Figure 4

Characterization of functional domains in the signal peptide of TβRI. Panel A: Schematic illustration of the TβRI mutants with deletions in the signal peptide. Using wild-type Tgfbr1 (WT) as a template, the regions 5' or 3' to the 9A stretch sequence or the 9A stretch itself were truncated serially by site-directed deletion mutagenesis and designated as DS1, DS3, and DS2, respectively. HEK 293 cells were either untransfected (UN) or transfected with individual variants or vector control (VC) plasmid, as indicated at the top. The cells were divided into two groups for Western blotting or RT-PCR analyses 24 h after transfection. Panel B: Western blotting. Each lysate (30 μg) was resolved on a 10% SDS-polyacrylamide gel, immunoblotted with HA-specific antibody, and reprobed with anti-β-Actin antibody as a loading control. Panel C: RT-PCR. Total RNA was extracted from each transfectant and reverse transcribed. PCR reactions using P3 primers, as indicated at the bottom of Panel A, were utilized and the resultant amplicons were resolved on 1% agarose gels. A β-Actin transcript and its protein were used as loading controls, as indicated on the right.