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Fig. 2. Adenovirus-mediated gene transfer of asTGF-ß1 mRNA. A, a schematic representation of the strategy used for blockage of TGF-ß1 synthesis. The primary sequence of rat TGF-ß1 consists of 390 amino acids. The signal sequence (amino acids 1–22), the latency-associated peptide (amino acids 23–278), and the COOH-terminal part encoding the mature TGF-ß1 (amino acids 279–390) are shown in black, white, and gray, respectively. The asTGF-ß1 is complementary to the 3' end of the mRNA of TGF-ß1, arresting translation by halting ribosome (RS) after duplex formation. B, Southern blot hybridization of mock- (Lane 1), Ad5-CMV-asTGF-ß1 (Lane 2), and Ad5-CMV-EGFP (Lane 3) infected 293 cells. High molecular weight DNA was analyzed by digestion with EcoRV/HindIII or EcoRV/BglII, followed by 1.2% agarose gel electrophoresis and Southern hybridization using the 32P-labeled 278-bp PvuII fragment of clone pET3d-rTGF-ß1. C, Northern analysis of total RNAs isolated from human MFBs infected with Ad5-CMV-EGFP (Lane 1) or Ad5-CMV-asTGF-ß1 (Lane 2). The blot was subsequently hybridized with the 32P-labeled 278-bp PvuII fragment of pET3d-rTGF-ß1 and a GAPDH-specific cDNA probe. D, Western blot analysis of TGF-ß1 expression in HSC (Lane 2) or in cells infected with Ad5-CMV-asTGF-ß1 (Lane 3), or Ad5-CMV-EGFP (Lane 4). Forty ng of recombinant human TGF-ß1 (Lane 1) with a calculated Mr of 12,794 served as a positive control for rat TGF-ß1 (Mr 12,810). E, immunoprecipitation of TGF-ß1 from total protein cell lysates of HSC (Lane 1), mock infected cells (Lane 2), or cells infected with Ad5-CMV-asTGF-ß1 (Lane 3). TGF-ß1 was resolved by SDS-PAGE under reducing conditions, and labeled TGF-ß1 was visualized by autoradiography for 17 days.