Total p44 42 mapk

Samples (30 μg proteins) were separated on 12% SDS polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) and electroblotted onto 0.2 μm nitrocellulose membranes. Membranes were incubated overnight at 4 °C with primary antibody recognizing Phospho-GSK3β (Ser21/9) or Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA). Membranes were then incubated with a horseradish peroxidase linked anti-rabbit secondary antibody (1:2000; GE Healthcare, Piscataway, NJ, USA). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Pierce, Rockford, IL, USA). The same membranes were stripped and reprobed with total GSK3β or total p44/42 MAPK (1:1000; Cell Signaling Technology Inc.), respectively. Finally, to obtain a further loading control, membranes were stripped and reprobed with a monoclonal primary antibody recognizing β-actin (1:1000; Sigma-Aldrich) and then with a horseradish peroxidase linked anti-mouse secondary antibody (1:2000; GE Healthcare). Data were analyzed by densitometry, using Quantity One software (Bio-Rad, Hercules, CA, USA).

RPMI 1640, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from GE healthcare (PAA, Pasching, Austria); Anti-CD54-FITC was purchased from Biolegend (San Diego, CA, USA). Anti-CD13-FITC, CD33-PE, anti-CD15-PE, and anti-CD11b- PE were purchased from Affymetrix (eBioscience, San Diego, CA, USA). Primary antibodies for p-MEK1/2, total MEK1/2, p-P44/42 MAPK, total P44/42 MAPK, p-JNK, total JNK, p-P38, total P38, ICAM-1, p-P65, and total P65 were all obtained from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies for p-STAT1 and total STAT1 were purchased from Beckton Dickinson (BD Biosciences, San Jose, CA, USA). Anti-β-actin and horseradish peroxidase coupled secondary antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Horseradish peroxidase coupled secondary antibody was purchased from Cell Signaling Technology (Danvers, MA, USA).
Dimethyl sulfoxide (DMSO), all-trans retinoic acid (ATRA), and celastrol were all purchased from Sigma-Aldrich (St Louis, MO, USA). ATRA and celastrol were dissolved to 100 mM in DMSO, stored at −20°C, and used within three month. ATRA and celastrol was further diluted with culture medium (for cell culture experiments) or PBS (for animal experiments) just before use.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail tablet (Roche Diagnostics) and 1 mmol/L Na3VO4. Equal amounts of protein were loaded and separated on a 4–12% PAGE gel (Invitrogen). Proteins were transferred to polyvinylidenedifluoride (PVDF) membranes, which were blocked in 5% nonfat dried milk. Membranes were then incubated with primary and secondary antibody and developed by ECL. Antibodies used to probe were NMT1 and GNAQ (Santa Cruz Biotechnology), phospho-p44/42 MAPK (ERK1/2), Total p44/42 MAPK (ERK1/2), phospho-Akt (Ser473), phospho-FAK (Tyr576/577), Na,K-ATPase, GAPDH, Caspase-3, and Cleaved PARP (Cell Signaling).

Clys were obtained from PEPTIDE Inc. (Osaka, Japan). sIL-6R was purchased from R&D Systems. Antibodies against phospho-p44/42 MAPK, total-p44/42 MAPK, phospho-JNK MAPK, total-JNK MAPK, phospho-p38, total-p38 and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). U0126 (mitogen-activated protein kinase kinase [MEK] inhibitor) and SB2013580 (p38 MAPK inhibitor) were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Western blotting was performed as described (37 (link)). Protein was extracted from frozen tissues in RIPA buffer and total protein content was determined using the BCA protein assay (Sigma, St. Louis, Mo) with BSA as a standard. Proteins were separated by SDS-PAGE, transferred onto PVDF membranes and incubated with an appropriate primary and secondary antibody. Equal loading and transfer was confirmed by staining, imaging and quantifying the original gel, using Biorad stain-free gel technology. Immunoblotting was performed for pS6 (#5364), total S6 (#2217), pAkt ^Ser473 (#4060), total Akt (#4691), p-p44/42MAPK^{Thr202/Tyr204} (#9101) total p44/42 MAPK (#4695), and β-catenin (#8480), all from Cell Signaling. Bands were visualized by chemiluminescence to first indication of pixel saturation using a Biorad Chemidoc bioimager and densitometry performed using Image Lab 4.1 (Biorad, Hercules, CA).