Bcl xl

SDS-Page electrophoresis and transfer of proteins to nitrocellulose membranes were performed according to standard procedures. Ponceau S (0.1%) staining of membranes verified transfer of proteins and equal loading. Membranes were incubated with antibodies to CD133 (Miltenyi Biotec, Auburn, CA, USA), cleaved and intact PARP, BAD, p-BAD, BCL-XL, MCL-1 (BioLegend, San Diego, CA, USA) cleaved active caspase-3, cleaved active caspase-9, BCL-2, active BAX (Novus Biologicals, Centennial, CO), AKT and p-AKT (Santa Cruz Biotech, Dallas, TX, USA), ERK, p-ERK, or to β-Actin (ProteinTech, Rosemont, IL, USA) as a loading control. Immunoblots were sequentially reprobed with other antibodies after stripping them of antibodies. Immune complexes were detected by incubation with horseradish peroxidase-conjugated antibodies to mouse or rabbit IgG (1:3000), followed by enhanced chemiluminescence (ECL; Pierce, Rockford, IL, USA) and imaging in a GE Healthcare Amersham Imager 600.

After treatment with PPD (40 µM) or EsA (40 µM), or co-culture with control eESCs (ESC-NK) or PPD-pretreated eESCs (ESC-PPD-NK), these eESCs were collected and the expression levels of Bcl-2, Bcl-xL, Bax, Ki-67, PCNA, and or CD82 (all from Biolegend, USA) were analyzed by FCM according to the manufacturer’s instructions. In addition, the expression levels of IFN-γ, IL-10, NKG2A, NKp30, and NKp40 (all from Biolgend) in CD56⁺NK cells were analyzed by FCM. Isotypic IgG antibodies were used as controls. The samples were analyzed using a FACS-Calibur flow cytometer (Becton Dickinson, USA) and Cellquest software (Becton Dickinson). Statistical analysis was conducted using isotype-matched controls as references.