Jnk inhibitor 2 sp600125

The following were purchased: antibodies, SEK1/MKK4 (#9152), phospho-p38 MAPK (T180/Y182) (#4631), p38 MAPK (#9212), phospho-SAPK/JNK (T183/Y185) (#4668), SAPK/JNK (#9258), phospho-HSP27 (#2401), HSP27 (#2402), and GAPDH (#2118), all from Cell Signaling Technology, and horseradish peroxidase-conjugated anti-mouse and anti-rabbit, from GE Healthcare Biosciences; plasmids, wild type and constitutive active MAP2K4 (#14615 and #14813; Addgene) and GFP (Vivid Colors pcDNA 6.2/N-EmGFP-GW/TOPO, Invitrogen); chemical inhibitors, p38 MAPK inhibitor, SB203580 (Sigma-Aldrich) and associated negative control, SB202474, and JNK Inhibitor II (SP600125), and JNK Inhibitor II Negative Control (N¹-Methyl-1,9-pyrazoloanthrone), all from EMD Millipore.

Purified LPS was purchased from InvivoGen (San Diego, CA). AT-406 was synthesized as described^{65 (link)}. Flag-M2 antibodies, Flag-M2 beads, necrostatin-1 (Nec-1), cycloheximide (CHX), thapsigargin (THAP), propidium iodide (PI) and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) were purchased from Sigma (St. Louis, MO). JNK inhibitor II (SP600125) and DMSO were purchased from Merck Millipore (Billerica, MA). Annexin V-Cy5 was obtained from BD-Biosciences (Franklin Lakes, NJ). Recombinant GM-CSF was purchased from R&D (Minneapolis, MN, USA). z-VAD-FMK was obtained from Bachem (Bubendorf, Switzerland). Recombinant mouse and human TNF were purchased from PeproTech (Rocky Hill, NJ, USA). Recombinant mouse IFNβ was purchased from PBL Assay Science (Piscataway, NJ, USA). BV6 was purchased from Selleck Chemicals (Houston, TX). Recombinant human Fas ligand (FLAG-FasL) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). CellTiter-Glo^® Luminescent Cell Viability Assay was purchased from Promega (Fitchburg, Wisconsin). Lipofectamine 2000 was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Constructs of p38α MAPK and active MKK3 (MKK3b(Glu¹⁸⁹, Glu¹⁹³)) were gifts of Dr. Jiahuai Han (Xiamen University, China).

The LNCaP, 22Rv1, PC-3, and DU145 cell lines were obtained from the Bioresource Collection and Research Center (Hsinchu, Taiwan) and maintained in RPMI 1640 medium with 10% fetal calf serum (FCS), as described previously [28 (link)]. 22Rv1 cells, an AR-positive but androgen-independent cell line, were derived from a xenograft and serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR22 xenograft [29 (link)]. The FCS was purchased from HyClone Laboratories (Logan, UT, USA), RPMI 1640 medium from Life Technologies (Gaithersburg, MD, USA), Matrigel from BD Biosciences (San Jose, CA, USA), methyltrienolone (R1881) from NEN Life Sciences (Boston, MA, USA), ERK inhibitor (PD0325901), p38 inhibitor (SB202190), AMPK inhibitor (dorsomorphin), and camptothecin from Sigma (St. Louis, MO, USA), CAPE from Selleckchem (Houston, TX, USA), and JNK inhibitor II (SP600125) from Merck Millipore (Burlington, MA, USA). For the androgen treatment study, the cells were incubated in phenol red-free medium (RPMI-PRF) containing 5% charcoal-dextran FCS (CD-FCS) to deplete endogenous steroid hormones.