Journal: PLoS ONE

Article Title: Molecular Identification of Carnosine N-Methyltransferase as Chicken Histamine N-Methyltransferase-Like Protein (HNMT-Like)

doi: 10.1371/journal.pone.0064805

Figure Lengend Snippet: Chicken HNMT-like protein was produced in COS-7 cells and purified to homogeneity by affinity chromatography on nickel-sepharose (HisTrap HP) as described under “ ”. For the SDS-PAGE analysis (A), 20 µl of reduced sample from each fraction was loaded onto a 10% gel, electrophoresed and the resulting gel was then stained with silver . For the Western-blot analysis (B), 1 µl of reduced sample from each fraction was loaded onto a 10% gel, electrophoresed and blotted to nitrocellulose membrane which was then sequentially probed with a mouse primary antibody against His6 tag and a horseradish peroxidase-conjugated goat anti-mouse antibody. Secondary antibody was detected through autoradiography using chemiluminescence. M, prestained protein marker; L, cell-free lysate of COS-7 cells overexpressing the recombinant enzyme; AL, 4-fold diluted lysate applied to the column; FT, flow through; W, wash; Fractions 30 to 300 were eluted with the indicated concentrations of imidazole. The pattern of prestained protein bands was copied from the blotting membrane onto ECL film using a set of felt-tip pens.

Article Snippet: For transfections, COS-7 cells (CLS Cell Lines Service GmbH, Germany) or HEK-293T (a kind gift of Dr. Maria Veiga-da-Cunha, de Duve Institute, Brussels) were plated in 100-mm Petri dishes at a cell density of 1.7×10 6 or 2.1×10 6 cells per plate, respectively, in Dulbecco's minimal essential medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% (v/v) fetal bovine serum, and grown in a humidified incubator under 95% air and 5% CO 2 atmosphere at 37°C.

Techniques: Produced, Purification, Affinity Chromatography, SDS Page, Staining, Western Blot, Membrane, Autoradiography, Marker, Recombinant

Journal: PLoS ONE

Article Title: Molecular Identification of Carnosine N-Methyltransferase as Chicken Histamine N-Methyltransferase-Like Protein (HNMT-Like)

doi: 10.1371/journal.pone.0064805

Figure Lengend Snippet: COS-7 and HEK-293T cells were transfected with either unmodified pEF6/Myc-His A vector (Control) or the same vector encoding chicken HNMT-like protein (HNMT-like) as described under “ ”. The cell-free lysates (2–3 µg of protein) were incubated for 0, 5, 10, 15 and 20 min in the reaction mixture containing 1 µM SAM (100 pmol), as well as 440×10 3 cpm of ( 3 H)SAM. The formation of radiolabeled anserine was determined after its chromatographic separation from ( 3 H)SAM. Values are the means ± S.E. of two separate transfection experiments. The presence of recombinant protein in tested lysates was verified by Western-blot analysis. Lysates (15 µg of protein) were loaded reduced onto a 10% gel, electrophoresed and blotted to nitrocellulose membrane which was then sequentially probed with a mouse primary antibody against His6 tag and a horseradish peroxidase-conjugated goat anti-mouse antibody. Secondary antibody was detected through autoradiography using chemiluminescence. COS, COS-7 cell lysate; HEK, HEK-293T cell lysate.

Article Snippet: For transfections, COS-7 cells (CLS Cell Lines Service GmbH, Germany) or HEK-293T (a kind gift of Dr. Maria Veiga-da-Cunha, de Duve Institute, Brussels) were plated in 100-mm Petri dishes at a cell density of 1.7×10 6 or 2.1×10 6 cells per plate, respectively, in Dulbecco's minimal essential medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% (v/v) fetal bovine serum, and grown in a humidified incubator under 95% air and 5% CO 2 atmosphere at 37°C.

Techniques: Transfection, Plasmid Preparation, Control, Incubation, Recombinant, Western Blot, Membrane, Autoradiography

Journal: Biochemical Journal

Article Title: Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration

doi: 10.1042/BCJ20220397

Figure Lengend Snippet: ( A ) COS7 cells were co-transfected with YFP-tagged PKCβII wild-type or mutants and MyrPalm-CFP . Translocation to plasma membrane was monitored by measuring FRET/CFP ratio changes after stimulation with 200 nM PDBu. Data for each cell were normalized to the max FRET ratio for that cell and represent at least three independent experiments and n ≥ 30 cells per condition. ( B ) Half-time of translocation was determined for each cell by fitting the data to a non-linear regression using a one-phase association equation. ( C ) COS7 cells were transfected with CKAR2 alone (endogenous, light blue) or co-transfected with indicated mCherry-tagged PKCβII construct. An amount of 1 µM of the PKC inhibitor Gö6983 was added after 3 min and PKC activity was monitored by measuring FRET/CFP ratio changes. Data were normalized to the assay end point and are from four independent experiments; n ≥ 32 cells per condition. ( D ) Basal activity from ( C ) was determined for each cell by plotting the initial FRET/CFP ratio after normalizing to the assay end point. ( E ) COS7 cells were co-transfected with YFP-tagged PKCβII and MyrPalm-CFP and plasma membrane translocation was measured as in ( A ). PKCβII-WT and R42E are reproduced from ( A ) for comparison (dashed lines). ( F ) Half-time of translocation was determined as in ( B ) and PKCβII-WT is reproduced from ( B ) for comparison. All data represent mean ± SEM. *** P < 0.001, **** P < 0.0001 by one-way ANOVA and Tukey post hoc test.

Article Snippet: 2 × 10 5 COS7 cells were seeded into plates (Corning, catalog no. 430165) containing glass cover slips (Fisherbrand, catalog no. 12545102) glued on using SYLGARD 184 Silicone Elastomer Kit (Dow, catalog no. 04019862) and cells were transfected 24 h after seeding.

Techniques: Transfection, Translocation Assay, Clinical Proteomics, Membrane, Construct, Activity Assay, Comparison

Journal: Biochemical Journal

Article Title: Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration

doi: 10.1042/BCJ20220397

Figure Lengend Snippet: ( A ) Western blot of whole-cell lysate from COS7 cells transfected with YFP-tagged PKCβII wild-type or indicated R42 mutants. Cells were treated with indicated concentrations of PDBu for 24 h before lysis. Blot is representative of three independent experiments. *, phosphorylated species; -, unphosphorylated species. ( B ) Quantification of percent phosphorylated PKC in DMSO-treated condition. ( C ) Quantification of percent change in PKC levels at 200 nM PDBu relative to 0 nM (DMSO) control for each transfection condition. Data represent mean ± SEM. ns = not significant, ** P < 0.01, *** P < 0.001 by one-way ANOVA and Tukey post-hoc test.

Article Snippet: 2 × 10 5 COS7 cells were seeded into plates (Corning, catalog no. 430165) containing glass cover slips (Fisherbrand, catalog no. 12545102) glued on using SYLGARD 184 Silicone Elastomer Kit (Dow, catalog no. 04019862) and cells were transfected 24 h after seeding.

Techniques: Western Blot, Transfection, Lysis, Control

Journal: Biochemical Journal

Article Title: Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration

doi: 10.1042/BCJ20220397

Figure Lengend Snippet: ( A ) Western blot of Triton-soluble lysate (left lanes) and YFP-PKCβII immunoprecipitated from COS7 cells using GFP-Trap® Agarose. Cells were pre-treated with 20 µM MG-132 for 3 h followed by 30 min of 200 nM PDBu treatment prior to lysis. Blots were probed with indicated antibodies. ( B ) Quantification of PDBu-induced ubiquitination of immunoprecipitated PKCβII. Relative ubiquitination was determined (Ubiquitin/PKC) for immunoprecipitated samples and each condition was normalized to DMSO-treated control (1.0) to determine fold-increase in ubiquitination after PDBu stimulation. Data represent mean ± SEM from four independent experiments. ns = not significant, **** P < 0.0001 by two-way ANOVA and Šídák's multiple comparisons test.

Article Snippet: 2 × 10 5 COS7 cells were seeded into plates (Corning, catalog no. 430165) containing glass cover slips (Fisherbrand, catalog no. 12545102) glued on using SYLGARD 184 Silicone Elastomer Kit (Dow, catalog no. 04019862) and cells were transfected 24 h after seeding.

Techniques: Western Blot, Immunoprecipitation, Lysis, Ubiquitin Proteomics, Control

Journal: Biochemical Journal

Article Title: Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration

doi: 10.1042/BCJ20220397

Figure Lengend Snippet: ( A ) Western blot of whole-cell lysate from COS7 cells transfected with YFP-tagged PKCβII WT, R42P, Q66N, or the double-mutant R42P/Q66N. Cells were treated with DMSO or 2 µM PDBu for 24 h before lysis. Blot is representative of five independent experiments. ( B ) Quantification of ( A ) showing percent change in PKC levels at 2 µM PDBu relative to DMSO control. ns = not significant, **** P < 0.0001 by one-way ANOVA and Tukey post-hoc test. ( C ) Proposed mechanism of C1A disruption by R42P mutation (middle panel) and restoration of domain dynamics through double mutant R42P/Q66N (right panel). Maps represent LSP data visualized using Gephi software. Each node represents an amino acid with the diameter corresponding to Degree Centrality. Yellow and blue nodes indicate hubs of residues participating in Zn 2+ binding as in . Gray, pink, and green nodes are R42 or P42, Q66 or N66, and V73, respectively. Figure created using BioRender.com.

Article Snippet: 2 × 10 5 COS7 cells were seeded into plates (Corning, catalog no. 430165) containing glass cover slips (Fisherbrand, catalog no. 12545102) glued on using SYLGARD 184 Silicone Elastomer Kit (Dow, catalog no. 04019862) and cells were transfected 24 h after seeding.

Techniques: Western Blot, Transfection, Mutagenesis, Lysis, Control, Disruption, Software, Binding Assay