pe conjugated mouse anti human adam 10 ic1427p antibody  (R&D Systems)

Journal: Journal of Cell Science

Article Title: Sequential and ?-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth

doi: 10.1242/jcs.060830

Figure Lengend Snippet: Detection of a faster-migrating BTC-ICD fragment in IMPE-BTC-WT cells upon constitutive and Ca2+-ionophore-induced BTC shedding. (A) Cell lysates were prepared from IMPE-BTC-WT and IMPE-Vector-alone (control) cells grown under serum-free conditions. (B) A431 cells were treated with or without the Ca2+ ionophore A23187 for 1 hour in the presence or absence of GI254023X (GI) prior to preparation of cell lysates. Left panel: cell lysates were directly analyzed by western blotting using an anti-human BTC cytoplasmic-domain antibody. Right panel: ionophore-induced A431 cell lysates were immunoprecipitated with control IgG or anti-BTC cytoplasmic-domain antibody prior to western blotting using the same antibody. Asterisks indicate IgG heavy (top) and light (bottom) chains. (C) IMPE-BTC-WT cells were treated with or without the Ca2+ ionophore A23187 for 1 hour in the presence or absence of GI254023X (GI) prior to preparation of cell lysates (upper panel: short exposure shows ionophore-induced ectodomain cleavage of BTC-FL and concomitant BTC-CTF production; lower panel: long exposure shows ionophore-induced generation of BTC-ICD). (D) IMPE-BTC-WT cells were treated overnight with or without metalloprotease inhibitor GI254023X or γ-secretase inhibitor PIX. CM was collected prior to preparation of cell lysates. Graph shows BTC shedding into CM, which was measured by BTC ELISA. Blot: all cellular BTC isoforms from IMPE cells were precipitated from cell lysates with anti-HA agarose and then analyzed in western blotting with anti-HA antibody.

Article Snippet: Antibodies and reagents The following antibodies and reagents were used: biotinylated goat anti-human BTC ectodomain antibody, mouse anti-human BTC ectodomain antibody and rat anti-mouse ADAM10 antibody (R&D Systems); rabbit anti-HA epitope tag antibody (Zymed Laboratories, Bethyl Laboratories); mouse anti-HA agarose beads (Sigma); mouse anti-GFP (Clontech); horseradish-peroxidase-conjugated donkey anti-rabbit IgG F(ab)2 fragment (Amersham Biosciences); mouse anti-actin anti-histone-2 (Millipore); and mouse anti-PolII antibody (Santa Cruz Biotechnology).

Techniques: Plasmid Preparation, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cell Science

Article Title: Sequential and ?-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth

doi: 10.1242/jcs.060830

Figure Lengend Snippet: ADAM10 is required for proBTC ectodomain cleavage and production of BTC-CTF and BTC-ICD. (A) WT MEFs or ADAM10−/− MEFs expressing proBTC were grown in serum-free DMEM for 24 hours in the presence or absence of GI254023X. BTC shedding into CM was measured by BTC ELISA. (B) WT MEFs and ADAM10−/− MEFs expressing proBTC were grown in serum-free DMEM for 24 hours in the presence or absence of GI254023X or PIX. Cell lysates were precipitated with anti-HA agarose and analyzed by western blot with anti-HA antibody. (C) WT MEFs or ADAM10−/− MEFs expressing proBTC were pre-incubated for 30 minutes in the presence or absence of GI254023X or PIX before treatment with or without A23187 for 1 hour. BTC shedding into CM was measured by BTC ELISA. (D) WT MEFs expressing proBTC were treated under the same conditions as in C. Cell lysates were precipitated with anti-HA agarose and analyzed by western blot with anti-HA antibody (upper panel: short exposure; lower panel: long exposure shows only BTC-CTF and BTC-ICD).

Article Snippet: Antibodies and reagents The following antibodies and reagents were used: biotinylated goat anti-human BTC ectodomain antibody, mouse anti-human BTC ectodomain antibody and rat anti-mouse ADAM10 antibody (R&D Systems); rabbit anti-HA epitope tag antibody (Zymed Laboratories, Bethyl Laboratories); mouse anti-HA agarose beads (Sigma); mouse anti-GFP (Clontech); horseradish-peroxidase-conjugated donkey anti-rabbit IgG F(ab)2 fragment (Amersham Biosciences); mouse anti-actin anti-histone-2 (Millipore); and mouse anti-PolII antibody (Santa Cruz Biotechnology).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation