Tmb stabilized substrate for hrp

The liver tissues of DIO-zebrafish were collected by surgical extraction. Lysate protein was prepared by homogenization and sonication in T-PER Tissue Protein Extraction Reagent (Thermo Scientific, Rockford, IL) with protease inhibitor cocktail (Thermo Scientific). The samples were centrifuged at 12,000 × g for 30 min after homogenization with the MM300 Mixer Mill (30 Hz for 2 min; Retsch, Haan, Germany). For western blot analysis, protein samples were separated by 4–15 % SDS–PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad), and blocked at 20 °C with TBS containing 5 % skim milk (Becton, Dickinson and Company, Sparks, MD, USA) for 90 min. The membrane was incubated with goat polyclonal to E2F8 (1:2000; Aviva Systems Biology, San Diego, CA, USA) at 4 °C for 16 h, and then washed with TBS containing 0.05 % Tween-20 (TBST) five times. Horseradish peroxidase (HRP) -conjugated rabbit anti-goat (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) secondary antibodies were used to detect E2F8. After five washes with PBST, immunoreactions were detected using TMB stabilized substrate for HRP (Promega, WI, USA) with a Molecular Imager Chemi Doc XRS Plus (Bio-Rad), and analysed using PD Quest Advanced/Basic Ver.8.0 (Bio-Rad).

MDCK cells in 35 mm dishes were infected with MFPT^r virus or the WT virus at 1 PFU/cell or mock infected with PBS. At 0, 5, 10 and 15 hours post-infection, the infected cells were washed 3 times with PBS and stored at -80°C. Using cell lysis buffer (0.05 M Tris-HCl pH 7.0, 0.15 M NaCl, 1% SDS, 1% Triton X-100), the cell extracts were prepared and subjected to 10% SDS-PAGE. The resulting gels were blotted on PVDF membranes and soaked with 2% BSA in TBS for blocking treatment. Blotted proteins were immuno-reacted with rabbit anti-Akt antibody (Cell Signaling Technology, MA, USA), anti-phospho-Akt (Ser473) (D9E) XP Rabbit mAb (Cell Signaling Technology), anti-cleaved caspase 3 (Cell Signaling Technology), anti-PARP-1 (Santa Cruz Biotechnology, TX, USA), anti-Actin(c-2) (Santa Cruz Biotechnology), anti-Influenza A m1 (Santa Cruz Biotechnology), or anti-Influenza A ns1 (Santa Cruz Biotechnology) antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibodies (Santa Cruz Biotechnology) were used as secondary antibodies. Bands of these proteins were detected using TMB Stabilized Substrate for HRP (Promega KK, Tokyo, Japan). Semi-quantifying the expression levels of these proteins were performed using an image analyzing software, ImageJ [12 (link)] and that of actin for data normalization.

The recombinant protein was detected by western blot analysis. Electrophoresed proteins (SDS-Page) were transferred to a nitro-cellulose membrane using a semi-dry method in a transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) at 90 mA for 60 min. The membrane was then blocked overnight at 4 °C in 5% skimmed milk in Tris-buffered saline buffer (TBS), pH 7.6. Then, the membrane was incubated in a 1:1,000 dilution of anti-histidine horseradish peroxidase antibody with gentle shaking for 1 h at room temperature. The membrane was washed with PBST (phosphate buffered saline/Tween 20) three times after each incubation period. Visualization was performed using TMB stabilized substrate for HRP (Promega Corporation, Madison, USA) (20 (link)).