Fig 2: SHBG expression in human prostate cell lines.A) RT-PCR analysis of SHBG expression in human prostate-derived cell lines, using primers that amplify a region common to all SHBG mRNA isoforms (Ex2-Ex5). B) RT-PCR analysis (Ex1A-Ex8) of exon 1A transcripts in LNCaP cells. The different bands observed correspond to: α (full length transcript), β (skipping of exon 7) and γ (skipping of exons 6 and 7). C) As previously reported [2], full-length TU-1B transcripts are detected by RT-PCR analysis (Ex1B-Ex8) in human prostate tissue and LNCaP cells. Negative controls (Ctrl) are performed with water instead of cDNA.

Fig 3: Predicted secondary structure for 5'UTR sequences of TU-1 and TU-1A.M-fold prediction of the 5'UTR mRNA secondary structure of TU-1 (A) and TU-1A (B). Predicted thermodynamic stability is shown, and the first SHBG codon is marked with a grey box. Junction between exons 1A and 2 is indicated with a solid black line in B. The uAUG is shown in a blue box in B. The predicted major hairpin is also indicated (HP).

Fig 4: Western blot analysis of SHBG protein in human prostate.A–B) Two bands sized 52- and 48-kDa were detected in human prostate tissue using the SHBG 11F11 antibody (A) and were identical in size to those detected in human plasma and human testis (B). C) SHBG constructs used to transfect LNCaP cells. In the Flag-Ex2-Ex8 construct, SHBG ATG was mutated to ATA to avoid disturbing the Flag translation start site. The nucleotides involved in the SHBG translation start site are shown in all the constructs. D-E) Western blot analysis of the SHBG protein using the 11F11 antibody in transfected and non-transfected human prostate-derived cell lines (D), and in the supernatant (E). In (E), plasma and the hepatocarcinoma cell line (HepG2) were used as positive controls for secreted SHBG. F) Western blot analysis of LNCaP cells transfected or not with Flag-SHBG and pDsRed-SHBG constructs.

Fig 5: Identification of uORFs in TU-1A and TU-1B.A) The upper panel contains a schematic representation of the SHBG gene. Coding exons are shown in black boxes and noncoding alternative first exons are in gray boxes, with their respective sizes (in bp) indicated above. Below are depicted the exon organization of mature mRNAs of TU-1A and 1B and the corresponding ORF organization. uAUGs (AUG1 and 2) are written in gray while SHBG AUG is written in black (AUG3). uORFs are indicated in gray boxes while SHBG ORF is shown in a black box. AUG1 generates an uORF of 2 codons while AUG2 generates an uORF of 11 codons. B). Exon 2 scheme, showing AUG2 and 3. AUG2 starts a uORF whose stop codon overlaps the SHBG translation start codon (AUG3).