Ab179513

Recombinant FGF10 (catalogue: 345-FG-025) was purchased from R&D Systems (Minneapolis, MN). Antibodies against FGF10 (ab115825), MAP-2 (ab5392), GFAP (ab7260), tubulin (ab179513) and t-PI3K (ab22653) were purchased from Abcam (Cambridge, MA). Antibodies against p65 nuclear factor-κB (NF-κB, sc-372), IκB (sc-371), TNF-α (sc1351) and IL-6 (sc-1265) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against p-Akt (2965), t-Akt (9272) and p-PI3K (4288) was purchased from Cell Signaling Biotechnology (Danvers, MA). Fluorescent terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit was purchased from Promega (Medison, WI). Caspase-3, −8 and −9 activity colorimetric kits were purchased from Abcam. 2,3,5-triphenyltetrazolium chloride (TTC), wortmannin and Akt1/2-KI were purchased from Sigma Chemical Company (St. Louis, MO)

SH-SY5Y cells and differentiated SH-SY5Y cells were fixed in 4% PFA for 10 min at room temperature. Each sample was washed three times with 0.1% PBS-T for 2 min. Following fixation, cells were incubated at room temperature for 2 h in 5% NGS-T blocking solution and incubated overnight at +4 °C with the mouse monoclonal anti-tubulin III primary antibody (#ab179513, Abcam plc., Camabridge, UK) at a final concentration of 1:1000. The primary antibody was then removed, and each sample was washed three times with 0.1% PBS-T. Rabbit anti-mouse secondary antibody Alexa Fluor 488 (#ab150113, Abcam plc., Cambridge, UK) was added at a final 1:2000 concentration and left to incubate at RT for 1 h. The secondary antibody was removed, and each sample was washed three times with 0.1% PBS-T. Samples were then mounted on to glass slides, using FluoroGel Mounting media (Genetex, Irvine, CA, USA). Samples were finally imaged by confocal microscopy within 24 h.

EPC cells seeded in six-well plates were infected with different IHNV strains (Sn1203, LN-15, QH-17 or Blk94) for 1 h at MOI = 0.1, washed with PBS three times, and treated with 0.5 µM bufalin for 24 or 48 h. The antiviral effect of bufalin was assessed by RT-qPCR, viral titer determination, western blotting, and IFA. For RT-qPCR, the total RNA of cultured cells was extracted with the TRIzol reagent (10296028, Invitrogen, Carlsbad, CA, USA), and viral mRNA was analyzed using the primers listed in Table 1. For viral titer determination, cell culture supernatants were collected, 10-fold diluted, and added to EPC in 96-well plates. After culture for 7 days at 15°C, viral titers were determined by the Reed−Muench method. For western blotting, cells were collected using lysis buffer, and protein expression was analyzed using rabbit anti-IHNV-G polyclonal antibody (43 (link)), rabbit anti-β-tubulin monoclonal antibody (ab179513, Abcam, Cambridge, UK) as primary antibody, and HRP-labeled goat anti-rabbit IgG antibody (ab6721, Abcam) as secondary antibody. For IFA, cells were fixed with 4% paraformaldehyde and permeabilized with Triton X-100. The anti-IHNV-G antibody was used as the primary antibody, and Cy3-tagged goat anti-rabbit IgG antibody (ab97075, Abcam) was used as the secondary antibody.

Cells were treated as described above and lysed in cell lysis buffer (10 mM Tris, pH 8.1, 1 mM EDTA, 150 mM NaCl, 0.65% IGEPAL CA‐630) supplemented with cOmplete™ protease inhibitor cocktail (Roche). Protein levels were determined by the BCA protein assay kit (Pierce) and subjected to SDS‐PAGE and immunoblotting using the following antibodies: α‐VP16 (Abcam, ab4808), α‐FLAG (F3165, Sigma‐Aldrich), α‐AICD (Y188, Abcam, ab32136), α‐β‐tubulin (Abcam, ab179513) and appropriate HRP‐conjugated secondary antibodies. The blots were developed using Amersham Biosciences HyperfilmTM ECL (GE Healthcare) or the CCD camera LAS‐3000 (FUJIFILM Life Science).

Sample preparation (20 flies were used per sample, three biological replicates per group) and Western blotting were performed as described in Scialò et al (2016). The primary antibodies employed together with the appropriate secondary antibodies were as follows: anti‐NDUFS3 (Abcam; ab14711) 1:1,000 diluted in 5% milk in PBS‐Tween 1x, anti‐SDHA (Abcam; ab209986) 1:500 diluted in 5% BSA in PBS‐Tween 1x; anti‐ATP5α (Abcam; ab14748) 1:100,000 diluted in 5% milk in PBS‐Tween 1x, anti‐UQCRC1 (Abcam; ab110252) 1:500 diluted in 5% BSA in PBS‐Tween 1x, anti‐MTCO1 (Abcam; ab14705) 1:500 diluted in 5% BSA in PBS‐Tween 1x, Porin (Abcam; ab14734) 1:1,000 diluted in 5% milk in PBS‐Tween 1x, anti‐beta‐tubulin [EPR16774] (Abcam; ab179513) 1:1,000 diluted in 5% milk in PBS‐Tween 1x, HRP Horse Anti‐Mouse IgG Antibody (Peroxidase; Vector laboratories, California; PI‐2000) 1:5,000 diluted in 5% milk, HRP Goat Anti‐Rabbit IgG Antibody (Peroxidase; Vector laboratories, California; PI‐1000) 1:5,000 diluted in 5% milk. Image quantification: ImageJ was used to quantify the protein band intensities. For each image, the bands were quantified and normalised by the corresponding tubulin intensity used as a loading control.