Fig. 1
Development of an anti-ADAM8 ADP2 IHC assay and breast CCM. A ADAM8 protein levels in breast lines. Protein extracts (30Ā Ī¼g) from breast lines MCF10A, SUM149, MB-231 and MB-231-LUC, and control lines HEK-A8 and HEK-EV, grown in 2D or 3D as indicated, were subjected to immunoblotting with an anti-ADAM8 antibody (LS-C20181), which detected precursor (Proform) and active ADAM8; Ī²-actin was used as loading control. A representative blot of two independent experiments with similar results is shown. To create a breast CCM with a gradient of active ADAM8, MCF10A-2D, MB-231-2D and MB-231-3D were selected; HEK-EV-2D and HEK-A8-2D were included as negative and positive controls, respectively. B ADPs did not detect ADAM8 in breast lines under IHC conditions optimized for the commercial anti-human ADAM8 LS-B4068 antibody. CCM slides were subjected to IHC using conditions for the rabbit LS-B4068 antibody and either LS-B4068 or our newly developed ADP antibodies. Representative images of staining in HEK-A8-2D and MB-231-3D cells are shown. Pink color is due to Matrigel used (only) in this early CCM. CāE ADP2 under ADP-optimized conditions provides superior ADAM8 detection compared to LS-B4068. CCM IHC staining was performed using the final optimized conditions for the ADP mAbs and either our top diagnostic mAb ADP2 (1:100) or IgG2b (1:100) isotype-matched control for non-specific staining (C). The CCM breast lines (red box) displayed ADAM8 levels consistent with those seen in Western blotting (Part A). Values given below were determined by densitometry of immunoblots with extracts from independent cultures loaded at various concentrations to ensure a linear range of quantitation, and are presented relative to MCF10A (set to 1.0). IHC of the CCM with LS-B4068 (1:50) and its optimal conditions shows weaker staining than that observed with ADP2 (D). IHC of CCM with LS-B4068 (1:100) using ADP conditions shows poorer staining than with its optimal conditions (E). Images are at 40Ć magnification. MB-231, MDA-MB-231