FIG. 6.

Type II hCGβ protein products. (A) Sequence alignment of hCGβ. Key residues are numbered relative to the signal peptide cleavage site (solid line). The Phe residue in the signal peptide sequence (underlined) is replaced by Leu in hCGβ5. Known N- and O-linked glycosylation sites are denoted by gray and black shading, respectively. (B) (Top) Immunoblot of extract of HeLa cells transfected with construct expressing β1a, β2a, or β2b mRNA or empty vector and probed with anti-hCGβ antibody. Extract was incubated in the presence or absence of PNGase F (PNGF) before electrophoresis. (Bottom) Longer exposure of the same immunoblot. An apparent hCGβ1-derived band is denoted by the white arrow. (C) Total RNA extracted from cells in panel B was DNase I treated, reverse transcribed in the absence or presence of reverse transcriptase (RT), and PCR amplified for 20 or 25 cycles. PCR products were visualized in an ethidium bromide-stained agarose gel. (D) Immunoblot of extract from HeLa cells transfected with C-terminal FLAG-tagged peptide β2b′ construct, ATG mutants, or empty vector. Blot was probed with anti-FLAG antibody. (E) Amino acid sequence of putative peptides translated from β2b or β1c mRNAs. Predicted subtilisin-like proprotein convertase (1) and N-Arg dibasic convertase (2) cleavage sites, as well as a peptide amidation site (3), are denoted by arrowheads. Positions of adenine mutations in ATG codons at nucleotides A175, A265, and A271 relative to the transcription start site are marked. (F) PCR product of RNA isolated from cell extract in panel D, amplified over 20 or 25 cycles.