Hybond p pvdf membranes

The western blot was used to determine levels of AGR2 and associated proteins. Cells were first washed with PBS and lysed in RIPA lysis buffer (APOLO, Hsinchu, Taiwan) containing 50mM Tris, 0.15M NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS supplemented with a protease inhibitor cocktail (MCE, Monmouth Junction, NJ, USA). Protein concentrations were detected using a protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins (30 μg) were subjected to sodium dodecyl sulfate 8% -12% polyacrylamide gel electrophoresis. Fractionated proteins were transferred to Hybond-P PVDF membranes (Millipore, Darmstadt, DE). Membranes were blocked with PBS containing 5% nonfat milk and 0.2% Tween 20. For the detection of human anti-AGR2 (Invitrogen, Vilnius, Lithuania) and anti-β-actin (Sigma-Aldrich, St Louis, MO), the membranes were incubated overnight at 4°C, followed by the addition of anti-mouse IgG or anti-rabbit IgG antibody linked to Horseradish peroxidase (Jackson, West Grave, PA, USA). Blots were finally developed using an enhanced chemiluminescence reagent (Millipore, Darmstadt, DE).

Equal amounts of protein were separated via electrophoresis on NuPAGE 4-12% gradient precast polyacrylamide gels (Life Technologies, Carlsbad, CA). Proteins were transferred onto Hybond-P PVDF membranes (Millipore, Billerica, MA) and blocked for 1 h at room temperature in 5% non-fat milk prepared in Tris Buffered Saline with 0.1%Tween-20 (vol/vol). Membranes were incubated with primary antibodies overnight at 4 °C. Primary antibody concentrations were as follows: GRP78 (1:1000; Cat#3177), Cl CASP3 (1:250; Cat#9664), GADD153 (1:500; Cat#5554), Cl PARP (1:1000; Cat#9541), ATF4 (1:500; Cat#11815), p-eIF2α (1:500; Cat#3398), NF-κB (1:1000; Cat#8242), XIAP (1:250; Cat#2042), and PDI (1:5000; Cat#3501) were obtained from Cell Signaling Technologies; XBP1s (1:500; Cat#37152) was obtained from Abcam; ATF6 (1:500; Cat#24169-1-AP) was obtained from Proteintech; MCL1 (1:1000; Cat#sc-819) was obtained from Santa Cruz Biotechnology; and NCOA3 (1:5000; Cat#PA1-845) was obtained from ThermoScientific. Following primary incubation, immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced-chemiluminescence detection system (ThermoScientific, Rockford, IL). Immunoreactive band density was then quantified using ImageJ software.