RCC cells were grown after thawing in normal medium (RPMI1640, 2 mM L-Glutamin, 100 μg/ml Streptomycin, 10% FCS, Biochrom, Berlin, Germany) for 7 days. After passaging cells were treated on day 2–4 with 0,125 μM 5-aza-2`desoxycytidine (Sigma, St. Louis, USA) or a mock solution (control cells) and cultured in normal medium on days 5–7 until harvesting. Isolation of total RNA, reverse transcription and relative quantitation of mRNA levels of internal controls as well CRHBP mRNA were carried out as described before [7 (link)].
For ectopic expression of CRHBP 3μg of human cDNA expression vector (NM_001882, Origene Technologies, Rockville, MD, USA) and PerFectin transfection reagent (T303007, Genlantis, San Diego, CA, USA) were applied according to the manufacturer´s instructions in a transfection volume of 1 ml for 6*105 cells
CHO cells were used as control for transfection and expression efficiency. For SDS-PAGE and Western blot analysis each 0,5 x 106 transfected cells were lysed in 100 μl 2x sample buffer (125 mM Tris-HCl pH 6,8, 20% Glycerol, 4% SDS, 5% Mercaptoethanol, 0,025% Bromphenolblue). Immunoprobing was carried out by use of the anti-Flag-POD antibody (A8592, Sigma-Aldrich, St. Louis, MO, USA).
For ectopic expression of CRHBP 3μg of human cDNA expression vector (NM_001882, Origene Technologies, Rockville, MD, USA) and PerFectin transfection reagent (T303007, Genlantis, San Diego, CA, USA) were applied according to the manufacturer´s instructions in a transfection volume of 1 ml for 6*105 cells
CHO cells were used as control for transfection and expression efficiency. For SDS-PAGE and Western blot analysis each 0,5 x 106 transfected cells were lysed in 100 μl 2x sample buffer (125 mM Tris-HCl pH 6,8, 20% Glycerol, 4% SDS, 5% Mercaptoethanol, 0,025% Bromphenolblue). Immunoprobing was carried out by use of the anti-Flag-POD antibody (A8592, Sigma-Aldrich, St. Louis, MO, USA).