Anti ev a71 3d

The procedures for protein sample collection and separation have been described elsewhere [30 (link)]. In brief, the equal amount of total proteins were heated for 3 to 5 min, followed by separation in the 12% SDS-PAGE under the reduced conditions. After the separation, the gel was then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA) for the primary and secondary antibodies incubation. The primary antibodies used were listed as the following: Rabbit anti-GST (GeneTex Inc, Irvine, CA, USA, 1:1000), anti-Flag (Sigma, 1:3000 dilution) and anti-EV-A71 3D (1:5000 dilution); for detection of Stau1 (GeneTex, 1:2000) and β-actin (Sigma, 1:10,000). The primary and secondary antibodies were diluted in the Super antibody mate solution (MDBio Co., Ltd, Taipei, Taiwan) followed by incubation with the membranes.

We performed SDS-PAGE and western blotting to detect the CREB5 and viral proteins. Total protein sample isolation and separation have been described elsewhere [25 (link)]. In brief, the cellular total proteins was boiled and separation in an 12% SDS-PAGE. The separated SDS-PAGE was then transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA) and then washed. The membrane was and blocking with fast blocking solution (Biofuture Co., Taoyuan, Taipei) for 3 min prior to the primary and secondary antibodies incubation. The primary antibodies used were listed as the following: rabbit anti-EV-A71 VP (GeneTex, 1:1000), anti-Flag (Sigma, 1:3000 dilution) and anti-EV-A71 3D (1:5000 dilution); for detection of CREB5 (GeneTex, 1:2000) and β-actin (Sigma, 1:10000). The secondary antibodies were purchased from – (a) Goat anti-rabbit HRP secondary antibody (Abcam ab205718); (b) Goat anti-rabbit HRP secondary antibody (Abcam ab205719). The primary and secondary antibodies were diluted in the Super antibody mate solution (MDBio Co., Ltd., Taipei, Taiwan) followed by incubation ECL (BioMan biotech., Taipei, Taiwan) with the membranes. The signals of interested were detected using the ImageQuant™ LAS 4000 biomolecular imager (GE, USA).