Phospho p38 thr180 tyr182

Western blot analyses were performed as previously described (Jiang et al., 2016 (link); Jiang et al., 2017a ; Jiang et al., 2017b (link)). The HDAC Antibody Sampler Kit (#9928), Phospho-HDAC4^Ser246/HDAC5^Ser259/HDAC7^Ser155 (#3443), HDAC2 (#5113), HDAC6 (#7558), H3K4me2 (#9725), H3K4me3 (#9727), H3K27me2 (#9728), H3K27me3 (#9733), Phospho-AKT^Ser473 (#4060), AKT (#4685), Phospho-mTOR^Ser2448 (#5536), mTOR (#2983), Phospho-AMPKα^Thr172 (#2535), AMPKα (#5831), Phospho-MEK1/2^Ser217/221 (#9154), Phospho-ERK1/2^{Thr202/Tyr204} (#4370), ERK1/2 (#4695), Phospho-JNK1/2 ^{Thr183/Tyr185} (#4668), JNK1/2 (#9258), Phospho-p38^{Thr180/Tyr182} (#4511), p38 (#8690), Phospho-β-Catenin^Ser675 (#4176), Non-phospho-β-Catenin^{Ser33/37/Thr41} (#8814), β-Catenin (#8480), Phospho-IKKα/β^Ser176/180 (#2697), IKKβ (#8943), Phospho-NF-κB p65^Ser536 (#3033), NF-κB p65 (#8242), Phospho-Smad2^Ser465/467 (#3108), Smad2 (#5339), Phospho-Smad3^Ser423/425 (#9520), Smad3 (#9523), Phospho-Smad1/5^Ser463/465 (#9516), and Smad1 (#6944) antibodies were obtained from Cell Signaling Technology. Antibodies against Histone H3 (ab1791), H3K36me2 (ab9049), H3K36me3 (ab9050), H4K20me3 (ab9053), H3K9me1 (ab9045), H3K9me2 (ab1220), H3K4me1 (ab8895), H3K23me1 (ab176132), H4K5ac (ab51997), H4K8ac (ab15823), H3K18ac (ab40888), H3K23ac (ab61234) and H4K12ac (ab46983) were purchased from Abcam.

For the protein isolation, 1 × 10⁶ cells were cultured on 100mm Petri dishes and treated with experimental medium containing of DON for 24 h. The protein isolation and Western blots were conducted as previously described [48 (link)]. 30 µg (60 µg for phospho-p44/42 (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), phospho-SAPK/JNK (Thr183/Tyr185) antibody) of protein samples was used for electrophoresis. SOD1 (#4266), SOD2 (#13141), p65 (#8242), p44/42 (#4695), SAPK/JNK (#9252), p38 (#8690), phospho-p44/42 (Thr202/Tyr204) (#4370), phospho-p38 (Thr180/Tyr182) (#4511), phospho-SAPK/JNK (Thr183/Tyr185) (#4668) (Cell Signaling Technology, Leiden, WZ, The Netherlands) antibodies were used according to manufacturer’s recommendations. The results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-59540) (Santa Cruz Biotechnology Inc, Dallas, TX, USA) as a reference protein.

Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% (v/v) NP-40, 5 mM EDTA pH 8.0, 5 mM EGTA pH 8.0, 20 mM NaF, 0.1 μM PMSF, 0.1 μM NaVO₃, plus complete protease inhibitor cocktail (Roche)) and cellular lysates were separated by SDS–PAGE and transferred to PVDF membranes. The following antibodies were used for protein detection: p38α MAPK (1/1,000, Cell Signaling, #9228), phospho-p38 (Thr180/Tyr182, 1/1,000, Cell Signaling, #9215), phospho-c-Jun (Ser63, 1/1,000, Cell Signaling, #9161), c-Jun (1/1,000, Cell Signaling, #L70B11), phospho-JNK (Thr183/Tyr185, 1/1,000, Cell Signaling, #9255), JNK (1/1,000, Cell Signalling, #9258), phospho-Smad2 (Ser465/Ser467, 1/1,000, Millipore, #AB3849), Smad2 (1/1,000, Cell Signalling, #3103), human CXCL12/SDF-1 antibody (1/1,000, R&D Systems, #AF-310-NA), tubulin (Sigma). For detection, we used Alexa Fluor 680- (1/5,000, Molecular Probes) or Li-Cor IRDye 800- (Rockland) labelled antibodies with the Odyssey Infrared Imaging System (Li-Cor).

Mouse polyclonal antibodies specific for IL-6, rabbit polyclonal antibodies specific for EGFR, phospho-c-Jun and c-Jun were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies specific for phospho-Raf-1 Ser338, Raf-1, phospho-ERK1/2 Thr202/Tyr204, ERK1/2, phospho-JNK Thr183/Tyr185, JNK, phospho-p38 Thr180/Tyr182 and p38 were from Cell Signaling (Danvers, MA). PD98059, SB203580, SP600125, U0126, GW5074 and AZD6244 were obtained from Calbiochem (San Diago, CA). Mouse polyclonal antibody specific for IL-6 was purchased from R&D systems (Minneapolis, MN). All chemicals were obtained from Sigma-Aldrich (St Louis, MO). IL-6 enzyme immunoassay kit was purchased from Cayman Chemical (Ann Arbor, MI).

Lipopolysaccharides (LPS; Escherichia coli 0111:B4) and synthetic bacterial lipopeptide (Pam3Cys-Ser-Lys4-OH) were from InvivoGen (San Diego, CA, USA). N-acetylcysteine (NAC), diphenyleneiodonium (DPI), BAY11-7082 (BAY), caffeic acid phenethyl ester (CAPE), U0126, SB203580, and SP600125 were from Calbiochem (San Diego, CA, USA). 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt (Tiron) and dimethyl sulfoxide (DMSO; added to the cultures at 0.05 % (v/v) as a solvent control) were from Sigma-Aldrich. Phospho-SAPK/JNK (Thr183/Tyr185), phospho-ERK1/2 (Thr202/Tyr204), and phospho-p38 (Thr180/Tyr182) were from Cell Signaling Technology (Cell Signaling; Beverly, MA, USA). β-actin (I-19) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cisplatin and TGF-β1 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in PBS and 10 mM Citric Acid (pH 3.0), respectively. Small inhibitors LY294002, SB203580, U0126, SP600125 (SelleckChemicals, Houston, TX, USA) were dissolved in DMSO. Phospho-Akt (Ser473), phospho-Smad3 (Ser423/425), phospho-ERK (Thr202/Tyr204), Erk, phospho-p38 (Thr180/Tyr182), SAPK/JNK, phospho-SAPK/JNK (Thr183/Tyr185), p15^INK4B, p21^Waf1/Cip1, bax, bcl2, and ki67 were purchased from Cell Signaling Technology (Beverly, MA, USA). P38, pan-Akt, and β-Actin were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Smad3 and c-myc were purchased from Abcam Co Ltd (Cambridge, UK). Horse radish peroxidase (HRP) conjugated secondary anti-rabbit and antimouse IgG antibodies were from Sigma-Aldrich Co., Ltd.

The CMFE sample used in this study was provided by CNC Korea (Seoul, Korea) and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA). Arbutin, mushroom tyrosinase, α-melanocyte-stimulating hormone (α-MSH), L-ascorbic acid, L-DOPA, methanol, MG132, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Phosphate-buffered saline (PBS) was purchased from Biosesang (Seongnam, Korea). Primary antibodies against MITF (microphthalmia-associated transcription factor), tyrosinase, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p38 and phospho-p38 (Thr180/Tyr182) were purchased from Cell Signaling (Beverly, MA, USA). HPLC-grade MeOH and water were purchased from Burdick & Jackson (Muskegon, MI, USA). Unless otherwise indicated, all other chemicals were purchased from Sigma-Aldrich.