Amersham hybond pvdf membrane

Cells were harvested by trypsinization and pelleted by centrifugation at 1000g for 2 min at 4°C. Cell pellets were resuspended in 500 µL lysis buffer (100 mM KCl, 0.1 mM EDTA, 20 mM HEPES-KOH pH 7.6, 0.4% NP-40, 10% glycerol, 1 mM DTT, complete mini EDTA-free protease inhibitors [Roche]) and clarified at 21,000g for 5 min at 4°C. A total of 250 µL supernatant was mixed with 20 µL 4× Bolt LDS sample buffer (Thermo Fisher Scientific), 8 µL 10× Bolt sample reducing agent (Invitrogen) and proteins were denatured at 75°C for 10 min. Protein samples were loaded into Bolt 4%–12% Bis-TRIS Plus gels (Thermo Fisher Scientific) and run at 160 V for ∼1 h. The gel was transferred onto an Amersham Hybond PVDF membrane (GE Healthcare), according to manufacturer's instructions. Primary antibodies were added at 1:10,000 concentration for α-V5 antibodies (Cat #V8012, Sigma-Aldrich), 1: 10,000 for α-puromycin antibodies (Cat #3RH11, Cedarlane), and 1: 5000 for α-tubulin antibodies (Cat #T5168 Sigma-Aldrich). α-mouse secondary antibody (Cat #7076, New England Biolabs) was used at 1:10,000. Blots were imaged using ECL Prime Western Blotting Detection Reagent (GE Healthcare) and exposed on Amersham Hyperfilm (GE Healthcare).

The tissues were homogenized and lysed in a buffer containing 40 mmol/L Tris/HCl (pH 7.4), 150 mmol/L NaCl, 2 mmol/L EDTA, 1 mmol/L dithiothreitol, 1% Triton X-100, 2 mmol/L sodium orthovanadate, 10 mmol/L NaF, and 10 mmol/L sodium pyrophosphate supplemented with a protease inhibitor cocktail mixture (Sigma, St Louis, MO, USA). Protein contents were measured by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and bovine serum albumin was used as a standard. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then proteins were transferred onto an Amersham Hybond PVDF membrane (GE Healthcare, Little Chalfont, UK). After a blocking procedure, the membrane was incubated with anti-mPGES-1 (No. 160140; Cayman Chemicals, Ann Arbor, MI, USA), anti-cPGES (No. 160150; Cayman Chemicals), anti-COX-2 (No. 160106; Cayman Chemicals), anti-COX-1 (NAB37401; R&D Systems, Minneapolis, MN, USA), or anti-β-actin (clone 2F3; Fujifilm Wako Pure Chemical, Osaka, Japan) antibody and then incubated with a secondary antibody coupled to horseradish peroxidase (Jackson ImmunoResearch Laboratories, PA, USA). After washing, protein was detected by enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK).

Equal amounts of proteins (20 μg), as determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA), were resolved on a 8% or on a 12% SDS-PAGE and electrotransferred to Amersham Hybond PVDF membrane (GE Healthcare, Milan, Italy). Membranes were then blocked for 1 h at 25 °C in PBS containing 5% nonfat dry milk and 0.1% Tween-20 (Sigma-Aldrich), and were incubated for 1 h at 25 °C or overnight at 4 °C, with the following primary antibodies: BRLF1 and BZLF1(1 : 200 and 1 : 100, respectively, both obtained from Argene Biosoft, Verniolle, France), BALF5 (1 : 100), LC3 (1 : 8000, L7543) and β-actin (1 : 5000) were purchased from Sigma, Beclin1(1 : 500; sc-10086) and ATG5 (1 : 200, sc-133158) purchased from Santa Cruz (DBA, Milan, Italy), and phospho-p70 S6 kinase (1 : 1000, 07-018-I Millipore, Merk Spa, Vimodrone, MI, Italy). Phospho ERK1/2 and ERK1/2 antibodies (1 : 500) were obtained from Cell Signaling (EuroClone, Milan, Italy). The membranes were then incubated with the appropriate secondary antibodies conjugated to HRP (1 : 7500, Bio-Rad). The specific signals, visualized by Amersham ECL Plus kit (GE Healthcare) were quantified by densitometric analysis by ImageJ free-share software (http://imagej.nih.gov/ij).