Figure 3.

Residues 260–600 of p125A are sufficient for binding to Sec31A. (A) a schematic illustration of p125A deletion constructs. cDNA fragments encoding various mutants were generated by PCR and validated by sequencing. The fragments were digested with SalI and NotI and then subsequently cloned into pDmyc-neo. KIAA0725p, the homologue of p125A, also known as myc-p125B (Nakajima et al., 2002), is also indicated. (B) myc-p125A deletion constructs were transfected into HEK293 cells. Cell lysates were prepared and subjected to pulldown using immobilized GST-Sec31A and GST. Proteins retained by the beads, along with cell lysates, were subjected to SDS-PAGE, transferred to PVDF membrane, and immunoblotted using anti-myc antibody. Red asterisk indicates the expected sizes of the various HA-tagged mutants; red hash indicates the smallest fragment that could be pulled down by GST-Sec31A. Solid red triangle indicates GST-Sec31A, and the empty red triangle represents GST. b, d, and f are Ponceau staining of the immunoblots. Molecular size markers are in kD.