goat anti adam10  (R&D Systems)

Journal: Neoplasia (New York, N.Y.)

Article Title: Trop‐2 cleavage by ADAM10 is an activator switch for cancer growth and metastasis

doi: 10.1016/j.neo.2021.03.006

Figure Lengend Snippet: Trop-2 cleavage by ADAM10. ( A ) Inhibition of ADAM10 activity by treatment of BxPC3 cells with chemical inhibitors. Western blot detections of ADAM10 ( top ) and Trop-2 ( bottom ) show accumulation of the mature ADAM10 and corresponding reduction of the 40-kD Trop-2 cleavage band only upon treatment with GI254023X (blue). Quantification of the 40-kD Trop-2 cleavage band upon treatment with protease inhibitors is shown on the right, as percentage of total Trop-2. Ponceau red staining, control of protein loading. ( B ) Reduction of Trop-2 cleavage upon treatment of MTE4-14/Trop-2 transfectants with the ADAM10 inhibitor GI254023X (blue). Quantification of the Trop-2 cleavage band was carried out, and the ratio between uncleaved (un, black) versus cleaved (cl, gray) Trop-2 is shown on the right panel. ( C ) ADAM10 mRNA levels (red) 48 h upon treatment with ADAM10 siRNA or control siRNA (human CD133) (black). Vector: vector-alone transfectants; A87-A88: mutagenized Trop-2 at the cleavage site. The 2 −ΔΔCT algorithm was used to calculate the relative changes in gene expression. RNA levels of ADAM10 in Vector, wt Trop-2 and A87-A88 Trop-2 cells are shown here for cell group cross-comparison, and are intended as reference data for the functional assays shown in the next figures. ( D ) ADAM10 protein inhibition by shRNA (top). Corresponding reduction of the Trop-2 cleavage is shown (mid). Ponceau red staining, control of protein loading (bottom). ( E ) Western blotting of Trop-2 cleavage in MTE 4-14/Trop-2 transfectants. ( left ) Cells were seeded at the indicated fraction of full confluency, as normalized to 100%, or ( right ) cells were seeded at equal confluency and then lysed at different subsequent time points. Ponceau red staining, control of protein loading. MW markers are indicated. ( F ) Western blotting of MCF7 human breast cancer cells grown in adhesion or in suspension and lysed 5 d after seeding for analysis of the Trop-2 cleavage. Red arrow shows cleaved Trop-2. Ponceau red staining, control of protein loading. MW markers are indicated. (Color version of figure is available online.)

Article Snippet: The AF650 polyclonal goat anti-Trop-2 antibody was from R&D Systems; the rabbit polyclonal anti-ADAM10 antibody was from Merck-Millipore (AB19026); and the goat polyclonal anti-ADAM10 antibody (sc-31853) was from Santa Cruz Biotechnology.

Techniques: Inhibition, Activity Assay, Western Blot, Staining, Control, Plasmid Preparation, Gene Expression, Comparison, Functional Assay, shRNA, Suspension

Journal: Neoplasia (New York, N.Y.)

Article Title: Trop‐2 cleavage by ADAM10 is an activator switch for cancer growth and metastasis

doi: 10.1016/j.neo.2021.03.006

Figure Lengend Snippet: Trop-2 proteolytic processing-defective mutant versus wild-type. ( A ) Western blotting analysis of KM12SM/Trop-2 transfectants was carried out using anti-Trop-2 antibodies against the cytoplasmic tail and extracellular domain, as indicated. The Trop-2 bands were recognized by both antiextracellular domain and anticytoplasmic tail antibodies, indicating that the Trop-2 signal belongs to transmembrane forms. Red arrow, cleaved Trop-2. Vector, vector-alone transfectants. Ponceau red staining, control of protein loading. MW markers are indicated. ( B ) Flow cytometry analysis of MTE 4-14/Trop-2 transfectants using the T16 anti-Trop-2 antibody. The A87-A88 Trop-2 mutant (red) and wt Trop-2 (blue) are expressed at comparable levels. Vector: vector-alone transfectants. ( C ) Trop-2 cleavage at the cell surface. Trop-2 cleavage was assessed upon cell-surface biotinylation on KM12SM metastatic colon cancer cells. Pull-down after cell-surface biotinylation was carried out to reveal the status of Trop-2 at the cell membrane. ( left ) total cell lysate, ( right ) cell membrane pulled-down material. Western blotting analysis was carried out with a rabbit polyclonal antibody directed against the extracellular domain of Trop-2, and shows that wt Trop-2 is completely cleaved. Quantitative cleavage reduction was observed for the A87-A88 proteolytic site mutant. Red arrow, cleaved Trop-2. Ponceau red staining, control of protein loading. MW markers are indicated. ( D ) Growth curves in vitro of KM12SM human colon cancer cells and MTE4-14 murine cells transfected with wt Trop-2 versus A87-A88 Trop-2 mutant. Bars, standard errors of the mean (SEM). ( E ) ADAM10 (left) and MMP9 (right) reduced RNA levels by siRNAs, as measured by real-time RT-PCR. ( F ) Cell growth curves of wt Trop-2, A87-A88 Trop-2 or vector-alone transfectants upon ADAM10 (red), MMP9 (blue) or control (black) siRNA-mediated inhibition. Bars, SEM. P value (ADAM10, left panel): ANOVA analysis. Stars indicate post-hoc Bonferroni's t test P values (***, P ≤ 0.001). (Color version of figure is available online.)

Article Snippet: The AF650 polyclonal goat anti-Trop-2 antibody was from R&D Systems; the rabbit polyclonal anti-ADAM10 antibody was from Merck-Millipore (AB19026); and the goat polyclonal anti-ADAM10 antibody (sc-31853) was from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Western Blot, Plasmid Preparation, Staining, Control, Flow Cytometry, Membrane, In Vitro, Transfection, Quantitative RT-PCR, Inhibition

Journal: Neoplasia (New York, N.Y.)

Article Title: Trop‐2 cleavage by ADAM10 is an activator switch for cancer growth and metastasis

doi: 10.1016/j.neo.2021.03.006

Figure Lengend Snippet: Breast cancer case series. ( A ) Frozen samples from a consecutive breast cancer case series were analyzed for Trop-2 expression and cleavage by Western blotting. Western blot lanes bear the original patient number in the case series. The latin “bis” indication refers to a second sampling of the same tumor case, upon local relapse. Red arrow: cleaved Trop-2. Ponceau red staining, control of protein loading. MW markers are indicated. The bottom right panel shows a Western blotting analysis of Trop-2 expression and cleavage in normal breast tissue samples from control individuals. ( B ) Distribution of the fraction of cleaved Trop-2 molecules in individual breast cancer samples, ordered from low to high Trop-2 cleavage. ( C ) Western blotting analysis of ADAM10 expression and of Trop-2 cleavage in samples from breast cancer and normal breast tissues. The samples were pooled from the individual cases shown in the panel A as based on the extent of Trop-2 cleavage. The pools are as follows: A = 5+22+78+109+187; B = 113+131+174+187+204; C = 78+131+198+231+233; D = 9+18+24+47+61; E = 65+67+70+73+88; F = 95+103+106+115+122; G = 2+8+12; H = 3+7+9. ADAM10 was detected as bands at 55-50 kDa, designated as precursor and activated forms of the protein. Red arrow: presence/absence of Trop-2 cleavage at R87-T88. (Color version of figure is available online.)

Article Snippet: The AF650 polyclonal goat anti-Trop-2 antibody was from R&D Systems; the rabbit polyclonal anti-ADAM10 antibody was from Merck-Millipore (AB19026); and the goat polyclonal anti-ADAM10 antibody (sc-31853) was from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Sampling, Staining, Control