Anti mouse igg hrp nxa931

DUSP4 (sc-1200), gp91-phox (sc-5827), Nox4 (sc-21860), and actin (sc-1616-R) antibodies were obtained from Santa Cruz (Santa Cruz, CA), p-p38 (4511S), p38 (9212S), p-ERK1/2 (9101S), ERK1/2 (9102S), p-JNK (4671S), JNK (3708S), cleaved caspase-3 (9664S), and MK-2 antibody sampler kit antibodies (9329S) from Cell Signaling (Cambridge, MA). Secondary anti-rabbit (NA934V) and anti-mouse (NXA931) IgG-HRP antibodies were purchased from GE Life Sciences (Piscataway, NJ). p38 inhibitor, SB203580, was purchased from Cayman Chemical (Ann Arbor, MI). Click-iT TUNEL Alexa Fluor 488 Imaging Assay Kit and dihydroethidium (DHE) were purchased from Life Technologies (Grand Island, NY).

phospho-Parkin wild-type or R104A (5 μM) buffer were incubated in ubiquitination buffer (30 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM ATP, 10 mM MgCl₂) with HsUBE1 (0.2 μM), UBE2L3 (2 μM) and Ub (20 μM). The reactions were quenched at indicated time points by addition of DTT- and iodoacetamide-containing LDS buffer and resolved on a 4-12% SDS NuPAGE gradient gels (Invitrogen) and transferred to a PVDF membrane (BioRad). Membranes were blocked in a 5% (w/v) milk solution in PBS-T (PBS + 0.1% (v/v) Tween-20) for 30 min and incubated overnight at 4 °C with a ubiquitin recognizing antibody (Ubi-1, NB300-130, Novus Biologicals) in 5% (w/v) BSA in PBS-T and 0.1% (w/v) sodium azide. The membrane was then washed with PBS-T, incubated for 1 h at room temperature with anti-mouse IgG-HRP (NXA931, GE Healthcare) in 5% (w/v) milk in PBS-T, washed in PBS-T and visualised using the Amersham Western Blotting Detection Reagent (GE Healthcare) and a ChemiDoc Touch Imaging System (BioRad).