Pierce western blotting substrate plus

293T cells were transfected with equal amounts of expression plasmids (250 ng PB2 and 500 ng PB1). At 16 h post-transfection, the cells were harvested with sample buffer and heated for 20 min at 100°C. The cell lysates were then separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore). Immunoblot analysis was performed using anti-influenza PB2 or PB1 antibody (GeneTex) and HRP-conjugated secondary antibody. The antibodies were visualized with Pierce Western Blotting Substrate Plus (Thermo Scientific) and exposed on Amersham Hyperfilm ECL (GE Healthcare). The band intensities were quantified by ImageJ software and total protein expression was calculated.

Transfected HEK293 cells were washed by PBS twice and lysed in SDS-sample buffer. Western blot analysis was performed using a semidry transfer cell (Bio-Rad) with PVDF membrane. Signals were detected using Pierce Western Blotting Substrate Plus (Thermo). We used the following primary antibodies: mouse monoclonal antibodies specific to FLAG (M2, 1:6000, Sigma) and α-tubulin (DM1A, 1:6000, Sigma).

Total protein extracts from the LG were separated by polyacrylamide gel electrophoresis, transferred to PVDF membranes using a dry blotting system (V20-SDB, SCIE-PLAS, UK), and incubated with antibodies. Immunolabeled proteins were detected using Pierce Western blotting Substrate Plus (Thermo SCIENTIFIC, Germany) and a Lumino-image analyzer (LAS-4000, FujiFilm, Japan). All bands were normalized to β-actin. The primary antibodies used for Western blotting were as follows; AMP-activated protein kinase [AMPK (Cell Signaling, Japan), Phospho-AMPK (Cell Signaling)], and β-actin (Sigma Aldrich). 6rats were used in each group.

Western blot analysis was conducted as previously reported [41 (link)]. Aortas were homogenized in ice cooled lysis buffer (1% Triton X-100, 50 mM Hepes [pH 7.4], 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, and protease inhibitor cocktail), followed by centrifugation at 15,000 rpm for 30 min at 4 °C and collection of supernatants (n = 5 for each group). Samples (2 to 10 μg) were resolved by 10% or 12.5% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. Thereafter, the membrane was blotted with 5% skim milk for 1 h, reacted with various primary antibodies at 4 °C overnight, and reacted with secondary antibody on the following day. The samples were then visualized using Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific Inc., Waltham, MA, USA). Images were obtained by the ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA) and quantified by PDQuest software (Bio-Rad, Hercules, CA, USA). For the primary antibody, anti-HO-1 antibody (Stress Gen Biotechnologies Inc., Victoria, BC, Canada), Sirt1 antibody (Merck Millipore, Billerica, MA, USA), and β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used, and for the secondary antibody, anti-rabbit IgG, HRP-linked whole antibody donkey (GE Healthcare, Buckinghamshire, UK) was used.

S2 cells were disrupted by incubating with a lysis buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, 50 μM ZnSO₄, 10 mM NaF, 0.4% Nonidet P-40 (NP40), and cOmplete Protease Inhibitor (Roche)) for 1 h at 4 °C. Lysates were prepared by centrifugation (15,000 rpm for 15 min) and denatured in the sample buffer. Samples were fractionated using 7.5% SDS–PAGE gel (e-PAGEL, E-T 7.5L) at 100 mV. Then blots were transferred to PVDF membranes (Life Technologies, IB401001), and reacted with rat anti-TRF2 (1:100; present study), rabbit anti-Fru Male (1:500)^{20 (link)} or mouse anti-α-actin (1:500; Abcam, ab3280) overnight, and subsequently with a horseradish peroxidase (HRP)-conjugated anti-rat, rabbit, or mouse IgG antibody (1:3000; Sigma) for 3 h. Pierce Western Blotting Substrate Plus (Thermo Scientific, NCI32132) was used according to the manufacturer’s instructions to detect chemiluminescence. Fluorescent images were obtained using an ImageQuant LAS 4000 system (Fujifilm).

Each sample was subjected to 7.5%, 12.5% or 15% (v/v) SDS-PAGE using a Tris-glycine buffer system, and the separated proteins were transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 3% (v/v) skim milk and 1% (v/v) bovine serum albumin (BSA) in PBS containing 0.1% (w/v) Tween-20 for 3 hours, followed by incubation at 4 °C overnight with the indicated primary antibody at an appropriate dilution (1:2,000) in PBS containing 3% (v/v) skim milk, 1% (v/v) BSA, and 0.1% (w/v) Tween-20. The membranes were washed and then incubated with a horseradish peroxidase-conjugated secondary antibody (1:2,000) (Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 1 hour. Signals were detected using Pierce Western Blotting Substrate Plus (Thermo Scientific, Southfield, MI, USA). The intensities of specific bands were quantitatively measured using an LAS-3000 mini imaging system (FujiFilm Co., Tokyo, Japan) equipped with Multi-Gauge Ver3.0 software (FujiFilm). As an internal control, the expression of β-actin was measured.