Anti rabbit igg hrp

Anti-RBX1 antibody (ab133565, 1:1000 dilution for immunoblotting and 1:100 for IHC) was purchased from Abcam. Anti-POLR2A antibody (sc-47701,1:5000 dilution for immunoblotting and 1:200 for IHC), anti-CUL1 antibody (sc-17775, 1:1000 dilution), anti-CUL2 antibody (sc-166506, 1:1000 dilution), anti-CUL4A/4B antibody (sc-377188, 1:1000 dilution), anti-CUL5 antibody (sc-373822, 1:1000 dilution) and anti-CUL7 (sc-53810, 1:1000 dilution) antibody were obtained from Santa Cruz. Anti-Ki67 antibody (#12075, 1:100 dilution), anti-cleaved Caspase-3 (Asp175) (#9664, 1:1000 dilution), Anti-SKP2 antibody (#2652, 1:1000 dilution), Anti-VHL antibody (#68547, 1:1000 dilution) and Anti-CUL3 (#2759, 1:1000 dilution) were purchased from Cell Signaling Technology. Anti-p53 (sc-126, 1:1000 dilution), anti-actin (sc-1616, 1:5000 dilution), HRP-anti-mouse IgG (sc-2055, 1:5000 dilution), HRP-mouse IgG kappa binding protein (sc-516102, 1:5000 dilution) and HRP-anti-rabbit IgG (sc-2054, 1:5000 dilution) were purchased from Santa Cruz.

For the analysis of protein expression, cell pellets were washed and resuspended in PBS. Cells were centrifuged and pellets were stored at −20 °C. Cell pellets were then lysed in Winman’s buffer containing 1% NP-40, 0.1 M Tris–HCl pH 8.0, 0.15 M NaCl, and 5 mM EDTA, complemented with protease inhibitor cocktail (Roche). The total protein content was quantified using the DC™ Protein Assay kit (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions. Protein lysate (20 µg) were loaded on 12% SDS-PAGE gel and transferred into a nitrocellulose membrane (GE Healthcare, Cleveland, OH, USA). The following primary antibodies were used: rabbit anti-PARP-1 (1:2000, sc-7150, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-Caspase 3 (1:2000, 05-654, Merck Millipore, Darmstadt, Germany), and goat anti-actin (1:2000, sc-1616, Santa Cruz Biotechnology). The corresponding secondary antibodies were: anti-rabbit IgG-HRP, anti-mouse IgG-HRP, or anti-goat IgG-HRP (1:2000, Santa Cruz Biotechnology). The Amersham™ ECL Western Blotting Detection Reagents (GE Healthcare), the High Performance Chemiluminescence Film (GE Healthcare), and the Kodak GBX developer and fixer (Sigma) were used for signal detection [35 (link),36 (link)].

Cell lysates were prepared by incubation with a lysis buffer (40 mM Tris-HCl pH 8.0, 120 mM NaCl, 0.1% Nonidet-P40) supplemented with protease inhibitors. Then proteins were then subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) before being transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL). Primary antibodies were used to detect the relevant protein, and β-actin was used as loading control. Blots were developed with horseradish peroxidase (HRP)-conjugated secondary antibodies and proteins were visualized using enhanced chemiluminescence (ECL) (Amersham, Arlington Heights, IL), according to the manufacturer's protocol. Secondary antibodies, anti-mouse IgG-HRP, anti-goat IgG-HRP and anti-rabbit IgG-HRP were purchased from Santa Cruz biotechnology (CA, USA).

Cells were washed in ice‐cold PBS and sedimented at 1000 g for 10 min at RT. Pellets were suspended in RIPA buffer (10 mm Tris/HCl, pH 7.4, 1% sodium deoxycholate, 1% Triton X‐100, 0.1% SDS) containing 1 mm PMSF and protease inhibitor cocktail (Sigma‐Aldrich). Protein concentration was quantified using Bicinchoninic acid (Thermo Scientific, Rockford, IL, USA). Total proteins were separated by SDS/PAGE and transferred to Porablot NCP membranes (Macherey‐Nagel, Düren, Germany). Blots were probed with anti‐FLAG (1 : 2000; Sigma‐Aldrich), anti‐E‐cadherin (1 : 2000; Cell Signaling Technology, Danvers, MA, USA), anti‐N‐cadherin (1 : 2000; Cell Signaling Technology), anti‐Snail (1 : 2000; Cell Signaling Technology), and β‐actin (1 : 2000; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. Primary antibody binding was detected with anti‐goat IgG‐HRP (1 : 2000; Santa Cruz Biotechnology), anti‐mouse IgG‐HRP (1 : 2000; Santa Cruz Biotechnology), or anti‐rabbit IgG‐HRP (1 : 2000; Santa Cruz Biotechnology). Membranes were revealed using the EZ‐ECL chemiluminescence kit (Biological Industries, Haemek, Israel) and the ChemiDoc Touch Gel Imaging System (Bio‐Rad, Hércules, CA, USA).

In western blotting, we used the following primary antibodies: mouse monoclonal anti-STAT3 (1:1000) (BD Transduction Laboratories, San Jose, CA, USA; 612356), mouse monoclonal anti-phosphorylate-STAT3 (Tyr705) (1:100) (Santa Cruz Biotechnology Inc., Heidelberg, Germany; sc-8059), mouse monoclonal anti-sequestome1 (SQSTM1) (1:500) (BD Transduction Laboratories; cat. no. 610833), and rabbit polyclonal anti-LC3 (1:1000) (Novus Biologicals, Cambridge, UK; NB100-2220SS). To assess EBV infection, rabbit polyclonal anti-BRLF1 antibody (1:100) (Bioss Antibodies, Woburn, MA, USA; bs-4542R) and anti-EBNA1 antibody (1:100) (Santa Cruz Biotechnology Inc., Heidelberg, Germany; sc-81581) were used. Mouse monoclonal anti-β-actin (1:10,000) (Sigma Aldrich; A5441) (1:10,000) was used to detect β-actin, serving as a loading control. The goat polyclonal anti-mouse IgG-horseradish peroxidase (HRP, Santa Cruz Biotechnology Inc., Heidelberg, Germany; sc-2005) and anti-rabbit IgG-HRP (Santa Cruz Biotechnology Inc., Heidelberg, Germany; sc-2004) were used as secondary antibodies. All the primary and secondary antibodies were diluted in PBS-0.1% Tween 20 solution containing 3% of BSA (SERVA, Reno, NV, USA; 11943.03).

Cells were lysed in 1 × Laemmli buffer and denatured at 95 °C for 5 min. Cell lysates were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Blots were incubated with anti-STING (D2P2F), Phospho-TBK1 (D52C2), Phospho-NF-κB p65 (93H1), cleaved caspases-3 (Asp175) (5A1E), p53 (1C12) (all Cell Signaling) and anti-TBK1 (108A429) (Novus Biologicals) (all 1:1000 dilution). As a secondary antibody anti-rabbit-IgG-HRP (1:2000 dilution) (Santa Cruz Biotechnology) was used. Anti-β-actin-HRP or GAPDH (both 1:5000 dilution) was used as control. ECL signal was recorded on the ChemiDoc XRS Biorad Imager and data were analysed with Image Lab (Biorad).