Gapdh bs 0755r

Cells were lysed in RIPA buffer (Beyotime Biotechnology, China) containing 1X Protease and Phosphatase inhibitor (Beyotime Biotechnology, China). An equal amount of protein samples was separated by 8% SDS/PAGE and transferred to 0.25 mm polyvinylidene fluoride membranes (Millpore, Germany). The membranes were blocked with 5% non-fat milk for 1 h, then incubated with the individual antibody at 4 ℃ overnight: Tubulin (YFB6011, YIFAN BIOLOGICAL), Rcl1 (15330-1-AP; Proteintech), GAPDH (bs-0755R, Bioss). The membranes were then incubated with the second antibody (bs-40295G-HRP, Bioss) at 37 ℃ for 1 h. Finally, protein bands were visualized using Omni ECL reagent (EpiZyme, China), and the gray intensity was acquired by using Fiji (NCBI, USA).

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).