A00683 1

Total protein was extracted from A549 cells and lung tissues using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China). After the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they were transferred to membranes, which were blocked with Protein Free Rapid Blocking Buffer (PS108, EpiZyme) and incubated with primary antibodies against CHOP (A5462, Bimake), BiP (11587-1-AP; Proteintech, Wuhan, China), ATF6 (D262665, Sangon, China), ATF4 (A5514, Bimake), XBP-1s (24868-1-AP, Proteintech), XBP-1u (25997-1-AP, Proteintech), IRE1α (A00683-1, Boster, Wuhan, China), phospho-IRE1α (S724, human) (ab124945, Abcam, Cambridge, UK), phospho-IRE1α (S724, mouse) (530878, ZEN-BIO, Chengdu, China), Vimentin (ET1610-39, HuaBio, Hangzhou, China), and E-cadherin (340341, ZEN-BIO) overnight. GAPDH (bs-0755R, Bioss, Beijing, China) was used as the internal reference protein. After incubation with HRP-conjugated goat anti-rabbit IgG (H + L) (AS014, ABclonal, Wuhan, China) and HRP-conjugated goat anti-mouse IgG (H + L) (AS003, ABclonal), the protein bands were visualized using a ChemiDoc™ Imaging System (Bio-Rad) and quantified using NIH ImageJ software (http://rsb.info.nih.gov).

Western blot was performed using a previously published protocol [43 (link)]. The primary antibodies used were as follows: anti-p-PERK (DF7576, Affinity), anti-PERK (ER64553, HuaBio), anti-p-IRE-1α (S724) (ab48187, Abcam), anti-IRE1α (A00683-1, Boster), anti-p-eIF-2α(S51) (ET1603-14, HuaBio), anti-eIF-2α (ET7111-34, HuaBio), anti-Col I (ab34710, Abcam), anti-LDHA (DF6280, Affinity), and β-actin (AC026, ABclonal). After supplementation with the primary antibodies, the lung tissues and cells samples were incubated with goat anti-rabbit or anti-mouse secondary antibodies (074-1506/074-1806; Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) at a concentration of 1:5000 for 1 h. The target bands were visualized using the ECL prime Western blotting detection reagent.