Anti phospho c jun

Thapsigargin (Tg), puromycin dihydrochloride, p38 inhibitor (SB203580), and JNK inhibitor (SP600125) were purchased from Sigma (St. Louis, MO, USA). AZD8055 and refametinib were procured from Cayman Chemical (Ann Arbor, MI, USA). The anti-puromycin and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Millipore (Burlington, MA, USA) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. The anti-phospho 4E-BP1 (Ser65), anti-4E-BP1, anti-phospho ERK 1/2, anti-ERK 1/2, anti-phospho p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho eIF2α, anti-eIF2α, anti-phospho c-Jun, anti-c-Jun, and horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor^® 488 AffiniPure Goat Anti-Mouse IgG (Fcγ fragment specific) was supplied by Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The Selleck Anti-Cancer Compound Library consisting of 414 drugs was purchased from the Department of Convergence Medicine, ASAN Medical Center, University of Ulsan College of Medicine (Seoul, Korea).

Anti-ICAM-1, anti-GAPDH, anti-S1PR1, anti-S1PR2, anti-S1PR3, anti-c-Src, anti-EGFR, anti-PDGFR, anti-JNK1, anti-p42, anti-p38, anti-c-Jun, and anti-c-Fos antibodies and ICAM-1 neutralizing antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-c-Src, anti-phospho-EGFR, anti-phospho-PDGFR, anti-phospho-JNK1/2, anti-phospho-p42/p44 MAPK, anti-phospho-p38 MAPK, anti-phospho-Akt, and anti-phospho-c-Jun antibodies were from Cell Signaling (Danver, MA). W123, JTE-013 and CAY10444 were from Cayman (Ann Arbor, MI). PP1, U0126, SP600125, SB202190, AG1478, AG1296, Genistein, Tanshinone IIA, and LY294002 were from Biomol (Plymouth Meetings, PA). BCECF/AM was from Molecular Probes (Eugene, OR). SDS-PAGE reagents were from MDBio Inc (Taipei, Taiwan). All other reagents were from Sigma (St. Louis, MO).

The following commercial antibodies were used: anti-ERK1/2, anti-phospho-ERK1/2, anti-JNK1/2, anti-phospho-JNK1/2, and anti-phospho-c-Jun (all Cell Signaling Technology, Danvers, MA, USA); anti-c-Jun, anti-phospho-Histone3, anti-p21 and anti-EGFR (Santa Cruz Biotechnology, Heidelberg, Germany); anti-phospho-EGFR and blocking anti-EGFR (Thermo Fisher Scientific, Rockford, IL, USA); anti-Paxillin (Abcam, Cambridge, MA, USA); and anti-β-Actin (Sigma-Aldrich, St Louis, MO, USA). Secondary antibodies were anti-rabbit IgG Horseradish peroxidase linked F(ab’)₂ I fragment (from donkey) (GE Healthcare, GE, Little Chalfont, United Kingdom), Horseradish peroxidase linked Rat anti-mouse IgG₁ (BD Pharmingen, Beckton Dickinson, Franklin Lakes, NJ, USA) and Alexa Fluor 488 conjugated anti-mouse IgG (from donkey) (Thermo Fisher Scientific, Rockford, IL, USA).

Anti-c-Jun (#9165), anti-phospho-c-Jun (#9261), anti-phospho-JNK (#9251), anti-Sox2 (#3579), anti-Nanog (#4903), anti-E-cadherin (#3195) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-β-actin (A1978) was from Sigma (St. Luis, MO, USA). Anti-JNK1 (sc-474), anti-JNK2 (sc-7345) and anti-K-Ras (sc-30) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-CD133 (W6B3C1) was from Miltenyi Biotech (Germany). SP600125 was purchased from Calbiochem (La Jolla, CA, USA) and was dissolved in DMSO to prepare a 50 mM stock solution.

Protein extraction solution PRO-PREP^TM was obtained from iNtRON Biotechnology (Jungwon, Gyeonggi, Korea). Polyvinylidene fluoride membranes and western chemiluminescent HRPV substrate were obtained from Millipore Corporation (Billerica, MA, USA). The above reagents were used according to the manufacturer’s protocol. The following antibodies from Cell Signaling Technology (Danvers, MA, USA) were used: EGFR antibody, anti-phospho-Src family (Tyr416), anti-phospho-AKT (Ser473), anti-phospho-Erk1/2, anti-p47^phox, anti-phospho-NF-κB p65, anti-phospho-c-Fos, anti-phospho-c-Jun, anti-p44/42 MAPK (ERK1/2), anti-NF-κB p65, and anti-β-actin monoclonal antibodies. The detailed western blot steps were described previously [13 (link)].