Quantification of gp120 Shedding by MβCD

gp120 shedding induced by MβCD alone (no PT present) was measured using Western blots as described previously.^{4 (link)} Briefly, MβCD was mixed with virus stocks (1:1, v/v) and incubated at 37 °C for 30 min before being spun for 2 h at 21130 × g at 4 °C. Sample supernatants were mixed 1:1 with Laemmli buffer and boiled at 95 °C for 5 min before being loaded onto 10% SDS-PAGE gels. After gel electrophoresis, the protein was transferred onto a 0.45 μm PVDF membrane (Immobilon-P, Millipore), blocked with 5% milk solubilized in PBS containing 0.1% Tween-20 and then stained with sheep anti-gp120 (D7324, Aalto), followed by donkey antisheep HRP (Invitrogen) antibodies. Luminol substrate (Advansta) was added and the protein bands exposed on chemiluminescence film (Blue Ultra, Genemate) and developed (M35-A X-OMAT processor, Kodak). The developed film was then scanned, and the corresponding image was analyzed with ImageJ (NIH) densitometry.