Westernbright ecl horseradish peroxidase substrate

The apparent molecular mass of either purified gp120 or unpurified gp120s in cell culture supernatants was determined by sodium dodecylsulfate polyacryamide gel electrophoresis (SDS-PAGE) on NuPAGE 4–12% Bis-Tris precast gels (Thermo Fisher Scientific, Invitrogen, Carlsbad, CA). Samples reduced by dithiothreitol (DTT) were run on gels in MES (2-(N-morpholino)ethanesulfonic acid) running buffer (Thermo Fisher) and stained with SimplyBlue stain (Thermo Fisher) For immunoblots, PAGE gels were transferred using iBlot 2 (Thermo Fisher). Membranes were blocked with 5% milk for 1 hour. The primary antibody, a polyclonal goat antibody raised against gp120, was incubated with the membrane at 1.5 ug/ml concentration in 5% milk. Blots were washed three times with PBS with 0.05% Tween (PBS-T). The secondary antibody used was the Peroxidase AffiniPure bovine anti-goat IgG (Jackson ImmunoResearch, West Grove, PA) and incubated with the membrane at a 1:5000 dilution in 5% milk. Blots were washed three times with PBS-T and three times with PBS. The chemiluminescent substrate used was WesternBright ECL horseradish peroxidase substrate (Advansta, Menlo Park, CA). Images were taken using a FluorChem Q imager (Alpha Innotech, San Leandro, CA).

Immunoblotting was carried out as previously described using antibodies against HUWE1, (ab70161, Abcam), c-MYC (ab32072, Abcam), GAPDH (ab181602, Abcam) and ubiquitin (sc-8017, Santa Cruz, Heidelberg, Germany) and secondary antibodies anti-mouse and anti-rabbit (DAKO, Cambridgeshire, UK). Ubiquitinated proteins were isolated using UbiQapture-Q (Enzo Life Sciences, Exeter, UK) to bind both mono- and poly-ubiquitinated proteins independent of lysine chain linkage, or tandem ubiquitin binding entity (TUBE) antibodies specific for either K48 or K63 linkages (UM607 and UM604, Life Sensors, Malvern, USA), according to the manufacturer’s instructions. A proportion (25 μg) of total cell lysate was retained for use as an input control and 250 μg of remaining lysate was bound to the appropriate affinity matrix to capture ubiquitinated proteins. Input controls and protein eluted from the matrix were subsequently analyzed by immunoblotting. Blots were visualized using WesternBright^™ ECL horseradish peroxidase substrate (Advansta, UK), scanned into the G:BOX gel doc system (Syngene, Cambridge, UK) and analyzed using GeneTools.