Amersham enhanced chemiluminescence plus western blotting detection system

Cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred onto pure nitrocellulose blotting membrane (Pall Gelman Laboratory, Ann Arbor, MI, USA). The membranes were incubated with primary antibodies against phospho-H2A.X (Ser139), CHOP, beclin-1, LC3B, caspase-3, caspase-9, ERK, JNK (Cell Signaling Technology, Beverly, MA, USA), GRP78, Bax, phospho-p38 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-IRE1 (Abcam, Cambridge, MA, USA), and actin (Sigma-Aldrich), followed by incubating with a corresponding secondary antibody (Pierce, Rockford, IL, USA). The Amersham enhanced chemiluminescence plus western blotting detection system (GE Healthcare Life Sciences, Buckinghamshire, UK) was used to detect the protein bands.

Cell and mouse skin lysates were subjected to SDS-PAGE, and the separated proteins were transferred to membranes and incubated with primary antibodies against phospho-H2A.X (Ser139), CHOP, phospho-PERK, beclin-1, LC3B, caspase-3, and caspase-9 (Cell Signaling Technology, Beverly, MA, USA), GRP78, Bax (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-IRE1 (Abcam, Cambridge, MA, USA), and actin (Sigma-Aldrich) followed by the incubation with a secondary antibody (Pierce, Rockford, IL, USA). Protein bands were detected using an Amersham Enhanced Chemiluminescence Plus Western Blotting Detection system (GE Healthcare Life Sciences, Buckinghamshire, UK).