated proteins; lane 4, DDX47-associated proteins treated with ribonuclease; lane 5, DDX47-associated proteins; lane 6, DDX18-associated proteins treated with ribonuclease; lane 7, DDX18-associated proteins; lane 8, control FLAG tag-associated proteins treated with ribonuclease; lane 9, control FLAG tag-associated proteins. Proteins which were identified by mass spectrometry are shown at the right of the gel image. ( c ) Baculovirus-produced NOP132 was mixed with GST (lane 1), GST-GRWD1 (lane 2), GST-DDX47 (lane 3), or GST-DDX18 (lane 4) bound to the glutathione-Sepharose-4B beads. Bound NOP132 was detected by western blotting using anti-NOP132N (upper panel). GST-fusion proteins stained with Coomassie brilliant blue are shown in the lower panel. Asterisks indicate the positions of the GST-fusion proteins.

Techniques Used: Transfection, Immunoprecipitation, Staining, SDS Page, Molecular Weight, Marker, FLAG-tag, Mass Spectrometry, Produced, Western Blot

39) Product Images from "Identification of Novel MAGE-G1-Interacting Partners in Retinoic Acid-Induced P19 Neuronal Differentiation Using SILAC-Based Proteomics"

Article Title: Identification of Novel MAGE-G1-Interacting Partners in Retinoic Acid-Induced P19 Neuronal Differentiation Using SILAC-Based Proteomics

Journal: Scientific Reports

doi: 10.1038/srep44699

Validation of the interaction between MAGE-G1 and VIME by GST pull-down and co-immunoprecipitation experiments. ( a ) GST or GST-VIME proteins were expressed in Escherichia coli BL21 respectively and purified with Glutathione-Sepharose 4B beads and washed, then beads were incubated with Flag-MAGE-G1 expressed in HEK293T. Flag-MAGE-G1 and GST-VIME were detected with indicated antibody. Full-length blots are included in a Supplementary Information . ( b ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Vime or pCMV-3 × Flag empty vector plus GFP- Vime expression plasmids. 25 μg of whole cell protein lysate was used as input to confirm the expression of the Flag-MAGE-G1 (with anti-Flag) or GFP-VIME (with anti-GFP) by immunoblotting (IB). The rest of cell lysates were incubated with anti-Flag-magnetics beads. The immunoprecipitated (IP) protein complex was resolved by SDS-PAGE and probed with antibodies against Flag or GFP. ( c ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Vime or pEGFP empty vector plus Flag- Mage - g1 expression plasmids. The experiment procedure was same as that mentioned above except that cell lysates were immunoprecipitaed with anti-GFP-magnetics beads. ( d ) Whole cell lysates from RA-treated P19 cells were immunoprecipitated (IP) with anti-MAGE-G1 antibody. IgG antibody was used as negative control of immunoprecipitation and 25 μg whole cell lysate was used as input. The immunoblotting (IB) were probed for the immunoprecipitated proteins with anti-VIME antibody.
Figure Legend Snippet: Validation of the interaction between MAGE-G1 and VIME by GST pull-down and co-immunoprecipitation experiments. ( a ) GST or GST-VIME proteins were expressed in Escherichia coli BL21 respectively and purified with Glutathione-Sepharose 4B beads and washed, then beads were incubated with Flag-MAGE-G1 expressed in HEK293T. Flag-MAGE-G1 and GST-VIME were detected with indicated antibody. Full-length blots are included in a Supplementary Information . ( b ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Vime or pCMV-3 × Flag empty vector plus GFP- Vime expression plasmids. 25 μg of whole cell protein lysate was used as input to confirm the expression of the Flag-MAGE-G1 (with anti-Flag) or GFP-VIME (with anti-GFP) by immunoblotting (IB). The rest of cell lysates were incubated with anti-Flag-magnetics beads. The immunoprecipitated (IP) protein complex was resolved by SDS-PAGE and probed with antibodies against Flag or GFP. ( c ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Vime or pEGFP empty vector plus Flag- Mage - g1 expression plasmids. The experiment procedure was same as that mentioned above except that cell lysates were immunoprecipitaed with anti-GFP-magnetics beads. ( d ) Whole cell lysates from RA-treated P19 cells were immunoprecipitated (IP) with anti-MAGE-G1 antibody. IgG antibody was used as negative control of immunoprecipitation and 25 μg whole cell lysate was used as input. The immunoblotting (IB) were probed for the immunoprecipitated proteins with anti-VIME antibody.

Techniques Used: Immunoprecipitation, Purification, Incubation, Transfection, Plasmid Preparation, Expressing, SDS Page, Negative Control

Validation of the interaction between MAGE-G1 and FSCN1 by GST pull-down and co-immunoprecipitation experiments. ( a ) GST or GST-FSCN1 proteins were expressed in Escherichia coli BL21 respectively and purified with Glutathione-Sepharose 4B beads and washed, then beads were incubated with Flag-MAGE-G1 expressed in HEK293T. Flag-MAGE-G1 and GST-FSCN1 were detected with indicated antibody. Full-length blots are included in a Supplementary Information . ( b ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Fscn1 or pCMV-3 × Flag empty vector plus pEGFP- Fscn1 expression plasmids. 25 μg of whole cell protein lysate was used as input to confirm the expression of the Flag-MAGE-G1 (with anti-Flag) or GFP-FSCN1 (with anti-GFP) by immunoblotting (IB). The rest of cell lysates were incubated with anti-Flag-magnetics beads. The immunoprecipitated (IP) protein complex was resolved by SDS-PAGE and probed with antibodies against Flag or GFP. ( c ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Fscn1 or pEGFP empty vector plus Flag- Mage - g1 expression plasmids. The experiment procedure was same as that mentioned above except that cell lysates were immunoprecipitaed with anti-GFP-magnetics beads. ( d ) Whole cell lysates from RA-treated P19 cells were immunoprecipitated with anti-MAGE-G1 antibody. IgG antibody was used as negative control of immunoprecipitation (IP) and 25 μg whole cell lysate was used as input. The immunoblotting (IB) were probed for the immunoprecipitated proteins with anti-FSCN1 antibody.
Figure Legend Snippet: Validation of the interaction between MAGE-G1 and FSCN1 by GST pull-down and co-immunoprecipitation experiments. ( a ) GST or GST-FSCN1 proteins were expressed in Escherichia coli BL21 respectively and purified with Glutathione-Sepharose 4B beads and washed, then beads were incubated with Flag-MAGE-G1 expressed in HEK293T. Flag-MAGE-G1 and GST-FSCN1 were detected with indicated antibody. Full-length blots are included in a Supplementary Information . ( b ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Fscn1 or pCMV-3 × Flag empty vector plus pEGFP- Fscn1 expression plasmids. 25 μg of whole cell protein lysate was used as input to confirm the expression of the Flag-MAGE-G1 (with anti-Flag) or GFP-FSCN1 (with anti-GFP) by immunoblotting (IB). The rest of cell lysates were incubated with anti-Flag-magnetics beads. The immunoprecipitated (IP) protein complex was resolved by SDS-PAGE and probed with antibodies against Flag or GFP. ( c ) COS-7 cells were co-transfected with either Flag- Mage - g1 plus GFP- Fscn1 or pEGFP empty vector plus Flag- Mage - g1 expression plasmids. The experiment procedure was same as that mentioned above except that cell lysates were immunoprecipitaed with anti-GFP-magnetics beads. ( d ) Whole cell lysates from RA-treated P19 cells were immunoprecipitated with anti-MAGE-G1 antibody. IgG antibody was used as negative control of immunoprecipitation (IP) and 25 μg whole cell lysate was used as input. The immunoblotting (IB) were probed for the immunoprecipitated proteins with anti-FSCN1 antibody.

Techniques Used: Immunoprecipitation, Purification, Incubation, Transfection, Plasmid Preparation, Expressing, SDS Page, Negative Control

40) Product Images from "Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct"

Article Title: Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

Journal: Cells

doi: 10.3390/cells9051208

Co-immunoprecipitation of aquaporin-2 (AQP2) and sorting nexin 27 (SNX27). ( A , B ) Immunoblotting of AQP2 in pull-down samples from rat kidney inner medulla tubule suspension using pre-immune immunoglobulin G (IgG) of mouse (mIgG), Dynabead M-280 with anti-SNX27 antibody, pre-immune IgG of rabbit (rIgG), or Dynabead M-280 with anti-AQP2 antibody, respectively. ( C , D ) Immunoblotting of SNX27 in pull-down samples from rat kidney inner medulla tubule suspension using pre-immune IgG of mouse (mIgG), Dynabead M-280 with anti-SNX27 antibody, pre-immune IgG of rabbit (rIgG), or Dynabead M-280 with anti-AQP2 antibody, respectively. ( E ) Immunoblotting of vacuolar protein sorting-associated protein 35 (Vps35) in pull-down samples from rat kidney inner medulla tubule suspension using pre-immune IgG of rabbit (rIgG) or Dynabead M-280 with anti-SNX27 antibody, respectively. ( F ) Human Embryonic Kidney 293T (HEK293T) cells were transiently expressed with hemagglutinin (HA)-tagged AQP2 (full length) plasmid alone or both HA-tagged AQP2 (full length) and FLAG-tagged SNX27 (full length) plasmid. Immunoblotting of AQP2 and SNX27. In the control condition (Con), cells were transfected only with p3XFLAG-CMV-10 and pcDNA3.1-HA. ( G ) Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-AQP2 antibody and anti-SNX27 antibody. ( H ) Schematic representation of SNX27 constructs. ( I ) Immunoblotting using anti-glutathione S-transferase (GST) antibody after purification of GST-tagged SNX27 constructs. ( J ) Immunoblotting using anti-AQP2 antibody after purification of histidine (His)-tagged carboxyl terminus of AQP2 (AQP2c). ( K , L ) GST-SNX27 fusion proteins were incubated with His-tagged AQP2c proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted with anti-glutathione S-transferase (GST) or anti-AQP2 antibody.
Figure Legend Snippet: Co-immunoprecipitation of aquaporin-2 (AQP2) and sorting nexin 27 (SNX27). ( A , B ) Immunoblotting of AQP2 in pull-down samples from rat kidney inner medulla tubule suspension using pre-immune immunoglobulin G (IgG) of mouse (mIgG), Dynabead M-280 with anti-SNX27 antibody, pre-immune IgG of rabbit (rIgG), or Dynabead M-280 with anti-AQP2 antibody, respectively. ( C , D ) Immunoblotting of SNX27 in pull-down samples from rat kidney inner medulla tubule suspension using pre-immune IgG of mouse (mIgG), Dynabead M-280 with anti-SNX27 antibody, pre-immune IgG of rabbit (rIgG), or Dynabead M-280 with anti-AQP2 antibody, respectively. ( E ) Immunoblotting of vacuolar protein sorting-associated protein 35 (Vps35) in pull-down samples from rat kidney inner medulla tubule suspension using pre-immune IgG of rabbit (rIgG) or Dynabead M-280 with anti-SNX27 antibody, respectively. ( F ) Human Embryonic Kidney 293T (HEK293T) cells were transiently expressed with hemagglutinin (HA)-tagged AQP2 (full length) plasmid alone or both HA-tagged AQP2 (full length) and FLAG-tagged SNX27 (full length) plasmid. Immunoblotting of AQP2 and SNX27. In the control condition (Con), cells were transfected only with p3XFLAG-CMV-10 and pcDNA3.1-HA. ( G ) Cell lysates were immunoprecipitated with anti-HA antibody and immunoblotted with anti-AQP2 antibody and anti-SNX27 antibody. ( H ) Schematic representation of SNX27 constructs. ( I ) Immunoblotting using anti-glutathione S-transferase (GST) antibody after purification of GST-tagged SNX27 constructs. ( J ) Immunoblotting using anti-AQP2 antibody after purification of histidine (His)-tagged carboxyl terminus of AQP2 (AQP2c). ( K , L ) GST-SNX27 fusion proteins were incubated with His-tagged AQP2c proteins and precipitated using Glutathione Sepharose 4B beads. Precipitates were immunoblotted with anti-glutathione S-transferase (GST) or anti-AQP2 antibody.

Techniques Used: Immunoprecipitation, Plasmid Preparation, Transfection, Construct, Purification, Incubation

Related Articles

In Vitro:

Article Title: An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu)
Article Snippet: .. In vitro phosphorylation activity was determined using RhoGDI2 immobilized on glutathione Sepharose beads as the substrate and purified Src kinase. .. One ug of Src kinase was used per reaction and incubated at 1 h at RT in kinase buffer (50 mM Hepes-NaOH, pH 7.5, 10 mM MgCl2 , 10 mM MnCl2 , 0.2 mM DTT, 1 mM ATP).

Purification:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies. ..

Article Title: TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction
Article Snippet: .. For the GST-LC3B[G120] affinity-isolation assay, HEK293 cells expressing TP53INP2[NLSΔ], TP53INP2[Δ1-28],[NLSΔ] TP53INP2W35,I38A [NLSΔ], TP53INP2[Δ67-111],[NLSΔ] or TP53INP2[Δ112-144],[NLSΔ] were lysed and the cell lysate was incubated with purified GST or GST-LC3B[G120] proteins at 4°C for 4 h. Then the glutathione-sepharose 4B beads were added to the mixture followed by incubation at 4°C for 2 h. Immunocomplexes were washed and used for western blot. .. Protein bands were detected with Coomassie Brilliant Blue and quantified using the ImageJ software.

Article Title: An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu)
Article Snippet: .. In vitro phosphorylation activity was determined using RhoGDI2 immobilized on glutathione Sepharose beads as the substrate and purified Src kinase. .. One ug of Src kinase was used per reaction and incubated at 1 h at RT in kinase buffer (50 mM Hepes-NaOH, pH 7.5, 10 mM MgCl2 , 10 mM MnCl2 , 0.2 mM DTT, 1 mM ATP).

Incubation:

Article Title: The Putative RNA Helicase HELZ Promotes Cell Proliferation, Translation Initiation and Ribosomal Protein S6 Phosphorylation
Article Snippet: .. Crude HeLa cell lysates were incubated with recombinant GST, the indicated GST-HELZ fragments, GST-HIF-1α530–826 as well as GST-Paip2106–127 and GST pull–down was conducted using glutathione–sepharose beads. .. Eluates were subjected to SDS–PAGE and immunoblotting. (TIF) Click here for additional data file.

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies. ..

Article Title: Tyr728 in the Kinase Domain of the Murine Kinase Suppressor of RAS 1 Regulates Binding and Activation of the Mitogen-activated Protein Kinase Kinase *
Article Snippet: .. The supernatant containing GST-tagged KSR1 protein was incubated with 1 ml of glutathione-Sepharose beads for 2 h at 4 °C with rotation. .. After incubation, the beads were washed three times with buffer containing 25 m m Tris-HCl (pH 7.6), 300 m m sodium chloride, 25 m m β-glycerophosphate, 25 m m sodium fluoride, 10 m m sodium pyrophosphate, 10 m m β-mercaptoethanol, 1 m m sodium orthovanadate, 10% (v/v) glycerol, 0.2% (v/v) Nonidet P-40, and standard proteinase inhibitors.

Article Title: TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction
Article Snippet: .. For the GST-LC3B[G120] affinity-isolation assay, HEK293 cells expressing TP53INP2[NLSΔ], TP53INP2[Δ1-28],[NLSΔ] TP53INP2W35,I38A [NLSΔ], TP53INP2[Δ67-111],[NLSΔ] or TP53INP2[Δ112-144],[NLSΔ] were lysed and the cell lysate was incubated with purified GST or GST-LC3B[G120] proteins at 4°C for 4 h. Then the glutathione-sepharose 4B beads were added to the mixture followed by incubation at 4°C for 2 h. Immunocomplexes were washed and used for western blot. .. Protein bands were detected with Coomassie Brilliant Blue and quantified using the ImageJ software.

Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking
Article Snippet: .. The soluble fraction of the lysed cells was incubated 2 h at 4 °C with 10 μg GST alone or GST-tagged golgin-160(1–393) that had been pre-conjugated to glutathione-Sepharose 4B beads. .. After washing the beads twice with lysis buffer, protein bound to the beads was separated by SDS-PAGE and β1AR was detected by immunoblotting with anti-FLAG antibody followed by ECL.

Article Title: Measles Virus Infection Inactivates Cellular Protein Phosphatase 5 with Consequent Suppression of Sp1 and c-Myc Activities
Article Snippet: .. The GST-Sp1 bound to the glutathione-Sepharose beads was incubated with the cell lysate for 2 h at 4°C, and the beads were then washed with kinase buffer. .. The beads were resuspended in 20 μl of kinase buffer supplemented with 4 μCi of [γ-32 P]ATP/μl (3,000 Ci/mmol) and incubated for 1 h at 30°C.

Activity Assay:

Article Title: An adventitious interaction of filamin A with RhoGDI2(Tyr153Glu)
Article Snippet: .. In vitro phosphorylation activity was determined using RhoGDI2 immobilized on glutathione Sepharose beads as the substrate and purified Src kinase. .. One ug of Src kinase was used per reaction and incubated at 1 h at RT in kinase buffer (50 mM Hepes-NaOH, pH 7.5, 10 mM MgCl2 , 10 mM MnCl2 , 0.2 mM DTT, 1 mM ATP).

Expressing:

Article Title: TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction
Article Snippet: .. For the GST-LC3B[G120] affinity-isolation assay, HEK293 cells expressing TP53INP2[NLSΔ], TP53INP2[Δ1-28],[NLSΔ] TP53INP2W35,I38A [NLSΔ], TP53INP2[Δ67-111],[NLSΔ] or TP53INP2[Δ112-144],[NLSΔ] were lysed and the cell lysate was incubated with purified GST or GST-LC3B[G120] proteins at 4°C for 4 h. Then the glutathione-sepharose 4B beads were added to the mixture followed by incubation at 4°C for 2 h. Immunocomplexes were washed and used for western blot. .. Protein bands were detected with Coomassie Brilliant Blue and quantified using the ImageJ software.

Western Blot:

Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Article Snippet: .. pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies. ..

Article Title: TP53INP2 contributes to autophagosome formation by promoting LC3-ATG7 interaction
Article Snippet: .. For the GST-LC3B[G120] affinity-isolation assay, HEK293 cells expressing TP53INP2[NLSΔ], TP53INP2[Δ1-28],[NLSΔ] TP53INP2W35,I38A [NLSΔ], TP53INP2[Δ67-111],[NLSΔ] or TP53INP2[Δ112-144],[NLSΔ] were lysed and the cell lysate was incubated with purified GST or GST-LC3B[G120] proteins at 4°C for 4 h. Then the glutathione-sepharose 4B beads were added to the mixture followed by incubation at 4°C for 2 h. Immunocomplexes were washed and used for western blot. .. Protein bands were detected with Coomassie Brilliant Blue and quantified using the ImageJ software.

Recombinant:

Article Title: The Putative RNA Helicase HELZ Promotes Cell Proliferation, Translation Initiation and Ribosomal Protein S6 Phosphorylation
Article Snippet: .. Crude HeLa cell lysates were incubated with recombinant GST, the indicated GST-HELZ fragments, GST-HIF-1α530–826 as well as GST-Paip2106–127 and GST pull–down was conducted using glutathione–sepharose beads. .. Eluates were subjected to SDS–PAGE and immunoblotting. (TIF) Click here for additional data file.

Chromatin Immunoprecipitation:

Article Title: Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP *
Article Snippet: .. When A375 cell lysate was passed through a column prepared by immobilizing GST-IRF-1 on glutathione-Sepharose beads, endogenous CHIP bound specifically to GST-IRF-1 and not to a GST alone control column ( A ). .. Additionally, CHIP was co-immunoprecipitated with IRF-1 from A375 cells in which both proteins were overexpressed ( B ).

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    GE Healthcare glutathione sepharose 4b
    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on <t>glutathione-sepharose</t> beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
    Glutathione Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 713 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare glutathione sepharose 4b beads
    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to <t>glutathione-Sepharose</t> 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
    Glutathione Sepharose 4b Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Journal: Molecular Therapy. Nucleic Acids

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

    doi: 10.1016/j.omtn.2018.02.011

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

    Article Snippet: Then the fusion protein GST-α-syn was purified on glutathione-sepharose 4B according to the manufacturer’s instructions (GE Healthcare, Boston, MA).

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

    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Journal: International Journal of Molecular Sciences

    Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

    doi: 10.3390/ijms15022929

    Figure Lengend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Article Snippet: The soluble fraction of the lysed cells was incubated 2 h at 4 °C with 10 μg GST alone or GST-tagged golgin-160(1–393) that had been pre-conjugated to glutathione-Sepharose 4B beads.

    Techniques: Purification, Binding Assay, Incubation, Staining, SDS Page

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0022107

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

    Article Snippet: Crude HeLa cell lysates were incubated with recombinant GST, the indicated GST-HELZ fragments, GST-HIF-1α530–826 as well as GST-Paip2106–127 and GST pull–down was conducted using glutathione–sepharose beads.

    Techniques: Binding Assay, Recombinant, Incubation, SDS Page

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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2016.02119

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

    Article Snippet: pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies.

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

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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2016.02119

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

    Article Snippet: pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies.

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

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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2016.02119

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

    Article Snippet: pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies.

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

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

    Journal: Frontiers in Microbiology

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

    doi: 10.3389/fmicb.2016.02119

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

    Article Snippet: pVIII Protein Interacts with eIFs Indirectly via Its Interaction with DDX3 To determine if the interaction of pVIII and DDX3 also affects the level of eIFs, the pellet and supernatant fractions of the rabbit-reticulo Lysates incubated with glutathione sepharose beads preloaded with purified GST.pVIII or GST were analyzed by Western blot using protein specific antibodies.

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