Anti rabbit hrp

Lysates were generated by incubating cells or ground tumors in RIPA buffer (Millipore). 20–30 μg of cleared lysate were analyzed by SDS-PAGE as previously described ^{11 (link)}. The following primary antibodies were used: Vinculin (1:5,000 dilution; Sigma, V9131), β-Actin (1:5,000; Sigma, A5441), Collagen I (1:500; Abcam, ab21286), Fibronectin (1:1,000; Abcam, ab2413), Smad4 (1:200; Santa Cruz, sc-7966), Smad2 p-S465/467 (1:1,000; Cell Signaling, 3108S), Smad2/3 (1:1,000; Cell Signaling, 3102S), GCN2 p-T899 (1:1,000; Abcam, ab75836), GCN2 (1:1,000; Cell Signaling, 3302S), ATF4 (1:200; Santa Cruz, sc-200), PC (1:1,000; Novus, NBP1–49536), S6K (1:1,000; Cell Signaling, 2708S), GLUL (1:1,000; Sigma, G2781), SMA (1:1,000; Millipore, CBL171). The following secondary antibodies were used: anti-rabbit HRP (1:5,000; GE, NA934V), anti-mouse HRP (1:5,000; GE, NA931). Quantification of band intensities of Collagen I relative to β-Actin or Vinculin in tumor allograft experiments was performed with Image Lab software v6.0 (Bio-Rad).

Three or more 7-wk old WT and BK5.EP4 mice were topically treated with 200 μl acetone or 2.5 μg TPA in 200 μl acetone, and total protein was isolated from the epidermis at 3, 12, and 24 h after treatment as previously described (Sung et al., 2006 (link)). Fifty to 75 μg epidermal lysates were separated on either an 8% or 15% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Thermo Scientific). Primary antibodies used were anti-MMP-9 (1:1000; Chemicon) for the 24 h samples, anti-pErk1/2 (1:1000), anti-Erk1/2 (1:1000), anti-pStat3 (1:1000), and anti-Stat3 (1:1000), all from Cell Signaling, Danvers, MA, for the 3 h samples, and anti-actin-HRP (1:1000; Santa Cruz Biotechnology) for all samples as loading controls. Antibodies against CYP1A1 and CYP1B1 (1:500, Santa Cruz Biotechnology) and aromatase (1:1000; Invitrogen, Carlsbad, CA) were also used. Secondary antibodies were anti-mouse-HRP (1:2500) and anti-rabbit-HRP (1:10000; GE Healthcare). Blots were blocked in 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST), primary antibodies were incubated overnight in 5% BSA in TBST at 4°C, and the secondary antibodies were incubated for an hour in 5% non-fat milk in TBST at room temperature. The membranes were washed, and SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Inc., IL) was used to detect antibody binding.

ELISAs were based on previously described methods (Bowley et al., 2007 (link); Moore et al., 2008 (link); Si et al., 2004 (link)) with several modifications. Briefly, 96-well microplates were coated overnight with sheep antibody D7324 at 5 μg/ml in PBS (AALTO Bioproducts, Ltd., Ireland). Wells were washed 3 times with PBS supplemented with 2.5% Tween-20 (PBS-T) and blocked for 1 hour at room temperature with PBS-T supplemented with 3% BSA. Supernatants were incubated in the wells for 3 hours, followed by 10 washes with PBS-T and 1 hour incubation with polyclonal rabbit anti-gp120 (American Biotechnologies Inc.). After 10 additional washes with PBS-T, wells were incubated with anti-rabbit-HRP (GE Healthcare). The reaction was developed using BM chemiluminescence ELISA substrate (Roche) according to the manufacturer’s instructions. The reaction was read on a luminometer (Viktor2, Perkin Elmer, Waltham, MA). Calculations of sgp120 concentrations were based on a standard curve of purified gp120 (Dunfee et al., 2006b (link)). Supernatant from cells transfected with empty pcDNA3 vector was used to determine background.