Journal: PLoS ONE

Article Title: Detection of Active Matriptase Using a Biotinylated Chloromethyl Ketone Peptide

doi: 10.1371/journal.pone.0077146

Figure Lengend Snippet: (A) Schematic structure of the biotin-RQRR-CMK peptide inhibitor. (B) The reactivity of biotin-RQRR-CMK was tested after 180 min of pre-incubation at 37°C (diamonds) or without preincubation (crosses). 0.2 µM matriptase SPD was incubated for 10 min at 37°C with (diamonds and crosses) or without (squares) 50 µM biotin-RQRR-CMK before addition of the chromogenic substrate to a final concentration of 300 µM. (C) The stability of 5 nM biotin-RQRR-CMK was further tested after the time points 0 (crosses), 60 (circles), 120 (stars), and 180 min (diamonds) of pre-incubation at 37°C and compared to a control not containing biotin-RQRR-CMK (squares). As described above, 0.2 nM matriptase SPD was added to each sample and incubated for 10 min at 37°C followed by addition of the chromogenic substrate to a final concentration of 300 µM. In all cases, the enzymatic activity of SPD was monitored by conversion of the chromogenic substrate (S2288). Each plot shows the change in optical density at 405 nm of the reaction mixture as a function of reaction time. The presence of active protease results in a continued release of a yellow cleavage product resulting in a linear color development in agreement with a pseudo 1 st order reaction due to the high molar excess of substrate to protease. Results shown are representative of 3 independent experiments.

Article Snippet: The antibodies used for detection of matriptase in Western blotting were monoclonal mouse anti-human matriptase antibody M32 or monoclonal mouse anti-human matriptase antibody M24 (1 μg/ml 1% milk in PBS-T), which detects a 70 kDa band (representing both active and zymogen matriptase both in boiled and non-boiled samples) and the 120–130 kDa complex of matriptase with HAI-1, which can only be detected in non-boiled samples ; monoclonal mouse anti-human matriptase antibody M69 (1 μg/ml 1% milk in PBS-T), which recognizes the 120–130 kDa complex of matriptase with HAI-1 ; monoclonal mouse anti-human HAI-1 antibody M19 (1 μg/ml 1% milk in PBS-T), which recognizes free 55 kDa HAI-1 and the 120–130 kDa matriptase-HAI-1 complex ; polyclonal rabbit anti-human matriptase (1.5 μg/ml 1% milk in PBS-T) raised against the SPD of matriptase recognizing a 70 kDa band (both active and zymogen matriptase) under non-reducing conditions and the 70 kDa zymogen form and the 30 kDa serine protease domain of cleaved matriptase under reducing conditions (Cat. no. IM1014, Calbiochem).

Techniques: Incubation, Concentration Assay, Control, Activity Assay

Journal: PLoS ONE

Article Title: Detection of Active Matriptase Using a Biotinylated Chloromethyl Ketone Peptide

doi: 10.1371/journal.pone.0077146

Figure Lengend Snippet: (A) Eleven days post-confluent Caco-2 cells grown on Transwell filters were labeled with 50 µM biotin-RQRR-CMK from the basolateral side for the times indicated (2–180 min) at 37°C (lanes 3–6) or for 180 min at 4°C (lane 7). As a measure of the steady state level of matriptase, membrane proteins on the basolateral plasma membrane of filter-grown Caco-2 cells were labeled by incubation with S-NHS-SS-biotin at 4°C for 30 min (lane 1). As a negative control, cells were labeled from the basolateral side with 50 µM control peptide; biotin-RQRR (lane 2). All cells were lysed and biotinylated proteins were precipitated using streptavidin-coated resin and were analyzed by non-reducing SDS-PAGE and Western blotting using the monoclonal matriptase antibody; M32. A tenth of the surface biotinylated sample was loaded (lane 1); whereas total sample volume was loaded for the other samples (lanes 2-7). (B) Caco-2 cells grown on Transwell filters were labeled with the biotin-RQRR-CMK peptide inhibitor from either the apical (lanes 1, 4, and 7) or the basolateral (lanes 2, 5, and 8) side for 180 min at either 4°C or 37°C. An overexposure of lanes 1–3 is shown in lanes 7–9. As a negative control, cells were labeled from the basolateral side with a peptide corresponding to the inhibitory peptide but lacking the CMK moiety (CTRL, lanes 3, 6, and 9). Cells were lysed and the lysates of multiple filters were pooled. Biotinylated proteins were precipitated using streptavidin-coated resin and the streptavidin pull downs were released by boiling in SDS-PAGE samples buffer and analyzed by SDS-PAGE and Western blotting using the monoclonal M32 antibody. Positions of the molecular weight markers (kDa) are indicated on the left. Results shown are representative of 3 independent experiments.

Article Snippet: The antibodies used for detection of matriptase in Western blotting were monoclonal mouse anti-human matriptase antibody M32 or monoclonal mouse anti-human matriptase antibody M24 (1 μg/ml 1% milk in PBS-T), which detects a 70 kDa band (representing both active and zymogen matriptase both in boiled and non-boiled samples) and the 120–130 kDa complex of matriptase with HAI-1, which can only be detected in non-boiled samples ; monoclonal mouse anti-human matriptase antibody M69 (1 μg/ml 1% milk in PBS-T), which recognizes the 120–130 kDa complex of matriptase with HAI-1 ; monoclonal mouse anti-human HAI-1 antibody M19 (1 μg/ml 1% milk in PBS-T), which recognizes free 55 kDa HAI-1 and the 120–130 kDa matriptase-HAI-1 complex ; polyclonal rabbit anti-human matriptase (1.5 μg/ml 1% milk in PBS-T) raised against the SPD of matriptase recognizing a 70 kDa band (both active and zymogen matriptase) under non-reducing conditions and the 70 kDa zymogen form and the 30 kDa serine protease domain of cleaved matriptase under reducing conditions (Cat. no. IM1014, Calbiochem).

Techniques: Labeling, Membrane, Clinical Proteomics, Incubation, Negative Control, Control, SDS Page, Western Blot, Molecular Weight

Journal: PLoS ONE

Article Title: Detection of Active Matriptase Using a Biotinylated Chloromethyl Ketone Peptide

doi: 10.1371/journal.pone.0077146

Figure Lengend Snippet: (A) Eleven days post-confluent filter-grown Caco-2 cells were treated with either a physiologically phosphate buffer pH 6.0 for 20 min (lanes 2, 4, 6, and 8) from both the apical and the basolateral side or left untreated (lanes 1, 3, 5, and 7). Cells were lysed and lysates were analyzed by Western blotting using antibodies against total matriptase (M24; lanes 1 and 2), matriptase SPD (IM1014; lanes 5 and 6), matriptase-HAI-1 complex (M69; lanes 3 and 4) and HAI-1 (lanes 7 and 8). Samples in lanes 1–4, 7, and 8 were not boiled to avoid dissociation of matriptase-HAI-1 complexes, while samples in lanes 5 and 6 were boiled and reduced to dissociate the S-S bridged SPD from the stem domain of activated matriptase in order to distinguish between the SEA domain-cleaved form (70 kDa) and the Arg614 cleaved form (25–30 kDa). Treatment with phosphate buffer pH 6.0 and DTT is indicated by +/−. Positions of the molecular weight markers (kDa) are indicated on the left. (B) A solution of 0.2 µM SPD was incubated for 10 min at 37°C with (crosses) or without (squares) 50 µM biotin-RQRR-CMK before addition the chromogenic substrate to a final concentration of 300 µM. All experiments were performed in 20 mM citric acid buffer pH 6.0, 140 mM NaCl and 0.1% BSA at 37°C. Results shown are representative of 3 independent experiments.

Article Snippet: The antibodies used for detection of matriptase in Western blotting were monoclonal mouse anti-human matriptase antibody M32 or monoclonal mouse anti-human matriptase antibody M24 (1 μg/ml 1% milk in PBS-T), which detects a 70 kDa band (representing both active and zymogen matriptase both in boiled and non-boiled samples) and the 120–130 kDa complex of matriptase with HAI-1, which can only be detected in non-boiled samples ; monoclonal mouse anti-human matriptase antibody M69 (1 μg/ml 1% milk in PBS-T), which recognizes the 120–130 kDa complex of matriptase with HAI-1 ; monoclonal mouse anti-human HAI-1 antibody M19 (1 μg/ml 1% milk in PBS-T), which recognizes free 55 kDa HAI-1 and the 120–130 kDa matriptase-HAI-1 complex ; polyclonal rabbit anti-human matriptase (1.5 μg/ml 1% milk in PBS-T) raised against the SPD of matriptase recognizing a 70 kDa band (both active and zymogen matriptase) under non-reducing conditions and the 70 kDa zymogen form and the 30 kDa serine protease domain of cleaved matriptase under reducing conditions (Cat. no. IM1014, Calbiochem).

Techniques: Western Blot, Molecular Weight, Incubation, Concentration Assay

Journal: PLoS ONE

Article Title: Detection of Active Matriptase Using a Biotinylated Chloromethyl Ketone Peptide

doi: 10.1371/journal.pone.0077146

Figure Lengend Snippet: Eleven days post-confluent Caco-2 cells grown on Transwell filters were labeled with 50 µM biotin-RQRR-CMK from the basolateral side for 180 min at 37°C. As a negative control, cells were labeled from the basolateral side with 50 µM control peptide; biotin-RQRR (CTRL), under the same conditions. Labeled proteases were precipitated using streptavidin-coated resin and the streptavidin pull downs were analyzed by reducing SDS-PAGE and Western blotting using the IM1014 antibody raised against matriptase SPD. Positions of the molecular weight markers (kDa) are indicated on the left and position of SEA domain-cleaved zymogen matriptase and matriptase SPD is indicated on the right. Results shown are representative of 3 independent experiments.

Article Snippet: The antibodies used for detection of matriptase in Western blotting were monoclonal mouse anti-human matriptase antibody M32 or monoclonal mouse anti-human matriptase antibody M24 (1 μg/ml 1% milk in PBS-T), which detects a 70 kDa band (representing both active and zymogen matriptase both in boiled and non-boiled samples) and the 120–130 kDa complex of matriptase with HAI-1, which can only be detected in non-boiled samples ; monoclonal mouse anti-human matriptase antibody M69 (1 μg/ml 1% milk in PBS-T), which recognizes the 120–130 kDa complex of matriptase with HAI-1 ; monoclonal mouse anti-human HAI-1 antibody M19 (1 μg/ml 1% milk in PBS-T), which recognizes free 55 kDa HAI-1 and the 120–130 kDa matriptase-HAI-1 complex ; polyclonal rabbit anti-human matriptase (1.5 μg/ml 1% milk in PBS-T) raised against the SPD of matriptase recognizing a 70 kDa band (both active and zymogen matriptase) under non-reducing conditions and the 70 kDa zymogen form and the 30 kDa serine protease domain of cleaved matriptase under reducing conditions (Cat. no. IM1014, Calbiochem).

Techniques: Labeling, Negative Control, Control, SDS Page, Western Blot, Molecular Weight

Journal: PLoS ONE

Article Title: Detection of Active Matriptase Using a Biotinylated Chloromethyl Ketone Peptide

doi: 10.1371/journal.pone.0077146

Figure Lengend Snippet: Murine keratinocytes were isolated from newborn wildtype (WT) or matriptase-deficient pups (KO) and cultured on collagen-coated plastic. The cells were grown until sub-confluent and then labeled with S-NHS-SS-biotin (lanes 1, 4, 7, and 10), or with 50 µM biotin-RQRR-CMK (lanes 2, 5, 8, and 11), or with 50 µM control peptide; biotin-RQRR (lanes 3, 6, 9, and 12). Cells were lysed and analyzed on Western blot (lanes 7–12). The remaining lysates were precipitated using streptavidin-coated resin, released from the beads by boiling under non-reducing conditions and lysates were analyzed by SDS-PAGE and Western blotting using the matriptase antibody AF3946. Results shown are representative of 2 independent experiments.

Article Snippet: The antibodies used for detection of matriptase in Western blotting were monoclonal mouse anti-human matriptase antibody M32 or monoclonal mouse anti-human matriptase antibody M24 (1 μg/ml 1% milk in PBS-T), which detects a 70 kDa band (representing both active and zymogen matriptase both in boiled and non-boiled samples) and the 120–130 kDa complex of matriptase with HAI-1, which can only be detected in non-boiled samples ; monoclonal mouse anti-human matriptase antibody M69 (1 μg/ml 1% milk in PBS-T), which recognizes the 120–130 kDa complex of matriptase with HAI-1 ; monoclonal mouse anti-human HAI-1 antibody M19 (1 μg/ml 1% milk in PBS-T), which recognizes free 55 kDa HAI-1 and the 120–130 kDa matriptase-HAI-1 complex ; polyclonal rabbit anti-human matriptase (1.5 μg/ml 1% milk in PBS-T) raised against the SPD of matriptase recognizing a 70 kDa band (both active and zymogen matriptase) under non-reducing conditions and the 70 kDa zymogen form and the 30 kDa serine protease domain of cleaved matriptase under reducing conditions (Cat. no. IM1014, Calbiochem).

Techniques: Isolation, Cell Culture, Labeling, Control, Western Blot, SDS Page