Anti p erk1 2 t202 y204

Anti-IRS-1, anti-pIRS-1 (S307), anti-AKT (T308), anti-AKT, anti-p-mTOR (S2448), anti-mTOR, anti-p-p70S6K (T389), anti-p70S6K, anti-p-S6 (S235/S236), anti-S6, anti-p-p38MAPK (T180/Y182), anti-p38MAPK, anti-p-ERK1/2 (T202/Y204), anti-ERK1/2, anti-p-SAPK/JNK (T183/Y185), anti-SAPK/JNK, anti-p-AMPK (T172), anti-AMPK, anti-ACC1, and anti-FASN monospecific antibodies were purchased from Cell Signaling (Danvers, MA). Insulin, Oil Red O, and anti-actin antibodies were obtained from Sigma-Aldrich (St. Louis, MO). Anti-SREBP1 antibody was bought from Becton-Dickinson (Franklin Lakes, NJ). Anti-SREBP2 and anti-ChREBP antibodies were bought from R&D Systems (Minneapolis, MN). Combined protease and phosphatase inhibitor cocktails (Halt™) and Tissue-Tek™ optimum cutting temperature (OCT) compound were bought from Thermo Fisher Scientific (Hampton, NH). Forward and reverse oligonucleotide primers (Additional file 1: Table S1) to carry out quantitative PCR analysis were designed according to a published program (OligoArchitectTM online, Sigma-Aldrich, St. Louis, MO). DNA primers were synthesized by Integrated DNA Technologies (Coralville, IA).

Rabbit anti-KDM4A, anti-ERK1/2, anti-p-ERK1/2 (T202/Y204), anti-c-Jun, and anti-p-c-Jun (S63) were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-KDM4B was purchased from Bethyl Laboratories (Montgomery, TX, USA). Rabbit anti-KDM4C was purchased from Novus Biological (Littleton, CO, USA). Rabbit anti-H3K9me3 was purchased from Active Motif (Carlsbad, CA, USA). Rabbit anti-IgG, and anti-p-Tyrosine were purchased from Millipore (Billerica, MA, USA). Mouse anti-CagA, and anti-IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse anti-β-Actin was purchased from Sigma. Lentiviral vector pLKO-control (pLKO), pLKO-shKDM4A (sh4A#1, TRCN0000234912; sh4A#2, TRCN0000234914), -shKDM4B (sh4B#1, TRCN0000018016; sh4B#2, TRCN0000379460), -shKDM4C (sh4C#1, TRCN0000235047; sh4C#2, TRCN0000235048) plasmids were purchased from The RNAi Consortium (TRC).

The primary antibodies used were anti-pan-ERK (1:10,000; BD Transduction Laboratories), anti-ALK (for immunofluorescence 1:1000; ab4061, Abcam), anti-ALK (D5F3, 1:5000; Cell Signaling Technology), anti-ALK mAb135 (1:2000; [Witek et al., 2015 (link)]), anti-pERK5 (1:1000; Cell Signaling Technology), anti-pALK-Y1278 (1:2000; Cell Signaling Technology), anti-pALK-Y1604 (1:2000; Cell Signaling Technology) and anti-pERK1/2-T202/Y204 (1:2000; Cell Signaling Technology), anti-FAM150A (1:4000, Atlas Antibodies), anti-HA (1:1000 for immunofluorescence, 1:6000 for immunoblotting; 16B12, Covance). The activating monoclonal antibody mAb46 and ALK monoclonal antibodies mAb13, mAb48 were a kind gift from M. Vigny and have been described previously (Moog-Lutz et al., 2005 (link)). The ALK inhibitor crizotinib was purchased from Chem Express (Shanghai).

The following antibodies were used: anti-Pellino-1 (F-7), anti-β-TrCP(H-85) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-actin, anti-Flag M2 (Sigma, St. Louis, MO, USA), anti-Slug, anti-Snail, anti-E-cadherin, anti-β-catenin, anti-Vimentin, anti-ERK1/2, anti-p-ERK1/2 (T202/Y204), anti-Akt, anti-p-Akt (S473), anti-GSK3β, anti-p-GSK3β (S9) (Cell Signaling Technology, Danvers, MA, USA), anti-HA, anti-Myc (Roche, Basel, Switzerland). The following reagents were used: LY294002, PD98059 (Calbiochem, San Diego, CA, USA), MG132, cycloheximide, dimethyl sulfoxide (DMSO) (AG Scientific, San Diego, CA, USA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma), EGF, mitomycin C (Sigma) and EGFR kinase inhibitor AG1478 (Selleckchem, Houston, TX).

To measure the extracellular signal-regulated protein kinase (ERK1/2), thyrospheres were lysed and subjected to Western blot analysis, as previously described (20 (link)). The following antibodies against total and phosphorylated ERK1/2 were purchased from Cell Signaling Technology (Beverly, MA, USA): anti-ERK1/2, anti-P-ERK1/2 (T202/Y204) and anti-Vinculin. Bands were detected digitally using the Odyssey Fe imaging system (Li-COR Bioscience, Lincoln, NE, USA) and the blots were then quantified using the Li-COR Image Studio software version 5.2.5.
The metal concentration used for evaluating ERK1/2 activation at 5, 15 and 30 minute time points was selected on the basis of the peak effect of each metal on BrdU incorporation.
ERK phosphorylation was also measured in cells exposed to metals in the presence of PD98059 (20 μM) that was kept in the culture medium throughout the entire period of exposure to metals.