Hrp conjugated anti flag

Cell lysates were prepared in lysis buffer containing 1% Nonidet P-40 and protease inhibitor cocktail (Roche) (Cao et al., 2003 (link)). Soluble proteins were immunoprecipitated using anti-Flag (M2, Sigma), anti-Myc (Sigma), or IgG of the same isotype from the same species as a negative control (Sigma). An aliquot of the total lysate (5%, vol/vol) was included as a control. Immunoblotting was performed with horseradish peroxidase (HRP)-conjugated anti-Myc (Sigma), HRP-conjugated anti-Flag (Sigma), HRP-conjugated anti-β-actin (Sigma), anti-VP35 (Creative Diagnostics), anti-IRF3 (Cell Signaling Technology), anti-phospho-IRF3 Ser396 (Cell Signaling Technology), anti-STING (Proteintech), or anti-NP (Sino Biological) antibodies. The antigen–antibody complexes were visualized via chemiluminescence (Immobilon Western Chemiluminescent HRP Substrate, Millipore). A PageRuler Western marker (Thermo) was used as a molecular weight standard.

High-binding 96-well plates (Corning) were coated with recombinant NiV-RBP-Fc, NiV-RBP, or HeV-RBP at 4 μg/ml and left to sit overnight at 4 °C. Subsequently, the plates were blocked with PBS containing 3% skim milk (w/v, Bio-Rad) at 37 °C for 1 h. The plates were washed with PBST three times, and polyclonal phages, monoclonal phages, or serially diluted antibodies were added, and plates were incubated at 37 °C for 1.5 h. Next, the plates were washed with PBST five times, and HRP-conjugated anti-M13 (Sino Biological, cat#11973-MM05T-H, 1:3000 dilution), HRP-conjugated anti-Flag (Sigma-Aldrich, cat#A8592, 1:2000 dilution), or HRP-conjugated anti-human IgG Fc antibody (Sigma-Aldrich, cat#A0170, 1:5000 dilution) was used as a secondary antibody. After 1 h of incubation, the plates were washed with PBST five times. The binding was measured with the subsequent addition of substrate diammonium 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS, Invitrogen), and the absorbance signal at 405 nm was measured with a BioTek Synergy H1 microplate reader (BioTek Instruments). The median effective concentration (EC₅₀) was determined by curve fitting using four-parameter nonlinear regression (GraphPad 8.0).

The mouse anti-Flag antibody was purchased from Applied Biological Materials (Richmond, BC, Canada). Rabbit anti-HA and rabbit anti-Snail antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The mouse anti-α-tubulin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). The rabbit anti-ERK3 antibody was purchased from Abcam (Cambridge, UK). The mouse anti-Myc antibody was purchased from Proteintec (Rosemont, IL, USA). Horseradish peroxidase (HRP)-conjugated anti-HA and HRP-conjugated anti-Flag antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). HRP-conjugated anti-Myc was purchased from Millipore (Burlington, VT, USA). The proteasome inhibitor MG132 and translation inhibitor cycloheximide were purchased from Calbiochem (SanDiego, CA, USA).

Cell lysates were prepared in prechilled Mammalian Protein Extraction Reagent (Thermo Scientific) containing protease inhibitor cocktail (Roche). Soluble proteins were immunoprecipitated using anti-Flag (Millipore, A2220) and anti-GFP (ABclonal, AE074) agarose beads or species-matched IgG of the same isotype as a negative control (Sigma-Aldrich, A0919 or A2909). An aliquot of the total lysate (5%, vol/vol) was included as a control. Immunoblotting was performed with horseradish peroxidase (HRP)-conjugated anti-Flag (Sigma, A8592, 1:2,000 dilution), HRP-conjugated anti-Myc (Sigma, SAB4200742, 1:2,000 dilution), HRP-conjugated anti-GFP (Santa Cruz, sc-8334, 1:500 dilution), HRP-conjugated anti-GST (Invitrogen, MA4-004-HRP, 1:1,000 dilution), anti-PRKACA (BD Biosciences, 610980, 1:1,000 dilution), anti-CREB1 (Proteintech, 12208-1-AP, 1:1,000 dilution), and anti-Phospho-CREB (Ser133) antibodies. The antigen-antibody complexes were detected using an enhanced chemiluminescence system (GE Healthcare) and Immobilon Western HRP Substrate (Millipore, WBKLS0100). PageRuler Prestained Protein Ladder (Thermo) was used as a molecular weight standard.