Western Blot Analysis of Protein Markers

Harvested cells were lysed in RIPA buffer (Thermo Fisher Scientific), and equal amounts of lysate proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA) using a Trans-Blot Turbo (Bio-Rad). After blocking with Bullet Blocking One (Nacalai Tesque, Kyoto, Japan), the membranes were incubated with primary antibodies against A1AT (1:2000; Dako, Tokyo, Japan), ERK1/2 (1:2000, #4695; Cell Signaling Technology [CST], Tokyo, Japan), phosphorylated (p-) ERK1/2 (1:2000, #4370; CST), p-38MAPK (1:2000, #8690; CST), p-p38MAPK (1:2000, #4511; CST), JNK (1:2000, #9252; CST), p-JNK (1:2000, #9251; CST), NFkB (1:2000, #4764; CST), IkB (1:2000, #4814; CST), p-IkB (1:2000, #2859; CST), CREB (1:2000, #9197; CST), p-CREB (1:2000, #9198; CST) hCGB (Thermo Fisher Scientific), or GAPDH (1:5000, 5A12; Fujifilm Wako Pure Chemical Corp.). After washing, the membranes were incubated with horseradish peroxidase-linked goat anti-rabbit or anti-mouse IgG (1:5000; Vector Laboratories, Burlingame, CA, USA). Immunoreactivity was detected with a chemiluminescence (Merck Millipore, Burlington, MA, USA) [7 (link)].