human mmp 15  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: Identification of the KLK14 hydrolysis sites within the CleavEx proMMP protein. CleavEx proMMP fusion proteins were separated using SDS-PAGE and electrotransferred for N-terminal sequencing. Identified sequences are represented in the bold font and the underscore denotes where the location of the expected activation cleavage P1-P1′ in the proMMP-derived sequence.

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D Systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D Systems), 0.5 μg proMMP16 (catalog no. 1785-MP-010, R&D Systems), and 1 μg proMMP17 (catalog no. 7796-MP-010, R&D Systems) were separately incubated in 10 μL with a range of KLK14 concentrations (25–250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Sequencing, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: N-terminal identification of KLK14-mediated processing of recombinant proMMPs. The KLK14 hydrolysis product sequences were analyzed by N-terminal sequencing using Edman degradation. The bold font denotes the amino acid sequences identified. The underscored residues represent changes to the native protein sequence, as reported by the manufacturer (R&D Systems, Abingdon, United Kingdom). KLK14 recognized the sequence 3-aa upstream of the native MMP17 activation site, likely because the native site was modified by the manufacturer. All residues are numbered according to the Uniprot reported sequence of the full-length proteins. Bands are labeled according to the notation explained at Figure 2 .

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D Systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D Systems), 0.5 μg proMMP16 (catalog no. 1785-MP-010, R&D Systems), and 1 μg proMMP17 (catalog no. 7796-MP-010, R&D Systems) were separately incubated in 10 μL with a range of KLK14 concentrations (25–250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Recombinant, Sequencing, Activation Assay, Modification, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: Gelatin zymography of proMMPs by KLK14-mediated processing. Activation of proMMPs by KLK14 results in a fully functional mature enzyme. Each proMMP was incubated with the indicated concentrations of KLK14 for 1 h at 37 °C. The reaction was stopped by the addition of KLK14-specific inhibitors, and the reaction mixture was analyzed by SDS-PAGE, followed by a zymogram with gelatin as a substrate. The proMMP2 ( A ) negative control was not activated. ProMMP14 ( B ), proMMP15 ( C ), and proMMP16 ( D ) were activated, whereas proMMP17 ( E ) did not show hydrolysis of gelatin; yet a shift corresponding to the loss of the profragment was observed (note that an amino acid substitution was introduced in proMMP17 by the manufacturer (R&D Systems, Abingdon, United Kingdom)). KLK = kallikrein-related peptidase; MMP = matrix metalloproteinase.

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D Systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D Systems), 0.5 μg proMMP16 (catalog no. 1785-MP-010, R&D Systems), and 1 μg proMMP17 (catalog no. 7796-MP-010, R&D Systems) were separately incubated in 10 μL with a range of KLK14 concentrations (25–250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Zymography, Activation Assay, Functional Assay, Incubation, SDS Page, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: Primers used for generating the proMMP CleavEx fusion proteins using three consecutive PCRs.

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D Systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D Systems), 0.5 μg proMMP16 (catalog no. 1785-MP-010, R&D Systems), and 1 μg proMMP17 (catalog no. 7796-MP-010, R&D Systems) were separately incubated in 10 μL with a range of KLK14 concentrations (25–250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Sequencing

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP associates with ZO-1 in polarized MDCK cells. (A) Western blot analysis of HA, E-cadherin and Rho-GDI in biotinylated cell lysates from Mock, MT2-MMP (MT2FL) and MT2-MMPWK (MT2WK) stable MDCK transfectants pulled down with streptavidin beads; input, unbound and bound fractions are shown (Inp, Unb, Biot). (B) Western blot analysis of ZO-1 and HA in cell lysates from Mock, MT2-MMP and MT2-MMPWK stable MDCK transfectants pulled down with anti-HA antibody; IgG immunoprecipitates and whole lysates (Input) are also shown as controls. A blot of the input lanes after a longer exposure is also shown. (C) Representative maximal projections from apical and basolateral stacks of confocal sections from polarized MDCK transfectants stained for HA (MT2-MMP, green), ZO-1 (red) and nuclei (Hoechst, blue). (D) Orthogonal x–z views of 3D confocal image stacks from C. (E) Representative peak intensity profiles from x–z views of 3D confocal image stacks from C. Graph to the right shows the quantification of MT2-MMP/ZO-1 Pearson correlation coefficient in polarized MT2-FL and MT2-WK MDCK transfectants. Values are mean±s.e.m. n=40 cells per condition in two independent experiments; *P<0.05.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Western Blot, Staining

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP overexpression induces aberrant apical epithelial cell accumulation in polarized MDCK monolayers. (A) Orthogonal x–z projections of 3D confocal image stacks of MDCK transfectants stained for F-actin (Phalloidin, gray), HA (MT2-MMP, green) and Hoechst (nuclei, blue). Scale bar: 10 μm. (B) Quantification of apical epithelial foci per field (left) and the percentage of foci having more than 8 nuclei (right). 10 fields were counted per condition in n=4 independent experiments. (C) Representative maximal projections are shown from subapical and complete stacks of confocal sections from polarized MDCK transfectants stained for E-cadherin (gray). The dotted yellow line marks apical foci. Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants. Data are represented as mean±s.e.m. and were tested by one-way ANOVA versus mock 1 followed by Dunnett's post-test in B and C. **P<0.01, ***P<0.001, ****P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Over Expression, Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: Apical epithelial cell accumulation depends on MT2-MMP catalytic activity. (A) Representative maximal projections from confocal sections of polarized MDCK transfectants stained for E-cadherin (gray) in the presence of GM6001 (50 μM) or vehicle (DMSO). Orthogonal x–z views are shown below. (B) Line and bar graphs show E-cadherin peak and average intensity, respectively, around the junctions formed by MDCK transfectants treated as in A. Bar graph at the bottom shows the number of apical events on the polarized MDCK monolayer in the presence or absence of DMSO. In the middle and bottom graphs, the difference between mock DMSO and MT2 FL were significant with P<0.01 and P<0.0001, respectively. (C) Representative maximal projections are shown from confocal sections of polarized MDCK transfectants (mock, MT2 and MT2EA) stained for E-cadherin (gray). Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants shown in C. Bar graph on the right shows the number of apical events occurring in polarized MDCK monolayers. Data are represented as mean± s.e.m. and were tested by one-way ANOVA followed by Sidak post-test in B. Dunnett's post-test was used in D. *P<0.05, **P<0.01, ****P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Activity Assay, Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: E-cadherin is cleaved by MT2-MMP after N445 in the EC5 loop. (A) In silico model of canine E-cadherin (green)/human MT2-MMP (blue) interactions in cis association at the plasma membrane; the catalytic MT2-MMP center and the E-cadherin peptide, GPIPEPRNMDFCQKNPQP, are shown in orange and red, respectively. (B) Scheme of E-cadherin structure with the peptide containing the predicted cleavage sites after N445 and N459 in the EC5 loop. (C) Representative extracted ion chromatograms of 3 independent experiments corresponding to the peptides detected following in in vitro digestion of the GPIPEPRNMDFCQKNPQP peptide in the absence or presence of the human MT2-MMP recombinant catalytic domain (rhMT2). (D) Western blot analysis of lysates recovered from MDCK transfectants cultured with different calcium concentrations. Results are representative of two independent experiments. (E) Representative orthogonal x–z views of confocal images for polarized MDCK transfectants co-immunostained for HA (MT2-MMP, green), E-cadherin (red) and nuclei (Hoechst, blue).

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: In Silico, Clinical Proteomics, Membrane, In Vitro, Recombinant, Western Blot, Cell Culture

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP disrupts apical E-cadherin-mediated signals. (A) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants stained for F-actin (Phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (B) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants (mock and MT2-MMP) in the presence of 4-HAP (500 nM) or vehicle (DMSO) for 72 h and stained for F-actin (phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (C) Quantification of apical/total MFI of myosin IIB in polarized MDCK transfectants treated with 4-HAP (500 nM) or vehicle (DMSO); n=5 independent experiments. (D) Quantification of cell circularity in MDCK cells presented in C. 25 cells per field were counted in 2 images per condition in 6 independent experiments. (E) Quantification of the number of apical events on polarized MDCK cells presented in panel B. 10 fields were counted per condition in n=4 independent experiments. Difference between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.0001 and P<0.05, respectively. Data are represented as mean±s.e.m. and were tested by one-way ANOVA followed by Sidak post-test. *P<0.05, **P<0.01, ****P<0.0001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Staining

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: Mislocalization of pSrc in polarized MT2-MMP-MDCK cells contributes to apical cell accumulation. (A) Percentage of cells in G0/G1, S, and G2/M phases of the cell cycle analyzed by flow cytometry in propidium-iodide-stained MDCK transfectants after 72 h of serum deprivation. Means±s.e.m. are shown for 3 independent experiments. (B) Orthogonal x–z projections of 3D confocal image stacks of polarized MDCK transfectants (mock and MT2-MMP) stained for F-actin (Phalloidin, green), pSrc (red), and nuclei (Hoechst, blue). Representative peak intensity profiles are shown on the right for pSrc (red) and F-actin (green). (C) Bar graphs show the apical (left) and junctional (right) pSrc intensity, relative to total mean fluorescence intensity (MFI) in 6 independent experiments. (D) Number of apical events occurring in polarized MDCK cells treated with PP2 or vehicle (DMSO). 10 fields were counted per condition in 3 independent experiments. Differences between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.001 and P<0.01, respectively. (E) Representative confocal images of 3D cysts formed by MDCK transfectants in Matrigel and stained for pSrc (green), F-actin (white), E-cadherin (red), and nuclei (Hoechst, blue). (F) Quantification of the percentage of lumenized cysts. Data are represented as the mean±s.e.m. and were tested by two-way ANOVA followed by Dunnett's post-test in A, two-tailed Welch-test comparison was used in B, and one-way ANOVA followed by Sidak post-test was used in C. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Flow Cytometry, Staining, Fluorescence, Two Tailed Test, Comparison

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP deficiency alters junctional E-cadherin and leads to decreased 3D colon organoid formation ex vivo and smaller crypts in vivo. (A) Representative confocal images of MT2-MMP-silenced colon organoids stained for MT2-MMP (green; top). Graph shows the normalized MT2-MMP mean fluorescence intensity (MFI) in stained organoids 72 h after siRNA transfection (bottom), n=6 images per condition from 3 independent experiments; Mmp15 mRNA levels decreased ∼20% in silenced organoids. (B) Bright-field microscopy images of MT2-MMP-silenced colon organoids. Bar graph shows the percentage of organoid generation efficiency 48 h after siRNA transfection (right) in 3 independent experiments. (C) Representative confocal images of MT2-MMP-silenced colon organoids stained for nuclei (Hoechst/Ho, blue), F-actin (red), E-cadherin (green) and β-catenin (white); magnified views of E-cadherin and β-catenin staining are shown in insets. (D) Representative confocal images of colonic tissues recovered from wild-type or MT2-MMP-null mice, and stained for Ki67 (red) and nuclei (Hoechst, blue). On the right, graph shows the percentage of Ki67-positive cells per crypt. 9–15 crypts were quantified per condition in 3 images taken from 2 mice per genotype (top). (E) Quantification of the cumulative frequency of crypt length (left) and width (right). Data are represented as mean±s.e.m. and were tested by unpaired Student's t-test in A and B and by two-tailed Welch-test comparison in D and E. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Ex Vivo, In Vivo, Staining, Fluorescence, Transfection, Microscopy, Two Tailed Test, Comparison