Western blots

MDA-MB-231 Br cells were seeded at a density of 10⁶ cells per well in 12 well plates and incubated for 24h to allow for cell adhesion. Cells were treated with Ch-1 at 10 µM for 10 min. before the addition of GPNMB (R&D Systems) at 50 ng/mL. After 1 hour of treatment, the cells were washed with cold PBS and cells were trypsinized, using RIPA buffer. Protein lysates were run on Western Blots (Bio-Rad) and ERK 1/2 kinase activation detected using an antibody from Abnova.

All participants were offered testing for HIV infection, Chlamydia trachomatis, Neisseria gonorrhea, and syphilis at baseline, 6- and 12-months. HIV tests were performed using enzyme immunoassays (Vironostika HIV-1 Microelisa System, bioMerieux, Durham, NC) or rapid tests (Oraquik) with confirmatory Western blots (Bio-Rad, Redmond, WA). Per clinic routine, participants also received pooled HIV RNA testing (Procleix HIV-1 Discriminatory Assay, Gen-Probe Inc., San Diego, CA) (24 (link)). Men testing positive by HIV RNA alone underwent subsequent confirmatory testing. We used culture to test for rectal gonorrhea, pharyngeal gonorrhea, and rectal chlamydial infection, and Aptima Combo2 (Gen-Probe Inc., San Diego, CA) to test for urethral gonorrhea and chlamydial infection. We tested for syphilis using RPR with confirmatory TPPA. Our composite endpoint of any HIV/STI included a new diagnosis of HIV, syphilis, or chlamydia or gonorrhea at any anatomical site.