Peroxidase labelled secondary antibody

In brief, cells were lysed in whole-cell lysate buffer containing 1% phosphatase inhibitor cocktail and NPC tumor tissues were lysed in RIPA buffer (Beyotime, Jiangsu, China) with 1% phosphatase inhibitor cocktail. Lysates containing 20-30 µg protein were loaded onto 8% or 12% sodium dodecyl sulfate-polyacrylamide gels for electrophoresis (SDS-PAGE) and the separated proteins transferred to poly vinylidene fluoride (PVDF) membranes (Pall, NY, USA). After blocking with 5% fat free milk for 1 h in Tris-buffered saline (TBS), the membranes were incubated with the primary antibody overnight at 4°C and then with the peroxidase labelled secondary antibody (agilent, CA, USA) for 1 h on the next day. WB bands were visualized with an enhanced chemiluminescence kit (EMD Millipore, MA, USA). The primary antibodies used were γH2AX, XRCC1, KDM5B, UB(1:2000 dilution, abcam, Cambridge, UK), HSP90, KDM5A, KDM5C, KDM5D(1:2000 dilution, proteintech, IL, USA), flag (1:2000 dilution, sigma, Louis, MO, USA ) and monoclonal anti-cleaved PARP-1, α-tubulin, H3K4 mono/di/tri-me, H3K4 mono-me, H3K4 di-me, H3K4 tri-me, H3(1:2000 dilution, Cell SignalingTechnology, Danvers, MA, USA).

Western blot analyses were performed as previously described 28 (link). In brief, cells were lysed in whole-cell lysate buffer containing 1% phosphatase inhibitor cocktail and NPC tumor tissues were lysed in RIPA buffer (Beyotime, Jiangsu, China) with 1% phosphatase inhibitor cocktail. Lysates containing 20-30 µg protein were loaded onto 8% or 12% sodium dodecyl sulfate-polyacrylamide gels for electrophoresis (SDS-PAGE) and the separated proteins transferred to poly vinylidene fluoride (PVDF) membranes (Pall, NY, USA). After blocking with 5% fat free milk for 1 h in Tris-buffered saline (TBS), the membranes were incubated with the primary antibody overnight at 4°C and then with the peroxidase labelled secondary antibody (agilent, CA, USA) for 1 h on the next day. WB bands were visualized with an enhanced chemiluminescence kit (EMD Millipore, MA, USA). The primary antibodies used were γH2AX (1:2000 dilution, Epitomics, Burlingame, CA, USA) and monoclonal anti-cleaved PARP-1, β-actin, p-AKT, p-mTOR, SQSTM1/p62, LC-3, P-P65 (1:2000 dilution, Cell Signaling Technology, Danvers, MA, USA).

Standard indirect immunoperoxidase procedures were used for immunohistochemistry
(IHC; ABC-Elite, Vector Laboratories, Burlingame, California). Slides were
dewaxed and rehydrated in distilled water. Endogenous peroxidase activity was
blocked using 0.5% H2O2. Sections were incubated with 10% normal goat serum
(DakoCytomation, Carpinteria, California) for 20 minutes and incubated with
primary antibody at room temperature (RT). Primary antibodies used were specific
for CD16 and OX40 (polyclonal anti-CD134/OX40, ab119904, Abcam, Cambridge,
United Kingdom; biotinylated anti-CD16 were purchased from DAKO (Glostrup,
Denmark) and Novocastra (Newcastle, United Kingdom). Fluorescein
isothiocyanate-conjugated anti-CD16, phycoerythrin-conjugated anti-CD16,
Cy5-conjugated anti-CD16, and isotype-matched monoclonal antibody were purchased
from BD Bioscience (San Jose, California), as previously published by our group.^{22 (link),23 (link)} Subsequently, sections were incubated with peroxidase-labelled secondary
antibody (DakoCytomation) for 30 minutes at RT. For antigen visualization,
sections were immersed in 3-amino-9-ethylcarbazole plus substrate-chromogen
(DakoCytomation) for 30 minutes and counterstained with Gill’s hematoxylin.