Kinase experiment:

To detect ASK1 activity, inactive GST-MKK6 (0.1 mg) is incubated (15 min, 25℃) with immunoprecipitated ASK1 in kinase assay buffer. Subsequently, GST-p38α (0.5 μg) is added to this reaction and incubated (15 min, 25℃) followed by detection of phospho-GST-p38 via immunoblot.

Bz 423 is a pro-apoptotic 1,4-benzodiazepine with therapeutic properties in murine models of lupus demonstrating selectivity for autoreactive lymphocytes, and activates Bax and Bak.

Bz-423 induces a larger superoxide response in Ramos cells, and causes rapid death of Ramos B cells. Bz-423 activates Bax and Bak, but does not activate ASK1/JNK in Ramos cells. In Ramos cells Bz-423-induced superoxide leads to Bax and Bak activation via changes in Mcl-1 expression and functional activation of BH3-only proteins[1]. Bz-423 rapidly increases superoxide levels in MEFs within 1 h and the magnitude of the increase is concentration-dependent. Bz-423-induced apoptosis dependents upon superoxide[2].

[1]. Blatt NB, et al. Bz-423 superoxide signals B cell apoptosis via Mcl-1, Bak, and Bax. Biochem Pharmacol. 2009 Oct 15;78(8):966-73. [2]. Blatt NB, et al. Bz-423 superoxide signals apoptosis via selective activation of JNK, Bak, and Bax. Free Radic Biol Med. 2008 Nov 1;45(9):1232-42.