Plasmids transfection
The DNA fragments corresponding to the full-length GRP78 were amplified
by polymerase chain reaction (PCR) from Marathon-Ready HeLa cDNA. Human
GRP78 cDNA encompassing full length, deletions lacking peptide-binding
region or ATPase domain were amplified by reverse transcription PCR and
cloned into pCR8/GW/TOPO (K280020, Invitrogen) as described before
(Goehring et al., 2014). The amino acid residue, Asp180, locates in the
ATPase domain of GRP78 and is essential for catalysis. The D180A mutants
of GRP78 were prepared with the QuikChange site-directed mutagenesis kit
(200523, Agilent, CO, USA), as previously described (Sawano & Miyawaki,
2000). All deletion and point mutations were made by PCR and confirmed
by DNA sequencing.