on of GPR30 in Human Seminoma represents its own control. The samples were frozen at 280uC before being ground in cold 
Tris containing protease inhibitors. Protein concentrations of the cell and tissue lysates were determined by the Bradford method. Equal amounts of the whole protein extract were resolved on a 12% SDS-polyacrylamide gel. The proteins were transferred to a polyvinylidene difluoride membrane, probed with anti-GPER Ab and detected using HRP-linked secondary Ab and the ECL System. After the blots were stripped, we verified equal loading of the protein by reprobing the same blots with anti-actin Ab. All experiments were performed in triplicate and the blots shown are representative. staining for GPER. In normal testis, GPER was localized in seminiferous tubules and also in Leydig cells. In seminoma, tumoural cells, which were recognized by their size and specific PLAP-staining, showed an intense staining for GPER. In the JKT-1 cells, the GPER protein was identified by membranous and cytoplasmic staining, whereas classical ERb had an intracytoplasmic and nuclear localization without co-localization with GPER. E2-BSA-FITC was also identified at the JKT-1 cell membrane but not testosterone-BSA-FITC used as negative control. The colocalization of GPER and E2-BSA at the JKT-1 cell membrane supported the notion that GPER could bind E2-BSA and could be a good candidate to SB-203580 mediate its proliferative effect previously reported in these cells. GPER expression in tumoural human cell lines and testis RNA extraction and cDNA synthesis Total RNA was extracted using TRIzol and processed using the RNeasy Mini Kit, according to the manufacturer’s instructions. The amount of RNA was estimated by spectrophotometry at 260 nm. Total cDNA was synthesized by reverse transcription of 10 mg of total RNA using random hexanucleotides as primers in the presence of dNTPs and Moloney murine leukaemia virus RT during 1 h at 37uC, following the manufacturer’s protocol. Expression of selected genes was quantified by real-time PCR using the StepOnePlusTM Sequence Detection System, following the manufacturer’s instructions. The gene-specific primers used for 8663121 gper and b-actin, which was used as a control gene to obtain normalized values, were as follows: 59-TCTAAACTGCGGTCAGATGTGGCT-39 and 59-TGTGAAGAGTGCAAGGTGACCAGT-39 and 59-TTGCTGATCCACATCTGCTG-39 and 59-GACAGGATGCAGAAGGAGAT-39, respectively. PCR amplification was performed according to the manufacturer’s instructions by first heating the mixture at 95uC for 10 min, followed by 55 cycles consisting of two steps: denaturation at 95uC for 30 s, annealing and extension at 60uC for 60 s ” and then a final cycle of three steps. The PCR product was electrophoresed on a 3% agarose gel in 16 TAE buffer and stained with the SYBRH Safe DNA gel stain. All experiments were performed in triplicate and the data shown are representative. Using Western blotting, we compared the expression of GPER between tumoural and normal tissues. Seminomas showed significantly higher GPER protein expression than normal peri-tumoural tissues. No significant difference was observed between non-seminomas and normal peritumoural tissues. These results were confirmed by analysing mRNA levels by RT-PCR, which revealed significantly higher GPER mRNA levels only in seminomas but not in non-seminomas, compared to normal peri-tumoural tissues. Analysis of mRNA by RT-PCR revealed that both JKT-1 and NCCIT cells expressed GPER. These results were confi
