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: Journal of Biochemistry

Article Title: Roles of CUB and LDL receptor class A domain repeats of a transmembrane serine protease matriptase in its zymogen activation

doi: 10.1093/jb/mvs118

Figure Lengend Snippet: Western blot analysis of the pro-CLS-matWT and pro-CLS-matS805A variants. Medium samples of COS-1 cells transfected with pSec-pro-CLS-matWT alone (CLS-WT), pSec-pro-CLS-matWT and pSec-HAI-1NIK1LK2 (CLS-WT + HAI-1), pSec-pro-CLS-matS805A alone (CLS-S805A) and pSec-pro-CLS-matS805A and pSec- HAI-1NIK1LK2 (CLS-S805A + HAI-1) were subjected to reducing SDS-PAGE. After western transfer, the blot was probed with Spr992 or S-protein–HRP. The position to which pro-CLS-matWT and pSec-pro-CLS-matS805A migrated is indicated as ‘Proforms’ to the right of the right panel. The position to which HAI-1NIK1LK2 migrated is indicated to the right of the right panel. The molecular masses of the marker proteins are indicated in kilodaltons to the left of the left panel.

Article Snippet: When a sample of media conditioned by COS-1 cells transfected with pSec-pro-CLS-mat WT alone was probed with Spr992, no signal at the position corresponding to 28 kDa was produced (left panel, lane CLS-WT).

Techniques: Western Blot, Transfection, SDS Page, Marker

Journal: Journal of Biochemistry

Article Title: Roles of CUB and LDL receptor class A domain repeats of a transmembrane serine protease matriptase in its zymogen activation

doi: 10.1093/jb/mvs118

Figure Lengend Snippet: Western blot analysis of the pro-LS-matWT and pro-LS-matS805A variants. Medium samples of COS-1 cells transfected with pSec-pro-LS-matWT alone (LS-WT), pSec-pro-LS-matWT and pSec-HAI-1NIK1LK2 (LS-WT + HAI-1), pSec-pro-LS-matS805A alone (LS-S805A) and pSec-pro-LS-matS805A and pSec-HAI-1NIK1LK2 (LS-S805A + HAI-1) were subjected to reducing SDS-PAGE. After western transfer, the blot was probed with Spr992 or S-protein–HRP. The 28-kDa position to which the SPCD part of two-chain pro-LS-matWT migrated is indicated as ‘SPCD of two-chain pro-LS-matWT’ to the right of the left panel. The 57-kDa position at which pro-LS-matS805A migrated is indicated as ‘pro-LS-matS805A’ to the right of the left panel. The 57-kDa position to which pro-LS-matWT and pro-LS-matS805A migrated is indicated as ‘Proforms’ to the right of the right panel. The position to which HAI-1NIK1LK2 migrated is indicated to the right of the right panel. The molecular masses of the marker proteins are indicated in kilodaltons to the left of both panels.

Article Snippet: When a sample of media conditioned by COS-1 cells transfected with pSec-pro-CLS-mat WT alone was probed with Spr992, no signal at the position corresponding to 28 kDa was produced (left panel, lane CLS-WT).

Techniques: Western Blot, Transfection, SDS Page, Marker

Journal: Journal of Biochemistry

Article Title: Roles of CUB and LDL receptor class A domain repeats of a transmembrane serine protease matriptase in its zymogen activation

doi: 10.1093/jb/mvs118

Figure Lengend Snippet: Western blot analysis of the pro-S-matWT and pro-S-matS805A variants. Medium samples of COS-1 cells transfected with pSec-pro-S-matWT alone (S-WT), pSec-pro-S-matWT and pSec-HAI-1NIK1LK2 (S-WT + HAI-1), pSec-pro-S-matS805A alone (S-S805A) and pSec-pro-S-matS805A and pSec-HAI-1NIK1LK2 (S-S805A + HAI-1) were subjected to reducing SDS-PAGE. After western transfer, the blot was probed with Spr992 or S-protein–HRP. The 28-kDa position to which the SPCD part of two-chain pro-S-matWT migrated is indicated as ‘SPCD of two-chain pro-S-matWT’ to the right of the left panel. The 40-, 36- and 31-kDa positions to which pro-S-matWT and pro-S-matS805A species migrated are indicated as ‘Proforms’ to the right of the left and right panels. The position to which HAI-1NIK1LK2 migrated is indicated to the right of the right panel. The molecular masses of the marker proteins are indicated in kilodaltons to the left of both panels.

Article Snippet: When a sample of media conditioned by COS-1 cells transfected with pSec-pro-CLS-mat WT alone was probed with Spr992, no signal at the position corresponding to 28 kDa was produced (left panel, lane CLS-WT).

Techniques: Western Blot, Transfection, SDS Page, Marker