Nitrocellulose membrane

Detection of PPV in leaf samples using dot-ELISA was performed as described previously [31 (link)]. Briefly, each apricot tree leaf sample (about 50 mg) was ground in 1 mL of 0.01 mol/L phosphate buffered saline (PBS), pH 7.4, followed by 3 min centrifugation at 5000 rpm at 4 °C. The resulting supernatant was used for PPV detection. A known PPV-infected and uninfected apricot tree leaf samples were used as the positive and negative controls. Each supernatant (2 µL) was loaded onto a nitrocellulose membrane (GE Healthcare, Bucks, UK) and the dotted nitrocellulose membrane was air-dried for 10 min at room temperature (RT). The nitrocellulose membrane was blocked for 30 min in a PBS solution containing 5% skimmed milk powder followed by 1 h incubation in a diluted anti-PPV MAb solution (first antibody). After three rinses in the PBS solution containing 0.05% Tween-20 (PBST), the membranes were probed again for 1 h in a diluted alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (second antibody) followed by three rinses in the PBST. Color reaction on the membrane was visualized after the addition of a nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate substrate solution (Sigma-Aldrich) for 10-20 min at RT.

Cells were harvested and lysed in TBS containing protease inhibitor and phosphatase inhibitor. After sonication and centrifugation at 13,000 × g for 15 min, each of the lysate samples (1.2 μg/spot) was loaded on a nitrocellulose membrane (0.45 μm pore size, GE Healthcare). Cell culture medium was collected and centrifuged at 800 × g for 3 min to remove debris. 500 μl supernatant from each sample was concentrated to 50 μl using Vivaspin (GE Healthcare), with a molecular weight cut-off of the filtration membrane of 10 kD. 2 μl of each concentrated sample was loaded on a nitrocellulose membrane. Membranes were blocked with 5% skim milk, hybridized with appropriate antibodies, and visualized using Western Lightning Plus-ECL (PerkinElmer, Waltham, MA). Images were acquired on ImageQuant LAS 4000 (GE Healthcare). The following primary antibodies were used: Tau12 (1:10,000, Millipore) and TOC1 (1:5,000).

The replication of HBV was detected by Southern blot analysis^{65 (link)}. Cells were harvested by scraping at 3 days post-transfection and lysed with 100 μL HEPES buffer. Viral capsids were precipitated with PEG buffer and digested with SDS buffer containing Proteinase K at 37 °C for 3 h. Total viral DNA was separated on a 1% agarose gel at 100 V for 3 h and transferred to a nitrocellulose membrane (GE healthcare). To detect HBV DNA, the membrane was hybridized with a highly purified [³²P]-labeled HBV probes. The level of HBV transcription was determined by Northern blot analysis as follows. Total RNA was extracted by using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. Total RNA (20 μg) was separated on a 1% formaldehyde agarose gel at 120 V for 3 h and transferred to a nitrocellulose membrane (GE healthcare) for 18 h. To detect HBV-specific RNAs, the membrane was hybridized with a highly pure [³²P]-labeled HBV probes. 18S RNA was used as a loading control. Relative levels of HBV replication and transcription were calculated using a phosphorimager.

The expression levels of proteins were confirmed as described elsewhere [19 (link)]. Proteins were separated and transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). Equal amounts of proteins were separated on a 10% SDS-polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). The membranes were blocked with tris-buffered saline/Tween 20 (TBST) containing 5% skim milk, and they were incubated for 12 h with primary antibodies. After washing with TBST, horseradish peroxidase-labeled secondary antibodies were added, and the blots incubated for 2 h. Protein bands were activated by chemiluminescence and visualized on an X-ray film.

A 10-µl aliquot of cell lysate was subjected to SDS/PAGE using a 10% (w/v) gel under reducing conditions. The separated proteins were electroblotted on to nitrocellulose membranes (GE Healthcare Bioscience, Piscataway, NJ, U.S.A.) with a bath-type blotter. After blocking for 1 h with 5% (w/v) skimmed milk in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (TPBS), the membranes were probed for 1 h with the respective antibodies (1:1000 for ACSL1, ACSL3, ACSL5, and ACSL6, 1:5000 for ACSL4, and 1:10000 for β-actin), followed by incubation with horseradish peroxidase-conjugated anti-rabbit (1:3000 for ACSL1, ACSL4, ACSL5, and ACSL6) and anti-mouse (1:3000 for ACSL3 and β-actin) IgG. After washing, the membranes were visualised using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Wellesley, MA, U.S.A.).

Protein lysates were collected in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris, 0.05% SDS, 1% triton) and quantified using a Pierce BCA assay kit (Thermo Scientific). Protein samples (20–30 µg) underwent routine electrophoresis on precast 4–12% Bis TRIS pre-cast gels (Invitrogen) and were transferred to nitrocellulose membranes (GE Healthcare). Blots were developed using enhanced chemiluminescence (GE Healthcare) and imaged with an Amersham Image 600 (GE Healthcare). Densitometry analysis was performed using ImageQuant TL software. E1A: Santa Cruz sc-430, 1:1000; Adenovirus: Abcam ab36851, 1:5000; Hsc70: Abcam ab36851, 1:10000; GAPDH: Santa Cruz sc-47724, 1:10000; phospho-Akt: Cell Signalling 9271L, 1:1000; Akt: Cell Signalling 9272; phospho-ERK1/2: Cell Signalling 4370, 1:1000; ERK1/2: Cell Signalling 9102; ARRB1/2: Cell Signalling 4674S, 1:500; ARRB1: Cell Signalling 12697S, 1:500.

Dot-ELISA is a modified ELISA technique based on sample-dotted nitrocellulose membranes. The Dot-ELISA procedure was carried out as described previously [40 (link)]. Briefly, 2 µL of maize leaf crude extract samples were pipetted evenly onto the nitrocellulose membranes (GE Healthcare, Bucks, UK) and incubated for 10 min at room temperature (RT), and a noninfected and a known MCMV-infected maize leaf crude extract were used as the negative and positive controls, respectively. Then, the membranes were blocked with PBST (a PBS solution containing 0.05% Tween-20) containing 5% skimmed milk powder for 30 min, followed by 1 h of incubation in a diluted anti-MCMV mAb solution at RT. After washing three times with PBST, alkaline phosphatase (AP)-conjugated goat anti-mouse immunoglobulin G (IgG) diluted in PBS containing 5% skimmed milk powder was added and incubated for another 1 h at RT. Afterward, the color reaction was created by adding a combination of NBT/BCIP (nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate) substrate solutions for 15 min at RT.