Hrp conjugated goat anti mouse igg h l

Antibodies against the viral nucleoprotein N were detected by an in-house ELISA and RVFV neutralizing antibodies in a microneutralization assay [14 ]. Briefly, for the detection of antibodies against N, sera were tested in duplicate in serial 3-fold dilutions starting at 1/50, in ELISA plates adsorbed with 100 ng/well of purified recombinant Thioredoxin-N (Trx-N) fusion protein produced in Escherichia coli and diluted in carbonate buffer (pH 9.6). Wells were blocked with 5% skimmed milk in PBS, 0.05% Tween 20, then bound antibodies were detected with goat anti-mouse-IgG (H+L)-HRP Conjugated (Bio-Rad, Hercules, CA, USA) and TMB (Thermofisher) was used as chromogen substrate. For neutralization, sera (in quadruplicates) were 2-fold diluted from 1/10, mixed with an equal volume of infectious virus containing 100 TCID₅₀ and incubated for 30 minutes at 37 °C. Then, a Vero cell suspension was added, and plates were incubated for 4 days. Monolayers were then controlled for the development of the cytopathic effect (CPE), fixed and stained. Anti-N titers are expressed as last dilution of serum (log10) giving an OD reading at 450 nm over 1.0 in ELISA; neutralization titers are expressed as the dilution of serum (log10) rendering a reduction in infectivity of 50%.

Total protein extracts were obtained as previously described [13 (link)], resolved with SDS-PAGE and transferred to Hybond membranes (Amersham Biosciences, NJ, USA). Non-specific binding sites were blocked for 2 h with 5% milk in Tris-Tween buffered saline (tTBS) (5 mM Tris pH 7.5, 15 mM NaCl, 0.1% Tween-20), washed three times with tTBS and incubated with antibodies. For the primary antibodies anti-GAPDH (7-B) (Santa Cruz Biotechnology, 1:1000), anti-β-Tubulin (Upstate, Lake Placid, NY, 1:2000), the secondary antibody goat-anti-mouse IgG (H+L)-HRP conjugated (BioRad 170-6516, 1:3000) was used. For the primary antibodies anti-WT1 (C19) (Santa Cruz Biotecnology, Santa Cruz, CA, USA, 1:500), anti-Akt (Cell Signaling Technology, Beverly, MA, USA, 1:1000), anti-Phospho-Akt Ser473 (Cell Signaling Technology, 1:1000), anti-p70 S6 Kinase (49D7) (Cell Signaling Technology, 1:1000), anti-Phospho-p70 S6 Kinase Thr389 (108D2) (Cell Signaling Technology, 1:1000), the secondary antibody goat-anti-rabbit IgG (H+L)-HRP conjugated (BioRad170-6515, 1:3000) was used. For detection, an ECL western blot detection system (Amersham Biosciences) was used. ZNF224 protein analysis was performed using the anti-ZNF224 (T3) antibody (Rabbit polyclonal antibody) (1) as previously described [13 (link)].

Cells were harvested in 2% sodium dodecyl sulfate (SDS)/phosphate-buffered saline (PBS) buffer containing a proteinase inhibitor cocktail (Roche) and sonicated. Protein concentrations were quantified using the Pierce^TM BCA protein assay kit (Thermo Fischer Scientific), and samples were mixed with loading sample buffer containing 10% β-mercaptoethanol before being boiled at 95 °C for 5 min. Subsequently, equal amounts of total protein extracts were subjected to SDS-PAGE, transferred to nitrocellulose membranes, blocked for 1 h in skimmed milk, and incubated overnight with primary antibody at 4 °C and then with for the corresponding secondary antibody for 1 h at room temperature. Blots were imaged on a ChemidocTM MP Imaging System (Bio-Rad, Hercules, CA). All blots were derived from the same experiment and were processed in parallel. Original uncut blots are shown in Supplementary Fig. 8. The primary antibodies used were mouse anti-β-Actin (MP Biomedicals 8691001, 1:5000), mouse anti-Parkin (Santa-Cruz Biotechnology, Dallas, TX, sc-32282, 1:500), and rabbit anti-PINK1 (D8G3, Cell signaling, Danvers, MA,1:1000). The secondary antibodies were HRP-conjugated goat anti-mouse IgG (H + L) (Bio-Rad, 1:10,000) and HRP-conjugated goat anti-rabbit IgG (H + L) (Bio-Rad, 1:10,000).