Alexa 555 secondary antibodies

APP-CTM1, APP-CT11, PS1_NT, Flotilin-2 and APP_NTH-452 homemade rabbit polyclonal antibodies were generously provided by Dr. Gopal Thinakaran (University of Chicago, Chicago, IL) as described previously (Deyts et al., 2012 (link); Vetrivel et al., 2009 (link)). Monoclonal anti-MAP2, GAP-43 (clone 7B10) and GAPDH were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal DCC (clone G97-449) antibody was purchased from BD Biosciences (San Diego, CA). Polyclonal phospho-(Ser/Thr) PKA substrate antibody and phospho-CREB (Ser133) antibodies were purchased from Cell Signaling Technology (Danvers, MA) and EMD Millipore (Billerica, MA), respectively. Monoclonal Alexa-647 and Alexa-555, and polyclonal Alexa-555 secondary antibodies were purchased from Invitrogen (Carlsbad, CA). IRDye 680 and IRDye 800CW-conjugated secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE). γ-secretase inhibitor Compound E was generously provided by Dr. Todd E. Golde (University of Florida, Gainesville, FL) (Seiffert et al., 2000 (link)). Cis-N-(2-phenylcyclopentyl) azacyclotridec-1-en-2-amine (MDL,12-330A) was obtained from Enzo Life Science (Farmingdale, NY). Tetrodotoxin was purchased from Tocris Bioscience (distributed by Fisher Scientific, Pittsurgh, PA). Unless indicated, all other reagents were purchased from Sigma-Aldrich.

To localise p65 protein expression in human AF and NP cells, the cells were plated on a glass-bottom confocal dish and exposed to IL-1β 10 ng/mL for 48 h. The disc cells were fixed with 4% paraformaldehyde and permeabilised with 0.2% Triton X-100 in PBS for 10 min at 25 °C. The cells were blocked with 5% bovine serum albumin (Millipore) in PBS and then incubated with the primary NF-κB p65 antibody (1:100; Sigma-Aldrich), followed by incubation with Alexa 555 secondary antibodies (1:200; Invitrogen). The samples were imaged using the EVOS FL auto cell imaging system (Thermo Fisher Scientific Inc., USA).

To evaluate the protein expression and localization, Immunocytochemical staining was performed. Nasal fibroblasts were seeded on a cell culture slide (SPL Life Sciences, Korea) with 5×10⁴ cells/mL. Before using the cell culture slide, poly-L-lysine was used to coat the culture slide for 4 hours, after which it was exposed to UV light for 6 hours. Fibroblasts were pretreated with or without MAPK inhibitors (SB203580, SP600125, U0126) and NF-kB inhibitor (BAY11-7082) and stimulated with apigenin (5 μM) for 72 hours. Fibroblasts were fixed with 4% paraformaldehyde and permeabilized with 0.01% Triton X-100 in 1% bovine serum albumin for 10 minutes. After that, fibroblasts were blocked with 3% bovine serum albumin for 1 hour, incubated with primary antibodies against phospho-p50, and then incubated with anti-rabbit Alexa 555 secondary antibodies (Invitrogen). Finally, fibroblasts were counterstained with 4'-6-diamidino-2-phenylindole for 10 minutes. Stained fibroblasts were visualized using a confocal laser scanning microscope (LSM700, Zeiss, Oberkochen, Germany).