Journal:

Article Title: Myxoma Virus M11L Prevents Apoptosis through Constitutive Interaction with Bak

doi: 10.1128/JVI.78.13.7097-7111.2004

Figure Lengend Snippet: (A) M11L and huBak constructs utilized for this study. Constructs were either left untagged or fused to Flag, TAP, or HA epitope tags as indicated. The TAP cassette is composed of three components; the protein A (ProtA) IgG-binding sequence and the calmodulin binding protein (CBP) domain are separated by a TEV protease cleavage site. (B) Table listing the binding partners associated with M11L during Flag pull-down assays and identified by mass spectrometry. Interaction frequency, percentage of 20 independent transfections in which the protein was identified. (C) Immunoprecipitation of Flag-M11L and identification of associated proteins. Cell lysates were immunoprecipitated with anti-Flag agarose, and bound proteins were eluted with Flag peptide. Eluted proteins were resolved on a 4 to 15% Tris-HCl SDS-gel and stained with Coomassie blue. Lane 1, molecular weight markers; lane 2, immunoprecipitation from untransfected control HEK293T cells; lane 3, immunoprecipitation from cells transfected with Flag-M11L. Each of the bands in the second and third lanes was excised, digested with trypsin, and analyzed by mass spectrometry. (D) Typical output from mass spectrometry of peptide sequence run. A short, 10-amino-acid residue peptide within the complete Bak sequence was identified as a Bak peptide (lowercased letters).

Article Snippet: The beads were washed three times with wash buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% CHAPS) and resuspended in 1 ml of tobacco etch virus (TEV) cleavage buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1 mM dithiothreitol) containing 200 U of TEV protease (Invitrogen).

Techniques: Construct, Binding Assay, Sequencing, Mass Spectrometry, Transfection, Immunoprecipitation, SDS-Gel, Staining, Molecular Weight

Journal:

Article Title: Myxoma Virus M11L Prevents Apoptosis through Constitutive Interaction with Bak

doi: 10.1128/JVI.78.13.7097-7111.2004

Figure Lengend Snippet: M11L specifically binds to Bak but not to other Bcl-2 family members by use of the TAP method. (A) TAP method for isolating M11L binding partners. The plasmid (M11L-C-TAP) was transiently transfected into 293T cells by the calcium phosphate method (step 1). The lysate with putative TAP M11L binding partner complexes was incubated with rabbit agarose IgG beads in order to bind the protein A of the TAP cassette. The beads were washed to remove unbound proteins and then incubated with TEV protease (step 2) to release bound complexes. The protein complex from cleaved products was allowed to bind via calmodulin binding protein (CBP) resin, and then the purified complex was eluted with calmodulin elution buffer containing EGTA (step 3). The eluants, containing proteins interacting with M11L, were separated by SDS-PAGE, and the proteins were analyzed (step 4). (B) Bak, but not Bad, Bid, Bax/p21, or Bcl-2, was detected as a binding partner of M11L in the M11L-C-TAP eluted products (lane 3), but not in control eluants of C-TAP alone (lane 2), following lysis with CHAPS buffer. Lane 1 confirms endogenous expression levels of the various Bcl-2 family members in 293T cell lysates. (C) Cell lysates of the C-TAP vector alone (lane 1) or the M11L-C-TAP bait (lane 2) were probed with anti-PAP before the TAP procedure to confirm the expression and size of the M11L-TAP fusion protein (34 kDa). (D) Eluants of the C-TAP vector alone (lane 1) or M11L-C-TAP (lane 2) were immunoblotted with anti-M11L, indicating that the M11L bait tagged with CBP (17 kDa) was recovered following TEV cleavage and elution. (E) huBak binds to untagged M11L. Shown are TAP eluants from lysates of cotransfections of pcDNA3-M11L plus N-TAP vector alone (lane 1) or pcDNA3-M11L plus N-TAP-huBak (lane 2) in 293T cells. Lane 3 (M11L and TAP vector) and lane 4 (M11L and N-TAP-huBak) represent corresponding cell lysates, confirming the expression of the appropriate cotransfected plasmids.

Article Snippet: The beads were washed three times with wash buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% CHAPS) and resuspended in 1 ml of tobacco etch virus (TEV) cleavage buffer (10 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1 mM dithiothreitol) containing 200 U of TEV protease (Invitrogen).

Techniques: Binding Assay, Plasmid Preparation, Transfection, Incubation, Purification, SDS Page, Lysis, Expressing

Journal: Microbial Biotechnology

Article Title: Enhancing the capability of Klebsiella pneumoniae to produce 1, 3‐propanediol by overexpression and regulation through CRISPR‐dCas9

doi: 10.1111/1751-7915.14033

Figure Lengend Snippet: Strains and plasmids used in this study.

Article Snippet: pRH2502 , Expression of dcas9 D10A H840A from a TetR‐regulated uvtetO promoter, Kan R , Addgene, USA.

Techniques: Mutagenesis, Derivative Assay, Plasmid Preparation, Cloning, Expressing