Binding Affinity of V-1520 in C6 Glioma Cells

Binding affinity of V-1520 was determined using C6 glioma cell lysates as previously reported (20 (link), 27 (link), 32 (link), 38 (link)). Lysates were incubated with N-(sec-butyl)-1-(2-chlorophenyl)-N-methyl-³H-isoquinoline-3-carboxamide ([³H]PK11195) (final concentration = 6 nM) and V-1520. The reaction was terminated by rapid filtration through a Brandel harvester and collection onto a filter presoaked with 0.3% polyethylenimine. Filters were then punched out into scintillation vials and bound radioactivity measured on a Beckman LS 6000 Scintillation Counter. All experiments were conducted in triplicate. Competition curves were analyzed with GraphPad Prism to obtain binding affinity (IC₅₀). K_i values were calculated using the equation K_i = IC₅₀/(1+[radioligand]/K_d), where IC₅₀ is determined from the competition curves, [radioligand] is the final concentration of [³H]PK11195 (6 nM) and K_d is the dissociation constant for [³H]PK11195 (5 nM).