mouse synthetic peptide  (Cusabio)

Journal: Developmental biology

Article Title: Spatiotemporal characterization of cyclooxygenase pathway enzymes during vertebrate embryonic development

doi: 10.1016/j.ydbio.2024.11.009

Figure Lengend Snippet: Antibodies used in the study.

Article Snippet: Cyclooxygenase-2 (COX-2) , 1:200 , Human, macaque monkey, mouse, ovine, rat , Rabbit IgG , Synthetic peptide corresponding to the C-terminal region of mouse COX-2 , Cayman Chemical 160126, , 20m PFA.

Techniques: Transduction

Journal: eLife

Article Title: Spinal V1 inhibitory interneuron clades differ in birthdate, projections to motoneurons, and heterogeneity

doi: 10.7554/eLife.95172

Figure Lengend Snippet: Antibodies.

Article Snippet: Prox1 , Synthetic peptide from the C-terminus of mouse Prox1 , Rabbit, polyclonal , Millipore Sigma , AB5475 , RRID: AB_177485 , 1:500.

Techniques: Recombinant, Variant Assay, Isolation

Journal: PLOS Biology

Article Title: Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1

doi: 10.1371/journal.pbio.3002156

Figure Lengend Snippet: ( A , B ) Co-immunoprecipitation assay. Intermolecular binding of full-length human Pxt1 to transiently expressed ( A ) or endogenous ( B ) Bak or Bax in HeLa cells was analyzed by immunoprecipitation and immunoblotting. All cells were treated with 20 μM z-VAD-fmk before transfection of plasmids. Full gel figures are available in . ( C ) SEC analysis. Recombinant Bak and the indicated human Pxt1 BH3 fragment were copurified and subjected to Superdex 200 increase 10/300 GL column (left). Separately purified recombinant Bax and the Pxt1 BH3 fragment were also mixed and incubated for 1 h, which were then subjected to the same column (right). The peak fractions were analyzed and visualized by SDS–PAGE and Coomassie staining. Full gel figures are available in . ( D ) The interaction between recombinant Bak and human Pxt1 BH3 was analyzed by ITC. The numerical data are included in . Bak, Bcl-2 antagonist/killer; Bax, Bcl-2-associated X; hPxt1, human Pxt1; ITC, isothermal titration calorimetry; PAGE, polyacrylamide gel electrophoresis; Pxt1, peroxisomal testis-specific 1; S, size marker; SDS, sodium dodecyl sulfate; SEC, size-exclusion chromatography.

Article Snippet: Synthetic peptides derived from mouse Pxt1 (residues 1–18), human Pxt1 (residues 76–101, 76–101 with L82A·L86A mutations, or 84–101), and human Bim (residues 141–166) were purchased from Peptron, Korea.

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Western Blot, Transfection, Recombinant, Purification, Incubation, SDS Page, Staining, Isothermal Titration Calorimetry, Polyacrylamide Gel Electrophoresis, Marker, Size-exclusion Chromatography

Journal: PLOS Biology

Article Title: Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1

doi: 10.1371/journal.pbio.3002156

Figure Lengend Snippet: ( A ) Structural representation. Bak is shown in electrostatic surface representation bound to the human Pxt1 BH3 fragment whose side chains are presented as sticks. Human Pxt1 is shown in green, except for the region absent in mouse Pxt1 that is shown in orange. The Pxt1 residues involved in the intermolecular interaction with Bak are labeled. ( B ) Intermolecular interactions. Pxt1 and Bak are shown in green and gray, respectively. Residues involved in the intermolecular interactions are presented as sticks with labels. Electrostatic interactions and hydrogen bonds between Arg87 and Asp91 of Pxt1 (red) and Ser117, Asn124, and Arg127 of Bak (blue) are marked with dashed lines. ( C ) Structural rearrangement of Bak induced by Pxt1 binding. Pxt1 is shown in green, while Bak is shown in wheat (apo; PDB code 2IMT) or violet (Pxt1-bound form). The Bak regions undergoing prominent conformational changes are marked by boxes and analyzed in detail. Box I, α3−α4 loop; box II, α2–α3 3 10 -helix and α3 helix; box III, a cavity region in the middle of the BH3-binding groove. The red dot in panel III indicates a water molecule. Bak, Bcl-2 antagonist/killer; Pxt1, peroxisomal testis-specific 1.

Article Snippet: Synthetic peptides derived from mouse Pxt1 (residues 1–18), human Pxt1 (residues 76–101, 76–101 with L82A·L86A mutations, or 84–101), and human Bim (residues 141–166) were purchased from Peptron, Korea.

Techniques: Labeling, Binding Assay

Journal: PLOS Biology

Article Title: Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1

doi: 10.1371/journal.pbio.3002156

Figure Lengend Snippet: ( A ) Structural alignments of 3 different BH3 domain-bound Bak molecules. The PDB codes are 5VWV for Bak bound to Bim and 5VWZ for Bak bound to Bim-h3Pc. ( B ) Details of the intermolecular interactions. Bak in a complex with Pxt1 is shown in an electrostatic surface representation, and 3 peptides accommodated in the hydrophobic groove region are shown together. The 5 BH3 consensus residues are shown as labeled sticks (top) and aligned (bottom). Ф, hydrophobic residue; Σ, small residue; Z, acidic residue; Γ, hydrophilic residue. The cavity region in the middle of the BH3-binding groove of Bak is marked by a rectangle (left) and shown in a separate box (right). ( C ) Structural comparison of intramolecular electrostatic networks of Bak in the apo or indicated BH3-bound forms. The 4 Bak residues mainly involved in the network are shown as labeled sticks. Dashed lines represent electrostatic interactions. The PDB codes are 2IMT for apo Bak, 7M5A for Bak bound to W3W5-mutant Bid BH3, 7M5B for Bak bound to M3W5-mutant Bid BH3, and 7M5C for Bak bound to Bak BH3. Bak, Bcl-2 antagonist/killer; Pxt1, peroxisomal testis-specific 1.

Article Snippet: Synthetic peptides derived from mouse Pxt1 (residues 1–18), human Pxt1 (residues 76–101, 76–101 with L82A·L86A mutations, or 84–101), and human Bim (residues 141–166) were purchased from Peptron, Korea.

Techniques: Labeling, Residue, Binding Assay, Comparison, Mutagenesis

Journal: PLOS Biology

Article Title: Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1

doi: 10.1371/journal.pbio.3002156

Figure Lengend Snippet: ( A ) Native gel electrophoresis. Recombinant Bak was incubated with the Bim or Pxt1 BH3 peptide with or without CHAPS. Appearance of a novel protein band can be observed in lanes 4 (100 μM Bak + 1 mM Bim BH3 + 1% CHAPS) and 6 (100 μM Bak + 1 mM Pxt1 BH3 + 1% CHAPS). A full gel figure is available in . ( B ) SEC analysis using a Superdex 75 10/300 GL gel filtration column. The elution positions of the standard protein size markers ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa) are indicated by arrowheads. Samples are prepared with the same concentration described in ( A ). The peak fractions from the mixture elution were analyzed and visualized by SDS-PAGE and Coomassie staining, shown on the left. Red arrows indicate the novel peak portion that appeared upon incubation with 100 μM Bak, 1 mM Pxt1 BH3, and 1% CHAPS. Full gel figures are available in . ( C ) Liposome release assay. Liposomes encapsulating self-quenching fluorescent dye were incubated with recombinant Bak and/or the indicated BH3 peptide. Release was normalized to detergent-solubilized liposomes. Experiments were performed in independent triplicate, and the numerical data are included in . Bak, Bcl-2 antagonist/killer; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PAGE, polyacrylamide gel electrophoresis; Pxt1, peroxisomal testis-specific 1; S, size marker; SDS, sodium dodecyl sulfate; SEC, size-exclusion chromatography.

Article Snippet: Synthetic peptides derived from mouse Pxt1 (residues 1–18), human Pxt1 (residues 76–101, 76–101 with L82A·L86A mutations, or 84–101), and human Bim (residues 141–166) were purchased from Peptron, Korea.

Techniques: Nucleic Acid Electrophoresis, Recombinant, Incubation, Filtration, Concentration Assay, SDS Page, Staining, Release Assay, Liposomes, Polyacrylamide Gel Electrophoresis, Marker, Size-exclusion Chromatography

Journal: PLOS Biology

Article Title: Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1

doi: 10.1371/journal.pbio.3002156

Figure Lengend Snippet: HeLa cells transiently expressing the indicated full-length constructs were used for MOMP analysis. ( A ) ΔΨm analysis using TMRE staining followed by flow cytometry. Numbers indicate the percentages of TMRE-negative (blue) or positive (red) mitochondrial populations at each designated hour (left) and are represented as graphs (right). Experiments were performed in 7 replicates *, P < 0.05; ****, P < 0.001 in the two-way ANOVA followed by Tukey’s HSD. The numerical data are included in . The gating strategies of flow cytometry are included in . ( B ) Localization of cytochrome c was analyzed using confocal microscopy. Mitochondria, cytochrome c , GFP, and DAPI were immunostained and visualized in HeLa cells fixed with 4% paraformaldehyde 8 h after transfection. The scale bars indicate 10 μm. The ratio of cells releasing cytochrome c upon expression of GFP only (control) or each of the indicated constructs was shown on the right. For each construct, 100–119 cells were counted, which were performed in 3 replicates. ns, nonsignificant; ****, P < 0.0001 in the one-way ANOVA followed by Tukey’s HSD. All cells were treated with 20 μM z-VAD-fmk before transfection of plasmids. The numerical data are included in . ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; DLA, alanine substitutions at Leu82 and Leu86; GFP, green fluorescent protein; hPxt1, human Pxt1; HSD, honestly significant difference; MOMP, mitochondrial outer membrane permeabilization; mPxt1, mouse Pxt1; Pxt1, peroxisomal testis-specific 1; TMRE, tetramethylrhodamine ethyl ester; ΔΨm, mitochondrial membrane potential.

Article Snippet: Synthetic peptides derived from mouse Pxt1 (residues 1–18), human Pxt1 (residues 76–101, 76–101 with L82A·L86A mutations, or 84–101), and human Bim (residues 141–166) were purchased from Peptron, Korea.

Techniques: Expressing, Construct, Staining, Flow Cytometry, Confocal Microscopy, Transfection, Control, Membrane

Journal: PLOS Biology

Article Title: Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1

doi: 10.1371/journal.pbio.3002156

Figure Lengend Snippet: HeLa cells transiently expressing the indicated full-length constructs were subjected to a cell death analysis. z-VAD-fmk was used to inhibit apoptosis. ( A , B ) Cell viability was measured using CellTiter-Glo assay. The effect of human Pxt1 expression on cell viability was compared to that of mouse Pxt1 ( A ) or the Bak binding-defective mutant form ( B ). Experiments were performed in 12 replicates ***, P < 0.001; ****, P < 0.0001 in the two-way ANOVA followed by Tukey’s HSD. The numerical data are included in . ( C ) Cleavage of Pro-caspase 3 was analyzed by immunoblotting after transfection of indicated Pxt1 constructs. *, nonspecific band. Full gel figures are available in . ( D – F ) Annexin V staining was analyzed by flow cytometry. HeLa cells expressing GFP-tagged hPxt1 ( D ), Bim ( E ), or mouse Pxt1 ( F ) were stained with APC-conjugated annexin V for 5 min, and sorted at 12 or 24 h posttransfection. In each quadrant, the ratio of cells positive (red) and negative (blue) for annexin V is shown at the top and bottom, and those positive and negative for GFP are shown on the right and left, respectively. The gating strategies of flow cytometry are included in . ANOVA, analysis of variance; APC, allophycocyanin; Bak, Bcl-2 antagonist/killer; DLA, alanine substitutions at Leu82 and Leu86; GFP, green fluorescent protein; hPxt1, human Pxt1; HSD, honestly significant difference; mPxt1, mouse Pxt1; Pxt1, peroxisomal testis-specific 1.

Article Snippet: Synthetic peptides derived from mouse Pxt1 (residues 1–18), human Pxt1 (residues 76–101, 76–101 with L82A·L86A mutations, or 84–101), and human Bim (residues 141–166) were purchased from Peptron, Korea.

Techniques: Expressing, Construct, Glo Assay, Binding Assay, Mutagenesis, Western Blot, Transfection, Staining, Flow Cytometry

Journal: PLOS Biology

Article Title: Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1

doi: 10.1371/journal.pbio.3002156

Figure Lengend Snippet: Bax −/− HeLa cells transiently expressing control GFP or GFP-tagged full-length hPxt1 were transfected with siNON or siBak, as indicated, and used for MOMP analysis. ( A ) ΔΨm analysis by flow cytometry. Numbers indicate the percentages of TMRE-negative (blue) or positive (red) mitochondrial populations at each examination (left) and are represented as graphs (right). Experiments were performed in 7 replicates. **, P < 0.01; ****, P < 0.0001 in the two-way ANOVA followed by Tukey’s HSD. The numerical data are included in . The gating strategies of flow cytometry are included in . ( B ) Confocal microscopy analysis of the release of cytochrome c . Bax −/− HeLa cells were transfected with siNON or siBak for 24 h, followed by transfection with GFP or GFP−hPxt1-expressing vector for additional 12 h. The mitochondria, cytochrome c , GFP, and DAPI were immunostained and visualized fixed with 4% paraformaldehyde posttransfection. The scale bars indicate 10 μm. The ratio of cells releasing cytochrome c upon expression of the indicated constructs was shown on the right. For each construct, 53–69 cells were counted, which were performed in 3 replicates. ns, nonsignificant; ****, P < 0.0001 in the one-way ANOVA followed by Tukey’s HSD. All cells were treated with 20 μM z-VAD-fmk before transfection of plasmids. The numerical data are included in . ANOVA, analysis of variance; Bak, Bcl-2 antagonist/killer; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; hPxt1, human Pxt1; HSD, honestly significant difference; MOMP, mitochondrial outer membrane permeabilization; TMRE, tetramethylrhodamine ethyl ester; ΔΨm, mitochondrial membrane potential.

Article Snippet: Synthetic peptides derived from mouse Pxt1 (residues 1–18), human Pxt1 (residues 76–101, 76–101 with L82A·L86A mutations, or 84–101), and human Bim (residues 141–166) were purchased from Peptron, Korea.

Techniques: Expressing, Control, Transfection, Flow Cytometry, Confocal Microscopy, Plasmid Preparation, Construct, Membrane

Journal: PLOS Biology

Article Title: Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1

doi: 10.1371/journal.pbio.3002156

Figure Lengend Snippet: Bax −/− HeLa cells transiently expressing control GFP or GFP-tagged full-length hPxt1 were treated with siNON or siBak, as indicated, and subjected to cell death analysis. ( A ) Cell viability was analyzed using the CellTiter-Glo assay. Experiments were performed in 9 replicates. ns, nonsignificant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in the two-way ANOVA followed by Tukey’s HSD. The numerical data are included in . ( B ) Annexin V staining-based cell death analysis using flow cytometry. HeLa cells in each condition were stained with APC-conjugated annexin V for 5 min and then sorted at the indicated time points posttransfection/treatment. The ratios of cells annexin V–positive and negative cells are shown in red and blue, respectively (top), and are represented as graphs (bottom). Experiments were performed in 7 replicates. ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in the two-way ANOVA followed by Tukey’s HSD. The numerical data are included in . The gating strategies of flow cytometry are included in . ANOVA, analysis of variance; APC, allophycocyanin; Bak, Bcl-2 antagonist/killer; FSC-A, forward scatter area; GFP, green fluorescent protein; hPxt1, human Pxt1; HSD, honestly significant difference.

Article Snippet: Synthetic peptides derived from mouse Pxt1 (residues 1–18), human Pxt1 (residues 76–101, 76–101 with L82A·L86A mutations, or 84–101), and human Bim (residues 141–166) were purchased from Peptron, Korea.

Techniques: Expressing, Control, Glo Assay, Staining, Flow Cytometry