Anti fap1

Cells or lung tissues were harvested and lysed in radio immunoprecipitation assay buffer and phenylmethanesulfonyl fluoride (100:1). The protein concentration was measured using bicinchoninic acid protein assay kit (Coolaber, China). After separation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were transferred to a polyvinylidene fluoride membrane, and the protein bands were blocked with 5% nonfat dry milk for 2 h, and mixed with anti-collagen III (1:1,000, Affinity, China), anti-collagen I (1:1,000, Affinity, China), anti-α-SMA (1:1,000, Affinity, China), anti-Vimentin (1:1,000, Affinity, China), Anti-GAPDH (1:10,000, Affinity, China), anti-ULK1 (1:1,000, Affinity, China), anti-FAP1 (1:1,000, Cell Signaling, United States), anti-TGF-β1, anti-ATG5, anti-DRAM2 (1:1,000, Cell Signaling, United States) 1,000, Bioss, China), anti-GABARAP (1:1,000, Affinity, China), anti-EZH2, anti-FOXK1, anti-STAT1 (1:1,000, Abcam, United Kingdom), anti-P62, anti-HuR (1:1,000, Proteintech, China)), anti-ATF3 (1:1,000, Proteintech, China), anti-LC3 polyclonal antibody and incubated overnight. Membranes were washed three times with 1× tris buffered saline tween and then incubated with goat anti-rabbit/mouse secondary antibody for 1 h. Finally, protein expression was detected by enhanced chemiluminescence kit (Spark Jade, China).

Tissues or cells were collected and lysed in radioimmunoprecipitation assay (RIPA, Solarbio, R0020) buffer and phenylmethylsulfonyl fluoride (PMSF, Solarbio, P0100) (RIPA buffer: PMSF = 100:1). The protein concentration was measured by bicinchoninic acid protein assay kit (Coolaber, SK1070). After separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred to polyvinylidenefluoride membranes. The protein was sealed for 2 h with 5% skim milk and incubated overnight at 4°C with antibodies: anti-collagen I (Affinity, AF7001), anti-collagen III (Affinity, AF0136), anti-vimentin (Affinity, AF7013), anti-α-SMA (Affinity, AF1032), anti-FAP1 (Cell Signaling Technology, 66562S), anti-S100A4 (Cell Signaling Technology, 13018S), anti-Myo1c (Abcam, ab194828), anti-YAP1 (Cell Signaling Technology, 14074S), anti-phospho-YAP1 (Cell Signaling Technology, 53749S), anti-F-actin (Abcam, ab130935), anti-GAPDH (Affinity, AF7021). 1×Tris buffered saline Tween was used to wash the membranes for three times. Then membranes were incubated for 1 h with secondary antibodies at room temperature. The expression of proteins was detected by enhanced chemiluminescence reagent kit (SparkJade, ED0015-B).