Phospho ire1

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).

The right lung samples and cell protein lysates were separated by 10% SDS polyacrylamide gel electrophoresis and immunoblots were developed as previously described (34 (link)). The membranes were blocked for 1 hours at room temperature with 5% nonfat milk and then incubated overnight at 4°C with the following antibodies: claudin-3 (Invitrogen, Carlsbad, CA, USA), claudin-4 (OriGene Technologies, Inc., Rockville, MD, USA), claudin-18 (Invitrogen), GRP78 (Abcam, Cambridge, MA, USA), phospho PERK (Taiclone, Taipei, Taiwan), phospho IRE1 (Abcam), ATF6 (Bioss Antibodies, Woburn, MA, USA), ATF4 (Bioss), GADD153 (CHOP, Santa Cruz Biotechnology, Dallas, TX, USA), B-cell lymphoma (Bcl)-2 (Santa Cruz Biotechnology), NF-κB p65, phospho-NF-κB p65, inhibitor of NF-κB (IκB)-α, cleaved caspase-3 and proliferating cell nuclear antigen (PCNA, Cell Signaling Technology), and β-actin (Sigma-Aldrich). All results were normalized to β-actin and expressed as the relative values to those for the control group.