Anti caprin 1

HONE-1 cells were washed with PBS and cultured with methionine-free DMEM (GIBCO-Life Technologies) at 37°C for 1 h prior to the addition of 2 μM tylophorine and 25 μM Click-iT® HPG (L-homopropargylglycine, Invitrogen) and incubation at 37°C for 24 h. The cells were then harvested and subjected to the Click Reaction with 20 μM TAMRA (Tetramethylrhodamine, Invitrogen) for labeling the de novo synthesized proteins in Click-iT® Protein Reaction Buffer according to the manufacturers' protocol (Invitrogen). The resultant cell lysates were immunoprecipitated with anti-caprin-1 (ProteinTech Group), anti-c-Myc (Cell Signaling Technology), anti-cyclin D1, anti-cyclin D2 (Santa Cruz Biotechnology), anti-TAMRA (Thermo Scientific) or GAR (Perkin-Elmer) respectively overnight at 4°C with constant agitation prior to incubation with protein G agarose (Millipore) at 4°C for another 2 h. After washes, the specific immunoprecipitated protein was eluted and analyzed by western immunoblot analysis with the antibody indicated.

Western blotting analyses were performed as described [24 (link)]. Primary antibodies used for western blotting were anti-p-p65 (Ser536) (Cell Signaling, Danvers, MA, USA, 3033), anti-p65 (Cell Signaling, 8242), anti-p-IκBα (Ser32) (Cell Signaling, 2859), anti-IκBα (Cell Signaling, 9242), anti-GAPDH (Cell Signaling, 2118), anti-Caprin-1 (Proteintech, Rosemont, IL, USA, 15112-1-AP), anti-HCoV-OC43-nucleocapsid protein (anti-HCoV-OC43-N) (Merck Millipore, Temecula, CA, USA, Mab9013), and anti-vinculin (GeneTex, Irvine, CA, USA, GTX109749); their correspondent secondary antibodies used were horseradish peroxidase coupled anti-rabbit IgG antibody (PerkinElmer, Boston, MA, USA, NEF812001EA) or horseradish peroxidase coupled anti-mouse IgG antibody (GeneTex, GTX213111-01).