Journal: BMC Biochemistry

Article Title: Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

doi: 10.1186/1471-2091-15-21

Figure Lengend Snippet: Domain architecture and sequences of human and mouse BNIP-2. A . Known or putative functional motifs indicated are: the Rho-binding domain (RBD), Cdc42/Rac1-Interactive Binding (CRIB)-like motif, BCH signature motif, BCH/BCH interaction motif, caspase and granzyme cleavage sites and kinesin-targeting motifs. Annotation of the RBD is based on close similarity to known RBDs in BNIP-2 homologs and Cdc42GAP. B . ClustalW multiple sequence alignment of human and mouse BNIP-2 splice variants, i.e., the database annotated hBNIP2 sequence [Swiss-Prot: Q12982] as well as short mBNIP-2 [Trembl: Q91VL0] and long mBNIP-2 [Swiss-Prot: O54940]. The black box highlights the P4-P9′ motif with the P4-P1 IEAD GrB cleavage motif and BNIP-2 domains indicated as in A .

Article Snippet: The TnT Quick Master Mix (40 μl) (pOTB7-hBNIP-2 (RZPD Imagenes, cat n° IRAUp969D059D, Germany) and pCMV-SPORT6-mBNIP-2 (RZPD Imagenes, cat n° IRAVp968F0522D, Germany)) – consisting of a rabbit reticulocyte lysate, reaction buffer, SP6 or T7 RNA polymerase, an amino acid mixture without methionine and RNasin Ribonuclease Inhibitor – was mixed with 2 μl [ 35 S]-methionine (20 μCi/ml), 1 μg of plasmid DNA template (1 μg/μl) and 7 μl nuclease-free water.

Techniques: Functional Assay, Binding Assay, Sequencing

Journal: BMC Biochemistry

Article Title: Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

doi: 10.1186/1471-2091-15-21

Figure Lengend Snippet: Autoradiographs showing GrB induced cleavage of murinized human BNIP-2 variants. P3′ (A) and P1′ (B) differing primed site residues following the identified P4-P1 cleavage site IEAD in hBNIP-2 were mutated to their corresponding mBNIP-2 amino acids. The in vitro transcribed and translated (mutated) BNIP-2 variants were incubated with varying concentrations (ranging from 1.95 nM to 1 μM (from left to right)) of human or mouse granzyme B. Black arrows indicate BNIP-2 precursor patterns, whereas dashed and grey arrows are indicative for BNIP-2 cleavage fragments. Percentages of cleavage are shown in the progression curves of hGrB and mGrB cleavage and indicated by full and dashed lines respectively.

Article Snippet: The TnT Quick Master Mix (40 μl) (pOTB7-hBNIP-2 (RZPD Imagenes, cat n° IRAUp969D059D, Germany) and pCMV-SPORT6-mBNIP-2 (RZPD Imagenes, cat n° IRAVp968F0522D, Germany)) – consisting of a rabbit reticulocyte lysate, reaction buffer, SP6 or T7 RNA polymerase, an amino acid mixture without methionine and RNasin Ribonuclease Inhibitor – was mixed with 2 μl [ 35 S]-methionine (20 μCi/ml), 1 μg of plasmid DNA template (1 μg/μl) and 7 μl nuclease-free water.

Techniques: In Vitro, Incubation

Journal: BMC Biochemistry

Article Title: Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

doi: 10.1186/1471-2091-15-21

Figure Lengend Snippet: GrB induced BNIP-2 cleavage in cellulo . Human and mouse GrB delivered by means of SLO to human HeLa and murine NIH/3T3 cells transfected with hBNIP-2 and mBNIP-2 respectively were examined for in cellulo BNIP-2 cleavage. The in vitro (IV) hGrB induced cleavage pattern of hBNIP-2 in transfected HeLa cell lysates as well as HeLa and NIH/3T3 cells transfected with the truncated hBNIP-2 and mBNIP-2 variants respectively are included for comparison. Black arrows indicate BNIP-2 precursor patterns whereas dashed and grey arrows are indicative for BNIP-2 cleavage fragments.

Article Snippet: The TnT Quick Master Mix (40 μl) (pOTB7-hBNIP-2 (RZPD Imagenes, cat n° IRAUp969D059D, Germany) and pCMV-SPORT6-mBNIP-2 (RZPD Imagenes, cat n° IRAVp968F0522D, Germany)) – consisting of a rabbit reticulocyte lysate, reaction buffer, SP6 or T7 RNA polymerase, an amino acid mixture without methionine and RNasin Ribonuclease Inhibitor – was mixed with 2 μl [ 35 S]-methionine (20 μCi/ml), 1 μg of plasmid DNA template (1 μg/μl) and 7 μl nuclease-free water.

Techniques: Transfection, In Vitro

Journal: BMC Biochemistry

Article Title: Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

doi: 10.1186/1471-2091-15-21

Figure Lengend Snippet: Upstream translation initiation sites (uTIS) in the 5’ leader sequences of BNIP-2. A. Domain architecture and BCH domains of human and mouse BNIP-2 variants. In-frame translation products possessing a BCH domain were taken into account (i.e., four mouse (1–4) and two human (5–6) BNIP-2 variants). From top to bottom: 1. UniProtKB accession long mBNIP-2 (O54940) complemented with the 5′ leader from ENSMUSG00000011958; 2. Short mBNIP-2 [Trembl: Q91VL0]; 3. Long mBNIP-2 [Swiss-Prot: O54940]; 4. G5E8U9 (Trembl), 5. J3KN59 (Trembl) and 6. Human BNIP-2 [Swiss-Prot: Q12982]. Red bars are indicative for 5′ leader sequences whereas A, B and C annotations refer to putative alternative upstream translation initiation sites deduced from ribosome profiling analyses of a human (C) and mouse (A and B) cell line. B. Assessing the influence of 5′ leader sequences on BNIP-2 precursor patterns. The involvement of upstream translation initiation (uTIS) in the appearance of multiple precursor bands was investigated by transfecting BNIP-2 with or without its 5′ leader sequence in human HeLa and mouse NIH/3T3 cells. Black arrows indicate BNIP-2 precursors.

Article Snippet: The TnT Quick Master Mix (40 μl) (pOTB7-hBNIP-2 (RZPD Imagenes, cat n° IRAUp969D059D, Germany) and pCMV-SPORT6-mBNIP-2 (RZPD Imagenes, cat n° IRAVp968F0522D, Germany)) – consisting of a rabbit reticulocyte lysate, reaction buffer, SP6 or T7 RNA polymerase, an amino acid mixture without methionine and RNasin Ribonuclease Inhibitor – was mixed with 2 μl [ 35 S]-methionine (20 μCi/ml), 1 μg of plasmid DNA template (1 μg/μl) and 7 μl nuclease-free water.

Techniques: Sequencing