Goat anti mouse igg horseradish peroxidase hrp

Microplates (Nunc, Rochester, NY, USA) were first coated overnight with 5 μg/mL inactivated EV71 and blocked with Tris-buffered saline containing Tween 20/1% bovine serum albumin (BSA) before adding the serum, nasal-wash, BALF, or faecal extract samples. After a 2-h incubation at room temperature, the plates were washed, followed by the addition of goat anti-mouse IgG horseradish peroxidase (HRP; 1:10000; Bethyl) or anti-mouse IgA HRP (1:5000; Bethyl). For measuring EV71-specific IgG1 or IgG2a, 1 μg/mL biotinylated anti-mouse IgG1 (1:5000; A85-1; Becton, Dickinson and Company, Franklin Lakes, NY, USA) or anti-mouse IgG2a (1:1000; R19-15; Becton, Dickinson and Company) was used; this was followed by incubation for 1 h with HRP-conjugated streptavidin (R&D Systems, Minneapolis, MN, USA). Subsequently, 100 μL of 3,3′,5,5′-tetramethylbenzidine substrate was added. After colour development in the dark at room temperature for 20 min, the reaction was stopped by adding 100 μL of 1 M H₂SO₄. The absorbance at 450 and 550 nm was measured using a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA), and the results are expressed in ELISA units (E.U.): E.U. = (A_sample − A_blank) / (A_positive − A_blank).

FKBP5 was cloned into pCMV-3Tag-8 (Agilent Technologies, Santa Clara, CA, USA, # 240203) to generate FKBP5-FLAG. Deletion mutants of FKBP5-FLAG were constructed using a Q5^® Site-Directed Mutagenesis Kit (New England Biolabs, Ipswich, MA, USA). Human IKKα was cloned into pCMV-3Tag-8 to generate IKKα-HA and IKKα-Myc.
Anti-β-actin (Abcam, Cambridge, MA, USA, # ab8227), anti-FLAG (Sigma, St. Louis, MO, USA, # F3165), anti-hemagglutinin (HA) epitope (Cell Signaling Technology, Danvers, MA, USA, # 3724), anti-Myc (Bethyl Laboratories, Montgomery, TX, USA, # A190-105A), anti-interferon-induced transmembrane protein 3 (IFITM3) (GeneTex, Irvine, CA, USA, # GTX63349), anti-FKBP5 (Abcam, # ab126715), anti-non-structural protein 1 (NS1) (Santa Cruz Biotechnology, Dallas, TX, USA, # sc-130568), anti- nucleoprotein (NP) (GenScript, Piscataway, NJ, USA, # A01506-40), anti-IKKα (Cell Signaling Technology, # 11930S) were also obtained, as were goat anti-mouse IgG-horseradish peroxidase (HRP) (Bethyl Laboratories, # A90-116P), and goat anti-rabbit IgG-HRP (Bethyl Laboratories, # A120-201P).