Goat anti mouse igg 680

Cells were washed with phosphate-buffered saline (PBS) and lysed using an ice-cold 1% NP-40 buffer containing a 1× protease inhibitor cocktail (Roche) and the supernatant was collected. Proteins from the lysate were denatured using an sodium dodecyl sulfate buffer and incubating at 70 °C for 10 min. The proteins were then separated in a 4–12% Bis-tris gel (Life Technologies) and transferred to a nitrocellulose membrane through a wet transfer method. The nitrocellulose was blocked with 5% non-fat milk in Tris buffered saline (TBS: blocking buffer) for 45 min at room temperature (RT). Immunoblotting was completed with respective antibodies and blots were visualized with infrared detection on the Odyssey (Licor). Primary antibodies were diluted in blocking buffer with Tween20 at various concentrations (1:200–1:1000 v/v). Secondary antibodies were diluted in blocking buffer with Tween20 at 1:10,000 v/v. Antibodies were purchased from: Cell Signaling Technologies 2506s (CDK5) and 9635s (14-3-3ε), Novus NB110-40878 (Perilipin-2), Bethyl A301-469A-M (KIAA0528), ABClonal A5776 (NudEL), Bioss bs-5522R (pSer231NudEL), Developmental Studies Hybridoma Bank E7-s (Tubulin-β), Life Technologies (goat anti-mouse IgG 680), and Li-cor (goat anti-rabbit IgG 800). Data shown here are representative of 2–3 separate experiments.

For IVIG fractions and their fragments, loaded 1 μg protein aliquots and Precision Plus protein standards (BIO-RAD, California, USA) onto 10% reducing SDS-PAGE gels. After electrophoresis, the separated proteins were transferred to nitrocellulose filter membranes. Rabbit anti-human IgG γ Fc region antibody (Dako, Copenhagen, Denmark), mouse anti-human Ig κ chainantibody (ZSGB-BIO, Beijing, China) and mouse anti-human Ig λ chain antibody (ZSGB-BIO, Beijing, China) at a dilution of 1:1,000 were used as primary antibodies.
For cell lysates of infected A549 or MDCK cells, they were prepared using RIPA buffer (Millipore, Massachusetts, USA), and the concentrations were determined with BCA protein assay kit (Thermo Scientific, Illinois, USA). About 20μg protein aliquots were separated and transferred to nitrocellulose filter membranes. Rabbit anti-Influenza A Virus Nucleoprotein antibody (GeneTex, California, USA) and mouse anti-β actin monoclonal antibody (ZSGB-BIO, Beijing, China) at a dilution of 1:1,000 were used as primary antibodies.
After incubation with appropriate secondary antibodies including Goat anti-rabbit IgG-680 (1:10,000; LI-COR, Nebraska, USA) and goat anti-mouse IgG-680 (1:10,000; LI-COR, Nebraska, USA), the blots were examined using a Odyssey imaging system (LI-COR, Nebraska, USA).

For cell lysates, scraped-off cells were washed with PBS and lysed in ice-cold lysis buffer (25 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP40, 1 mM EDTA, 1 mM PMSF, 1 mM Na₃VO₄, and 1 × protease inhibitor cocktail from Roche) and the supernatant collected. The proteins in cell lysate were separated in tris-glycine gel. Samples were denatured before loading to the gel using 1:1 (v/v) Laemmli sample buffer and heating at 100 °C for 5 min. The proteins were transferred onto nitrocellulose membrane by wet transfer method and the transferred membrane was blocked with either 5% non-fat milk or bovine serum albumin solution in Tris buffered saline with tween 20 (TBST) for an hour at room temperature. Immunoblotting was done with the respective antibodies and subsequently visualized with infrared detection in Odyssey instrument (Licor). The primary antibodies were diluted in the blocking medium (1:1,000 v/v) and the secondary antibodies were diluted in TBST (1:10,000 v/v). The antibodies were purchased from abcam-56676 (tubulin-α), Santa Cruz Biotechnology, CA, USA (LIS1 sc-15319 , NUDE1 sc-100328), Bethyl (NUDEL1, rabbit-polyclonal ), BD (dynactin/p150 Glued antibody, cat# 610473), Life Tech (Goat anti-mouse IgG 680) and Licor( goat anti-rabbit 800 nm).