Phospho erk 4370

REH (#ACC-22), TF-1 (#ACC334) and L929 (#ACC-2) cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany), KYM-1 cells were kindly provided by Harald Wajant (University of Wuerzburg). All cell lines were grown RPMI 1640 medium (PAN Biotech, Aidenbach, Germany) supplemented with 10% (v/v) fetal calf serum (Sigma, Steinheim, Germany). TF-1 cells were additionally supplemented with 5 ng/ml human granulocyte–macrophage colony-stimulating factor (Immunotools, Friesoythe, Germany). Antibodies used in the study: TNF #3707, p38 #9212, phospho-p38 #9211, ERK #4695, phospho-ERK #4370 (Cell Signaling, Beverly, MA, USA); tubulin #MS-581: Dunnlab (Asbach, Germany); NFAT5 #PA1-023: Thermo Fisher Scientific (Waltham, MA, USA). Chemicals: MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide): Biomol (Hamburg, Germany); zVAD-fmk (carbobenzoxy-valyl-alanyl-aspartyl-(Omethyl)-fluoromethylketone): Bachem (Bubendorf, Switzerland); BIRB796, PH797804, SB203580, BV6, LCL161 and birinapant: Selleck Chemicals (Houston, TX, USA). Necrostatin-1 (Nec-1): Stress-Marq (Victoria, Canada); Etanercept was obtained from Pfizer (Berlin, Germany).

Human lung cancer cell lines (A549, NCIH1299, NCIH23, NCIH226, NCIH460, NCIH520, NCIH1703, SW1271, SW1573 and NCIH358) and blood cancer cell lines (KMS11 and RCH-ACV) were purchased from ATCC, JCRB and DSMZ. Antibodies were obtained from the following sources: NSD2 39879, H3K36me2 61019 and H3K36me2 39255 from Active Motif, NSD3 11345-1-AP from Proteintech, phospho ERK 4370 and H3K36me2 2901 from Cell Signaling, total ERK from BD Biosciences (anti-ERK1 554100 and anti-ERK2 610103), H3K36me2 ab9049 from Abcam, H3 07-690 and H3K27me3 07-449 from Merck Millipore and ACTB A5441 from Sigma Aldrich. Plasmid containing human NSD2 cDNA was purchased from Source BioScience (IMAGE ID: 100066394) and subcloned into pMSCV-FLAG-puro. The E1099K mutation was introduced using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies).