Rabbit anti phospho jnk antibody

Human ovarian adenocarcinoma cells (SKOV-3) were purchased from American Type Culture Collection (Manassas, VA, USA). RES was purchased from MilliporeSigma (Burlington, MA, USA). MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from MilliporeSigma. Guava^® Cell Cycle Reagent was purchased from Luminex Corporate (Austin, TX, USA). Annexin V was purchased from ImmunoTools (Friesoythe, Niedersachsen, Germany). Propidium iodide was purchased from MilliporeSigma. Rabbit anti-phospho p38 antibody, rabbit anti p38 antibody, rabbit anti-phospho ERK1/2 antibody, mouse anti-ERK1/2 antibody, rabbit anti-phospho JNK antibody, rabbit anti-JNK antibody, rabbit anti-phospho AKT (Ser473) antibody, mouse anti-AKT antibody, rabbit anti-cyclin A2 antibody, rabbit anti-cyclin B1 antibody, rabbit anti-cyclin E1 antibody, rabbit anti-caspase-9 antibody, rabbit anti-caspase-3 antibody, rabbit anti-PARP antibody, mouse anti-β-actin antibody, and DAPI (4, 6-diamidino-2-phenylindole, dihydrochloride) were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-mouse IgG-IRDye^®800CW and goat anti-rabbit IgG-IRDye^®680RT were purchased from Li-COR Biosciences (Lincoln, NE, USA). Goat anti-rabbit conjugated with Alexa488 and Goat anti-mouse conjugated with Alexa594 were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

JNK3a220 (link)23 (link), and MKK4 and MKK717 (link)20 (link) were expressed in E. coli and purified as previously described40 . The effect of arrestin-3 and MBP-T1A on the phosphorylation of JNK3α2 by MKK7 or MKK4 was analyzed by an in vitro kinase assay, as described19 (link)20 (link). Briefly, the assays were conducted in 10 μL containing the following final concentrations: 50 nM active MKK7 or MKK4, 1 μM JNK3α2, and indicated concentrations of arrestin-3 or T1A. The mixtures were incubated individually at 30 °C for 10 sec. The reactions were stopped by the addition of 15 μL of Laemmli SDS sample buffer (Sigma) and 2 μl of total reaction sample was subjected to SDS-PAGE (8%) and transferred polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). Phosphorylated JNK3α2 was visualized by rabbit anti-phospho JNK antibody (Cell Signaling) and the level of JNK phosphorylation was quantified.