Figure 2

Mactinin binds to a Hsp90 containing complex on the surface of monocytoid cells. A: Mactinin binds directly to peripheral blood monocytes. Monocytes incubated without (plot 1) or with (plot 3: shaded area) 100 nM mactinin were analyzed by flow cytometry to detect mactinin bound to the cell surface. The control condition (cells incubated with mactinin and stained with isotype matched IgG₁ antiserum) is shown in plot 2. One of four representative experiments is shown. B: Mactinin binds specifically in a high molecular weight complex on monocytoid HL-60 cells. HL-60 cells were pre-incubated with or without excess unlabeled (cold) mactinin, and then with [¹²⁵I]mactinin. Cell lysate was resolved by 12% SDS-PAGE under non-reducing conditions, and [¹²⁵I]mactinin detected using autoradiography. Lane 1: high molecular weight complex on cells incubated with [¹²⁵I]mactinin alone. Lane 2: marked reduction in bound [¹²⁵I]mactinin in cells pre-incubated with unlabeled mactinin. C: Mactinin binds to monocytoid HL-60 cells. HL-60 cells were biotinylated and then incubated for 1 h at 4°C with 100 nM [¹²⁵I]-mactinin, washed, and incubated without or with the cross-linkers DSP or BS³, lysed, and resolved by 6–20% gradient SDS-PAGE under non-reducing or reducing conditions. Lane 1: 31 kD radiolabeled mactinin alone, reducing conditions (no cells). Lane 2: radiolabeled mactinin bound to a high molecular weight complex on HL-60 cells, non-reducing conditions, no cross-linker. Lane 3: retention of most of the radiolabeled mactinin in the complex in presence of the impermeable, non-cleavable cross-linker BS³, reducing conditions. Lane 4: 31 kD radiolabeled mactinin separated from the binding complex under reducing conditions, cleavable cross-linker DSP. Lane 5: retention of nearly all of the radiolabeled mactinin in the complex in presence of the cross-linker DSP, non-reducing conditions. D: Mactinin binds to 3 distinct proteins on HL-60 cells. HL-60 cells were biotinylated (to detect cell surface proteins that bind to mactinin) and incubated with 100 nM mactinin. The HL-60 cell protein-mactinin complex (the material in lane 5 in Fig. 2C) was electroeluted, reduced, run over a mactinin column, and the bound proteins were resolved by 6–20% gradient SDS-PAGE. Biotinylated HL-60 proteins (88, 79 and 68 kD) that bound to the mactinin affinity column were detected by Western immunoblotting. E: Identification of Hsp90 in the mactinin-binding complex. The membrane shown in Fig. 2D was stripped and re-probed using an anti-Hsp90 antibody. A single 90 kD band was detected (lane 1). Recombinant Hsp90 was run in lane 2.