Pe rat igg 2a

For the analysis of CD49f and Sca-1 expression in the Akt transformed stem cell line, Cy5.5-CD49f (Biolegend, CA) and
pacific blue-Sca-1 antibodies (Biolegend, CA) were used. Cy5.5-rat IgG-2a (Biolegend, CA) and pacific blue-rat IgG-2a
(Biolegend, CA) were used for control antibodies. Cells (50,000) were suspended in staining buffer (5% FBS in PBS) and human
TruStain FcX™ (Biolegend, CA) was added to block Fc receptors. Cells were incubated at room temperature for 10 min
followed by incubation with antibodies or control IgG for 30 min on ice in the dark. Cells were analyzed in the RSLII flow
cytometer (BD Sciences, NJ). Proximal prostatic ductal cells were sorted by FACS (Moflo XDP, Beckman Coulter, NJ) into various
fractions (Sca-1^high, Sca-1^med/low and Sca-1^neg) according to the mean fluorescence intensity
(MFI) of Sca-1 expression by the cells after addition of PE-Sca-1 antibody (BD Sciences, NJ). PE-rat IgG-2a (BD Sciences, NJ)
was used as a negative control. Sca-1+ cells with fluorescent intensities in the upper one-third were defined as
Sca-1^high cells, the remaining Sca-1+ cells served as the Sca-1^med/low population while the
unlabeled cells served as the Sca-1^neg population. Cells (5×10⁴) were implanted with UGM
(2×10⁵) sub-renal capsule and grafts examined after 8 weeks.

For FACS sorting of HFSCs and BL cells, the protocol has been described previously (Lee et al., 2016 (link)). The epithelial cells were isolated from the back skin through trypsin digestion. The cell suspensions were labeled with anti-CD34-Biotin (1:50, no. 13-0341-85, eBioscience), α-Streptavidin-APC (1:100, no. 554067, BD Biosciences) and anti-α6-integrin-PE (1:40, CD49f, no. 555736, BD Biosciences) to isolate HFSCs (CD34+, α6-integrin+) and BL cells (CD34−, α6-integrin+). Propidium iodide (1:1,250–1:2,500 of 1 mg mL⁻¹ stock, S7109, Sigma) was used to rule out the dead cells. For negative controls to gate the fluorescence-labeled cells, we used only α-Streptavidin-APC for CD34+ cells and PE-Rat-IgG2a (1:40, no. 555844, BD Biosciences) for α6-integrin+ cells. The cells were isolated with BD FACSAria located in the Flow Cytometry Core at Cornell University.
For gene expression analysis, RNA was extracted from either 1 cm² of total skin using the RNeasy Fibrous Tissue Mini Kit (no. 74704, QIAGEN) or the FACS-sorted cells using the mirVana miRNA Isolation Kit (AM1591, Ambion). The cDNA were synthesized using iScript (no. 1708841, Bio-Rad) cDNA synthesis kit and qRT-PCR analysis was described previously (Lee et al., 2016 (link)).