Semi dry transfer device

NSC cultures were seeded in 10 cm² dishes at a density of 100, 000 cells/cm². NSCs were infected (MOI 10) or stimulated 16 h after plating, lysed in RIPA buffer containing 50 mM Tris-HCL, pH 7.6; 150 mM NaCl; 0.1% sodium deoxycholate, 0.1% sodium dodecylsulfate, 0.1% NP-40, 1 mM EDTA, and a protease inhibitor cocktail (all from Sigma). Lysates were subjected to SDS-PAGE with 4 to 12% Tris-Tricine gels (Life Technologies). Proteins were blotted onto nitrocellulose membranes (GE-Healthcare, Pittsburgh, PA, USA) using a semi-dry transfer device (Biorad, Hercules, CA, USA). Western blot was performed using Tris-Buffered-Saline (TBS) containing 0.1% Tween-20 as the wash buffer, TBS containing 5% non-fat dry milk and 3% BSA as the blocking buffer, and primary or horseradish peroxidase-conjugated secondary antibodies diluted in blocking buffer. Detection was carried out by using a chemoluminescence kit (Sigma). Analysis was performed with a Chemidoc system (Bio-Rad, Hercules, CA) using conditions when signals were not saturating.

Equal amounts (500 ng) of HPV16 L1 protein (Abcam), purified L1:P18I10 and L1:T20 VLPs were mixed with 2× Laemmli sample buffer containing 5% 2-ME and boiled at 95 °C for 5 min. Samples were separated by 8–16% TGX Stain-free protein gels and then transferred to a PVDF membrane (Millipore, Burlington, MA, USA) using a Semi-Dry transfer device (Bio-Rad). The membrane was blocked with 5% skim milk in TBST. Then, the membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000, anti-HIV-1 gp120 V3 loop mAb (NIBSC, EVA3012) at a dilution of 1:40 and HIV1 gp41 (2F5) mAb (NIBSC, ARP3063) at a dilution of 1:4000, respectively. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate (Sigma-Aldrich) at a dilution of 1:4000. The Western ECL substrate kit (BIO-RAD) was used for signal development. The blot images were acquired by using Odyssey Fc imaging system at a chemiluminescence channel.