Fig. 2. Bz-423 does not activate ASK1/JNK in Ramos cells.

(A) After treatment with Bz-423 (10 µM) for the indicated times, Thx was immunoprecipitated from cellular lysates, and the immune complexes blotted for ASK1 and Thx. (B) Lysates prepared from cells treated with Bz-423 (10 µM) were immunoprecipitated for ASK1 and then blotted to detect total ASK1 and phospho-ASK1. (C) ASK1 was immunoprecipitated from Ramos cell lysates treated with H₂O₂ (1 mM, 1 h) and reacted with MKK6 and p38 as described in the Methods section. Lane A: no lysate + inactive MKK6 + inactive p38; lane B: no lysate + active MKK6 + inactive p38; lane C: control lysate + inactive MKK6 + inactive p38; lane D: H₂O₂-treated lysate + inactive MKK6 + inactive p38. (D) Lysates prepared from Ramos cells treated with Bz-423 (10 µM) were immunoblotted to detect total and phosphorylated JNK. (E) Following pre-treatment with SP600125 (1 µM, dashed line) or vehicle (solid line), Ramos cells were incubated with Bz-423 and viability (24 h) was determined by PI exclusion.