Anti xbp1

Lung homogenates or cell lysates were subjected to denaturating SDS-PAGE, followed by electroblotting and immunoblotting for anti-ATF4, anti-GRP78, anti-CHOP (Santa Cruz Biotechnology, Dallas, TX), anti-ATF6α, anti-IRE1 (Enzo Life Sciences, Farmingdale, USA), anti-XBP-1 (Novus Biologicals, Littleton, CO), anti-eIF2α, anti-phospho eIF2α, anti-phospho PERK, anti-PERK, anti-phospho AKT(Thr308), anti-phospho p70S6K (Cell Signaling Technology, Danvers, MA) or anti-phospho IRE1 (Abcam, Cambridge, MA). Blots were developed using corresponding HRP-conjugated secondary antibodies and detected using a chemiluminescent system (Amersham ECL Plus; GE Healthcare, Piscataway, NJ). Band intensities were quantified with a LAS-1000 plus system (Fuji Film, Japan).

To evaluate protein expression on Western blot membranes, the following antibody were used: rabbit polyclonal anti-ATF6 (1:600) (Proteintech, 24169-1-AP), rabbit polyclonal anti-XBP1 (1:1000) (Novus Biologicals, NB100-80861), rabbit polyclonal anti-phospho-eIF2alpha (Ser51) (1:1000) (Cell Signaling, 9721), rabbit polyclonal anti-eIF2alpha (1:4000) (Cell Signaling, 9722), rabbit polyclonal anti-BiP/GRIP78 (1:5000) (Proteintech, 11587-1-AP), rabbit polyclonal anti-CHOP (GADD153) (1:1000) (Proteintech, 15204-1-AP), mouse monoclonal anti-DUSP5 (1:200) (Santa Cruz Biotechnology Inc., Dallas, TX, USA sc-393801), mouse monoclonal anti-p-ERK (Santa Cruz Biotechnology Inc., sc-7383), rabbit polyclonal anti-ERK1 (Santa Cruz Biotechnology Inc., sc-93), rabbit polyclonal anti-ERK2 (Santa Cruz Biotechnology Inc., sc-154), rabbit monoclonal anti-Mcl1 (1:1000) (Cell Signaling, 39224), mouse monoclonal anti-p53 (1:100) (clone DO-1, Santa Cruz Biotechnology Inc., sc-126). Mouse monoclonal anti-β-actin (1:10,000) (Sigma Aldrich) was used as the loading control. The goat antimouse IgG-HRP (1:30,000) (Bethyl Laboratories, A90-116P) and goat antirabbit IgG-HRP (1:30,000) (Bethyl Laboratories, A120-101P) were used as secondary antibodies. All primary and secondary antibodies were diluted in PBS–0.1% Tween20 solution containing 3% of BSA (SERVA).

The following primary antibodies were used for Western blot analysis: rabbit polyclonal anti-PARP1(1:1000) (Proteintech, Rosemont, IL, USA, #13371-1), rabbit polyclonal anti-phospho STAT3 Tyr705 (1:500) (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, #sc-8059), mouse monoclonal anti-STAT3 (1:100) (BD Transduction Lab, Franklin Lakes, NJ, USA, #610189), rabbit polyclonal anti-LC3I/II (1:1000) (Novus, CO, USA, #NB100-2220), mouse monoclonal anti-p62/SQSTM1 (1:300) (BD Transduction Lab, #610832), rabbit polyclonal anti-XBP1 (1:1000) (NovusBio, #NBP1-77681SS), rabbit polyclonal anti-ATF6 (1:200) (Cell Signaling Technology, Danvers, MA, USA, #65880), rabbit polyclonal anti-phospho eIF2α (Ser15) (1:200) (Cell Signaling, #3398), rabbit polyclonal anti-eIF2α (1:500) (Cell Signaling, #9722), mouse monoclonal anti-β-actin (1:10000) (Sigma Aldrich, #A5441) and anti hsp70 (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, #sc-32239) were used as loading control. Goat anti-rabbit IgG-horseradish peroxidase HRP (1:10000) (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, sc-2004), goat anti-mouse IgG-horseradish peroxidase HRP (1:10000) (Santa Cruz Biotechnology, Inc. Heidelberg, Germany, sc-2005) were used as secondary antibodies. All primary and secondary antibodies used in this study were diluted in a PBS-0.2% Tween 20 solution containing 3% BSA.