Pherk

The DCs were lysed in a RIPA buffer (Cell Signaling Technologies, Danvers, MA, USA) in the presence of a cocktail of protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of total proteins were loaded onto SDS-PAGE gel and blotted onto a nitrocellulose membrane (BIO-RAD, Hercules, CA, USA). The membranes were incubated overnight, at 4 °C, with the following primary antibodies: HIF-1α (BD Biosciences, San Jose, CA, USA); phmTOR, mTOR, phULK1 ser 757, SQSTM1/p62, phAKT and AKT, Mcl-1, Vps34, Beclin-1, Atg5, phERK, α-Tubulin (Cell Signaling Technologies, Danvers, MA, USA); ULK1 and LAMP1 (Santa Crutz Biotechnology, Dallas, TX, USA) and β-actin (Sigma-Aldrich). Anti-mouse IgG HRP and anti-rabbit IgG-HRP (Cell Signaling Technologies, Danvers, MA, USA) were used as secondary antibodies. Images were acquired by ChemiDoc™ MP System and blot quantification was performed by using Image Lab software (BIO-RAD, Hercules, CA, USA).

TCL and NCF, and histones were resolved on 10 and 18 % polyacrylamide SDS-PAGE, respectively, and transferred to PVDF membrane. Proteins on PVDF membrane were hybridized with anti-H3 (Upstate-06-755; 1:2000 dilution), H4 (Millipore-07-108; 1:4000 dilution), H3S10ph (Millipore-06-570; 1:7000 dilution), H4K16ac (Millipore-07-329; 1:8000 dilution), H4K20me3 (Abcam-9053; 1:4000 dilution), β-actin (Sigma-A5316; 1:10,000 dilution), MSK1 (Santacruz-9392; 1:2000 dilution), ph-MSK1 (Abcam-31190; 1:3000 dilution), ERK1/2 (Santacruz-292838; 1:2000 dilution), ph-ERK (Cell signaling-9910; 1:2000 dilution), p38 (Santacruz-728; 1:2000 dilution), ph-p38 (Cell signaling-9910; 1:2000 dilution), and anti-flag (Sigma-F3165; 1:5000 dilution). Signal was visualized using horseradish peroxidase-conjugated anti-rabbit/mouse secondary antibody and ECL plus chemiluminescence kit (Amersham). Wherever required, the densitometry analysis was done on immunoblot and membrane to determine their mean intensities using ImageJ software. For native proteins, mean intensity of immunoblot was normalized with the stained PVDF membrane; for phosphorylated forms, mean intensity of immunoblot was normalized with immunoblot of native proteins. The resulted value was used to express their mean relative levels in resection margin and tumor.