Chemiluminescent substrate

Immunoblot analysis was performed as previously described (8 (link)). Briefly, membrane fractions were collected and 10 μg of protein samples were separated by SDS-PAGE using a 10% Laemmli system (21 (link)). Anti-myc HRP-conjugated antibody (anti-myc-HRP, 1:5000, Novex®) was used to detect expression of the Pho84^WT/MUT-myc constructs. The (−) control was cut from the blot to avoid visualization of irrelevant samples, but detection was performed simultaneously with the mutant sample blot. As a loading control, 10 μg of total cell extract was loaded onto a separate gel according to the previously described protocol, and anti-β-actin HRP-conjugated antibody (1:5000) (Abcam, UK) was used for detection. After 1 min of incubation with chemiluminescent substrate (GE Healthcare, UK), the membrane-enriched sample blot was exposed to X-ray film for 1.5 min. The control blot was visualized using the Bio-Rad ChemiDoc^TM MP imaging system with an exposure time of 30 s. The molecular masses of the separated proteins were determined by their mobility relative to the pre-stained protein markers (Fermentas, Germany). Figures were cropped and no further image processing was applied.

Western blot was performed as described previously [36 (link)]. Briefly, VLPs and recombinant proteins were solubilized in RIPA Buffer (Sigma, St. Louis, MO) and then in 2X Laemmli Buffer (Bio-Rad, Hercules, CA). After boiling the samples for5 minutes, we loaded them into a 10% SDS-PAGE gel and proceeded with electrophoresis for 2 hours at 100 volts. The protein was transferred to nitrocellulose for 2 hours at 90 volts, 4°C. Ponceau S stain (Sigma, St. Louis, MO) was used to verify protein transfer and the membrane was incubated overnight at 4°C with primary antibody, human monoclonal antibody to V3 of HIV-1 Env (447-52D; NIH AIDS Reagent Program). The following day, the membrane was washed 3 times in TBST (Tris-buffered saline plus Tween 20) and incubated for 2 hours at room temperature with anti-human HRP-conjugated secondary antibody (Southern Biotech, Birmingham AL). The secondary antibody was removed and the membrane washed 5 times with TBST, incubated with chemiluminescent substrate (GE, Schenectady, NY), and exposed to X-ray film (Denville Scientific, Metuchen, NJ). The film was developed with a Kodak X-GMAT 2000 (Eastman Kodak, Rochester, NY).

HEC-1B cells were seeded at 1 × 10⁴ cells/well in six-well plates in complete media. At 24 h post-seeding, the cells were treated with NF-conditioned media, CAF-conditioned media, and/or AMD3100 (200 or 500 ng/ml) for 1 h. Cell lysates or immunoprecipitates from cell lysates were subjected to SDS-PAGE and were transferred to polyvinylidene fluoride membranes. The membranes were incubated with the following primary antibodies: rabbit anti-human Akt, phospho-Akt, Erk, phospho-Erk, and GAPDH (Cell Signaling Technology, USA), followed by horseradish peroxidase-conjugated secondary antibody. The immunoreactive polypeptides were visualized using a chemiluminescent substrate (GE Life sciences).