Fig. 2: Interaction of Hsp90 with C9.
From: Cooperation between Hsp90 and mortalin/GRP75 in resistance to cell death induced by complement C5b-9
a–d Hsp90β-C9 binding was tested by co-sedimentation through sucrose gradients. Recombinant Hsp90β was incubated with C9 (1 µg each) for 1 h at 37 °C. The samples were layered on top of 13 ml of 10–30% sucrose density gradients that were subjected to unltracentrifugation for 18 h at 40,000 rpm. Fractions (300 µl) were collected from the gradient top. Samples (90 µl) from each fraction were analyzed by dot blotting with anti-Hsp90 (a, b) or anti-C9 (c, d) antibody and a peroxidase-conjugated secondary antibody. Representative dot blots are shown (a, c). Density of each scanned dot was quantified in arbitrary units (A.U.). Relative distribution of Hsp90 (b) and C9 (d) in fractions 1–24 of the gradients is shown. e, f Microtiter plate wells were coated with recombinant C9 or BSA as control. Then, K562 cells lysates (e) or recombinant Hsp90β (f) were added to the wells for 60 min at 37 °C. Binding of cytosolic (e) and recombinant (f) Hsp90 was quantified (optical density, A450) with anti-Hsp90 antibodies, followed by peroxidase-conjugated secondary antibodies. Increasing quantities of C9 yielded higher Hsp90 binding (P < 0.001, one-way ANOVA)
