cbd  (New England Biolabs)

Journal: Cancer research

Article Title: High levels of Nrf2 determine chemoresistance in type II endometrial cancer

doi: 10.1158/0008-5472.CAN-10-0713

Figure Lengend Snippet: Stable knockdown of Nrf2 by overexpression of Keap1 increased the susceptibility of SPEC-2 cells to chemotherapeutic drugs. (A) Two SPEC-2-derived cell lines, control and Keap1 + , stably expressing the control vector or Keap1-CBD were established using a retrovirus-based system. mRNA extracted from these two cell lines were subjected to real-time RT-PCR. (B) Cell lysates from these two cell lines were subjected to immunoblot analysis with antibodies against Nrf2, CBD (for detection of Keap1), NQO1, γ-GCS, and GAPDH. (C) The NQO1 enzymatic activity was measured by reduction of DCPIP. (D) The intracellular glutathione level was measured by using the QuantiChrom Glutathione Assay Kit (E) Cell viability was assessed by the MTT assay following 48 h treatment with the indicated doses of cisplatin and paclitaxel. The data are presented as means ± SD, *P

Article Snippet: Overexpression of Keap1-CBD was confirmed by immunoblot analysis with an anti-CBD antibody ( , Keap1-CBD panel).

Techniques: Over Expression, Derivative Assay, Stable Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Activity Assay, Glutathione Assay, MTT Assay

Journal: Cancer research

Article Title: High levels of Nrf2 determine chemoresistance in type II endometrial cancer

doi: 10.1158/0008-5472.CAN-10-0713

Figure Lengend Snippet: Keap1 + -tumors had a reduction in cell proliferation and an induction of apoptosis in response to cisplatin treatment. (A) Protein levels of Nrf2 and Keap1-CBD in tumor tissues. Each lane contained the tumor tissue lysate extracted from individual mouse. Three tumor tissues per group were used for the immunoblot analysis. (B) Reduced Nrf2 expression in Keap1 + -tumors was confirmed by IHC staining. (C) Keap1 + tumor tissues had reduced proliferation in response to cisplatin treatment, as measured by IHC-Ki67. (D) Keap1 + tumor tissues had reduced proliferation in response to cisplatin treatment, as measured by TUNEL staining.

Article Snippet: Overexpression of Keap1-CBD was confirmed by immunoblot analysis with an anti-CBD antibody ( , Keap1-CBD panel).

Techniques: Expressing, Immunohistochemistry, Staining, TUNEL Assay

Journal: Analytical chemistry

Article Title: Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins

doi: 10.1021/ac400949t

Figure Lengend Snippet: Antibody/fused-tag experiments. Measurements of AFM binding forces for: (A) CBD versus anti-CBD antibody and (B) MBP versus anti-MBP antibody. These experiments were performed using I C (with both fused domains CBD and MBP, according to ) with

Article Snippet: Anti-MBP and anti-CBD monoclonal antibodies were used for protein immobilization (New England Biolabs, Ipswich, MA).

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding

doi: 10.1128/JVI.00965-17

Figure Lengend Snippet: Profolding activity of vIL-6. (A) HEK293T cells were cotransfected with vectors expressing a GFP-fused version of the NHK folding variant of α1-antitrypsin (NHK-GFP) and vIL-6 or with NHK-GFP expression plasmid and empty vector (vec). At 48 h posttransfection, cell lysates were generated using NP-40-containing buffer, material microcentrifuged, and pellet (insoluble [insol.]) and supernatant (soluble [sol.]) fractions were denatured, SDS-PAGE fractionated, and immunoblotted for detection of GFP and also vIL-6 (to confirm expression) and ER luminal soluble protein BiP (to ensure appropriate separation of soluble and pellet fractions). The relative amounts of soluble to insoluble NHK-GFP are shown in the chart. (B) An independent assay was undertaken using secreted HHV-8 glycoprotein L (gL), fused to CBD, as the readout for folding. CBD-tagged HHV-8 gH was coexpressed with gL-CBD to enable secretory trafficking, and either vIL-6 expression plasmid or empty vector (vec) was cotransfected into HEK293T cells with the glycoprotein expression plasmids. After 48 h, cells and media were harvested for preparation of lysates and chitin bead precipitates (for concentration of secreted gL-CBD). Cell lysates (1% of total) and media precipitates (10%) were analyzed by immunoblotting for detection of gL-CBD; gH, vIL-6, and β-actin (loading control) were also analyzed in the lysate fractions. (C) An experiment equivalent to that of panel A was carried out in native HEK293T cells or VKORC1v2- or UGGT1-knockout (KO) derivatives. CBD- and KDEL motif-tagged hIL-6 (h6-C′-K) was included as a negative control along with empty vector (vec) for comparison with vIL-6 (CBD-fused, v6-CBD). Calculated ratios of NHK-GFP in the soluble and insoluble fractions of cell lysates are shown below each corresponding set of immunoblots. Digitally captured data from all immunoblots (panels A to C) were quantified using GeneTools (Syngene) analysis software.

Article Snippet: Commercially acquired antibodies used for immunoblotting and immunoprecipitation were as follows: CBD (New England BioLabs, catalog no. E8034S), S-tag (Cell Signaling Technology, catalog no. 8476S), GFP (Santa Cruz Biotechnology, catalog no. sc-8334), Strep II (Qiagen, catalog no. 34850), Flag (Sigma, catalog no. F7425), hemagglutinin (HA; Sigma, catalog no. H6908), β-actin (Sigma, catalog no. A5316), BiP (BD Biosciences, catalog number 610978), STAT3 (Santa Cruz Biotechnology, catalog number sc-482), pSTAT3 (Cell Signaling Technologies, catalog number 9131S), and gp130 (Santa Cruz Biotechnology, catalog number sc-655).

Techniques: Activity Assay, Expressing, Variant Assay, Plasmid Preparation, Generated, SDS Page, Concentration Assay, Knock-Out, Negative Control, Western Blot, Software

Journal: Journal of Virology

Article Title: Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding

doi: 10.1128/JVI.00965-17

Figure Lengend Snippet: Interactions of vIL-6 and VKORC1v2 with GlucIIα and UGGT1. (A) Coprecipitation assays were carried out using lysates of cells cotransfected with expression plasmids for CBD-fused vIL-6 and S-tag-fused GlucIIα or CBD-fused VKORC1v2 (v2-CBD) and StrepII-tagged UGGT1 to test the interactions between these protein pairs. CBD- and KDEL ER retention motif-linked hIL-6 (hIL-6-CBD.K) and CBD-tagged VKORC1v1 (v1-CBD) were used as negative controls in the respective experiments. Protein complexes were precipitated with CBD-binding chitin beads, and SDS-PAGE-fractionated and membrane-transferred proteins in precipitates and cell lysates were identified by immunoblotting with CBD antibody to visualize “baits” vIL-6 and VKORC1v2 or with S-peptide or StrepII antibody for detection of their candidate binding partners. β-actin probing of cell lysates provided a loading control. (B) Further precipitations were undertaken to test whether vIL-6 and VKORC1v2 could interact with UGGT1 and GlucIIα, respectively. In this experiment, GlucIIα-S and StrepII-UGGT1 were coexpressed with CBD-tagged vIL-6 or VKORC1v2, or negative controls hIL-6-CBD.K (hIL-6-C′.K) or VKORC1v1-CBD (v1-CBD). Chitin bead precipitates and cell lysates were analyzed as described previously. (C) Immunoprecipitation (IP) of vIL-6 from BCBL-1 PEL cell lysates and subsequent immunoblotting for GlucIIα and UGGT1. A sample of the lysate (10% of amount used for IP) was analyzed alongside material precipitated with vIL-6 antiserum (vIL-6) or control rabbit serum.

Article Snippet: Commercially acquired antibodies used for immunoblotting and immunoprecipitation were as follows: CBD (New England BioLabs, catalog no. E8034S), S-tag (Cell Signaling Technology, catalog no. 8476S), GFP (Santa Cruz Biotechnology, catalog no. sc-8334), Strep II (Qiagen, catalog no. 34850), Flag (Sigma, catalog no. F7425), hemagglutinin (HA; Sigma, catalog no. H6908), β-actin (Sigma, catalog no. A5316), BiP (BD Biosciences, catalog number 610978), STAT3 (Santa Cruz Biotechnology, catalog number sc-482), pSTAT3 (Cell Signaling Technologies, catalog number 9131S), and gp130 (Santa Cruz Biotechnology, catalog number sc-655).

Techniques: Expressing, Binding Assay, SDS Page, Immunoprecipitation

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

Article Title: PKN delays mitotic timing by inhibition of Cdc25C: Possible involvement of PKN in the regulation of cell division

doi:

Figure Lengend Snippet: PKN does not enhance Wee1 activity in vitro . Wee1 activity was assayed by using the substrate Cdc2 (N133A)/cyclin B complex. Affinity-purified GST/Wee1 was incubated with GST/PKN () or GST/PKN ()-K644E. The Cdc2 (N133A)/cyclin B complex was then added, and samples were incubated for 15 min at 25°C. Phosphorylation of Tyr-15 of Cdc2 was analyzed by Western blotting with antiphospho-Tyr-15 antibody ( Upper ). The same blot was reprobed with anti-Cdc2 antibody ( Lower ). GST/Wee1, GST/PKN (), and GST/PKN ()-K644E are indicated as Wee1, PKN, and PKN(KN), respectively.

Article Snippet: Antiphospho-Tyr-15 of Cdc2 polyclonal antibody and anti-Cdc2 monoclonal antibody were obtained from New England Biolabs and Santa Cruz Biotechnology, respectively.

Techniques: Activity Assay, In Vitro, Affinity Purification, Incubation, Western Blot

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

Article Title: PKN delays mitotic timing by inhibition of Cdc25C: Possible involvement of PKN in the regulation of cell division

doi:

Figure Lengend Snippet: Exogenous addition of PKN delays activation of Cdc2/cyclin B histone H1 kinase in egg-cycling extracts. Samples withdrawn from extracts at each time point were assayed for Cdc2/cyclin B histone H1 kinase activity and phosphorylation of Tyr-15 of Cdc2. Oscillation of histone H1 kinase activity in the cycling extracts containing buffer ( A ), GST/PKN () ( B ), and GST/PKCɛ (386−737) ( C ) are shown. A Lower and B Lower show phosphorylation of Tyr-15 of Cdc2 in extracts containing buffer or GST/PKN () by Western blotting with antiphospho-Tyr-15 antibody.

Article Snippet: Antiphospho-Tyr-15 of Cdc2 polyclonal antibody and anti-Cdc2 monoclonal antibody were obtained from New England Biolabs and Santa Cruz Biotechnology, respectively.

Techniques: Activation Assay, Activity Assay, Western Blot

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

Article Title: PKN delays mitotic timing by inhibition of Cdc25C: Possible involvement of PKN in the regulation of cell division

doi:

Figure Lengend Snippet: PKN inhibits Cdc25C activity in vitro . Cdc25C tyrosine phosphatase activity was assayed by using the Cdc2 (N133A)/cyclin B complex purified from Sf9 cells as the phosphorylated substrate. Affinity-purified GST/Cdc25C was incubated with GST/PKN () or GST/PKN ()-K644E; then Cdc2 dephosphorylation was initiated by addition of the Cdc2 (N133A)/cyclin B complex. Dephosphorylation of Tyr-15 of Cdc2 was analyzed by Western blotting with antiphospho-Tyr-15 antibody ( Upper ). The same blot was reprobed with anti-Cdc2 antibody ( Lower ). GST/Cdc25C, GST/PKN (), and GST/PKN ()-K644E are indicated as Cdc25C, PKN, and PKN(KN), respectively.

Article Snippet: Antiphospho-Tyr-15 of Cdc2 polyclonal antibody and anti-Cdc2 monoclonal antibody were obtained from New England Biolabs and Santa Cruz Biotechnology, respectively.

Techniques: Activity Assay, In Vitro, Purification, Affinity Purification, Incubation, De-Phosphorylation Assay, Western Blot

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

Article Title: PKN delays mitotic timing by inhibition of Cdc25C: Possible involvement of PKN in the regulation of cell division

doi:

Figure Lengend Snippet: Protein preparations. An aliquot of each recombinant protein was resolved by SDS/PAGE and silver stained. Lane 1, GST/PKCɛ (); lane 2, GST/PKN ()-K644E; lane 3, GST/PKN (); lane 4, GST/Wee1; lane 5, GST/Cdc25; and lane 6, the complex of GST/cyclin B and (His) 6 /Cdc2 (N133A).

Article Snippet: Antiphospho-Tyr-15 of Cdc2 polyclonal antibody and anti-Cdc2 monoclonal antibody were obtained from New England Biolabs and Santa Cruz Biotechnology, respectively.

Techniques: Recombinant, SDS Page, Staining