Anti cyp11a1

Intracellular cytokine and CYP11A1 staining was performed as described previously [33 ]. The cells were labeled with anti-human CD3 and CD4 antibodies (eBioscience) and stained for intracytoplasmic CYP11A1 (Abcam, Cambridge, MA), IL-13, and IFNγ (eBioscience) using an intracellular staining kit (eBiosciences) according to the manufacturer’s protocol. Cells were analyzed on an LSR II (BD Biosciences, San Jose, CA) using the FlowJo software (Tree Star, Ashland, OR). Slides were prepared from cultured PBMCs using cytospin. Numbers of cultured cells expressing CD4 and CYP11A were identified by ICC staining using anti-human CD4 (eBiosciences) and anti-CYP11A1 (Abcam) and analyzed using an LSM 700 confocal microscope (Carl Zeiss, Thornwood, NY). Quantitative analysis was performed by counting CYP11A1⁺ and CD4⁺ cells under the LSM 700 confocal microscope.

For western blot analysis, cells were harvest in a 10 mM Tris (pH 7.4) buffer containing 1% SDS and 1 mM Na3VO4 on ice and 20 µg of cell lysate were separated by 10% SDS-PAGE and electro-transferred onto a nitrocellulose membrane as described elsewhere. The blots were then washed in Tris-buffered saline (20 mM Tris-HCl, pH 7.6 containing 137 mM NaCl and 0.05z (vWv) Tween 20), blocked with 5% skim milk for 1 h, incubated with primary antibody (1:1000) at 4°C for overnight. Primary antibodies used for immunoblotting were anti-actin (Santa Cruz, USA; #sc-47778), anti-CYP11A1 (Abcam, USA; #ab175408), anti-CYP17A1 (Abcam, USA; #ab125022) and anti-STAR (Abcam, USA;#ab203193). After then the membranes were rinsed with TBST three times (10 min each) and then incubated with secondary antibody (1:5000) labelled with horseradish peroxidase (HRP) at room temperature for 1 h. After the reaction the membranes were rinsed with TBST three times (10 min each), the signals were detected using the enhanced chemiluminescence Western blot kit (EzWestLumi plus) from ATTO Corporation (Motoasakusa, Tokyo, Japan) and analyzed using the ImageQuant LAS 4000 mini (GE Healthcare) machine. The experiments were performed in triplicate and data were quantified by Image J program.