Nuclear and cytosolic extracts were stored at −80°. Protein concentration was determined as above. Whole-cell or nuclear extracts were mixed 1 : 1 with Laemmli sample buffer and heated at 95° for 5 min. Proteins were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE)

using Tris/Glycine29 or Tris/Tricine30 buffer systems. Resolved proteins were electro-transferred to PVDF or nitrocellulose membranes, blocked with 5% BSA (RPN412; Amersham) in TBS (20 mm Tris, pH 7·6, and 140 mm NaCl) containing 0·02% v/v Tween 20 (blocking solution) and probed with antibodies as indicated (see results). Immunoreactive bands were detected by ECL using a G:Box Chemi-XT CCD gel imaging system and GeneSnap image acquisition software (Syngene, Cambridge, UK). Relative band LY2835219 research buy intensities were quantitated using GeneTools image analysis software (Syngene). Total RNA was extracted from 3 × 106 cells using an RNeasy Plus Mini kit (Qiagen, Hilden, Germany). Purified RNA was quantified spectrophotometrically, aliquoted and stored at −80°. RNA (1 μg)

was converted to cDNA using Superscript III reverse transcriptase and 2·5 μm oligo(dT)20 primer in 20 μl, according to the manufacturer’s specifications. Real-time PCR was performed on a Bio-Rad Mini-Opticon thermal cycler using 15 ng of reverse-transcribed RNA and 200 nm specific forward and reverse primers in 25 μl, using SybrGreen

Sirolimus in vivo qPCR Super Mix. PCR conditions were 3 min at 95°, with 50 cycles of 15 seconds at 95° and 30 seconds at 60°. All samples were Thalidomide assayed in triplicate. mRNA levels were normalized using TATA binding protein (TBP) and ribosomal protein L13A (RPL13A) as internal controls31 using genex software (Bio-Rad). Melting point analysis was carried out for all runs. To measure PCR efficiency, serially diluted, reverse-transcribed mRNA (from 0·1 pg to 200 ng) was amplified with each set of primers, and linear standard curves obtained by plotting the log of the serial dilutions against the cycle threshold (CT) value. The slope of each curve was used to calculate efficiency for primer sets using the formula E = 10−1/slope. The relative expression of the tested genes in untreated and treated cells was determined using the 2−ΔΔCT formula.32 Amplification products for all tested genes were analysed on ethidium bromide-stained agarose gels to ensure single amplification products of the expected size. Primers were designed using Primer3 (http://frodo.wi.mit.edu/primer3/) and synthesized by MWG (Martinsried, Germany). IL-2 mRNA (NM_000586) was amplified from position 38 to 264, with primers: forward 5′-acctcaactcctgccacaat-3′ and reverse 5′-gccttcttgggcatgtaaaa-3′. IL-2RA mRNA (NM_000417) was amplified from 892 to 1072, with primers: forward 5′-ggctgtgttttcctgctgat-3′ and reverse 5′-gcgaccatttagcacctttg-3′.