Erp29

Cell lysates were harvested using RIPA cell lysis buffer. An equal amount (15 ug) of total cellular protein was separated by 12.5% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were then blocked with 5% non-fat milk for 2 h and incubated overnight with primary antibodies against ERp29 (Abcam), Akt, p-Akt, ERK, p-ERK(Cell Signaling Biotechnology), E-cadherin, vimentin, N-cadherin (Santa Cruz). After incubated with HRP conjugated-secondary antibody for 2 h at room temperature, the proteins were visualized by enhanced chemiluminescence (ECL).

After two washes with PBS, cells were lysed with buffer containing 2% phenylmethylsulfonyl fluoride and 1% protease inhibitor cocktail. Protein concentration was measured with a bicinchoninic acid assay kit. Equal amounts of protein (~30 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Merck Millipore, Billerica, MA, USA) for ~100 min. After blocking with 5% milk, the membrane was incubated overnight at 4 °C in a 1.25% milk solution containing primary antibodies including anti-BIP, anti-AKT and anti-pAKT, (1:1000; Cell Signaling Technology, Danvers, MA, USA; Cat: #3183; #4060; #4691), ERp29 (1:2000) and G6Pase, (1:500; Abcam, Cambridge, MA, USA; Cat:ab176573; ab83690), PEPCK, (1:2000; Proteintech Group, Chicago, IL, USA; Cat:16754-1-AP). The membrane was washed thrice with Tris-buffered saline containing 0.1% Tween-20, and incubated for 2 h at room temperature with a 1.25% milk solution containing horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000; Beyotime Institute of Biotechnology, Shanghai, China; Cat:A0208). Protein bands were developed by enhanced chemiluminescence (Fude Bio, Hangzhou, China) and signal intensity was determined using Image Lab software (Bio-Rad, Hercules, CA, USA).