Antibody buffer

The protein used for WB is derived from the supernatant or cell lysate. Cell culture medium was concentrated by Amicon Ultra-4 Centrifugal Filter Devices (Amicon Ultra 50 K device, 50,000 MW CO, Millipore, USA) after centrifugation at 7500 × g for 40 min. The condensed samples (approximately 200 µl) were mixed with 5× loading buffer, boiled for 3 min and then analyzed by WB. Cell lysates obtained after treatment of cells with different inhibitors. AT2 cells were treated with PS1145 at 20 μM for 30 min, or JSH-23 (#S7351, Selleck Chemicals, China) at 6 μM for 1 h, or C646 at 20 μM for 1 h, or Anacardic Acid (#S7582, Selleck Chemicals, China) at 10 μM for 1 h, respectively. After 48 h, cell lysates were collected. The protein was separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes (Millipore, USA), and blocked with 5% non-fat dry milk in TBST. After three times of washing with TBST, following primary antibodies dissolved in antibody buffer (Keygentec, China) were used: anti-ECM1 (sc-365335, SANTA CRUZ, USA), anti-p-p65 (S536) (#3033, CST, USA), anti-Ac-p65 (K310) (#3045, CST, USA), anti-t-p65 (#8242,CST, USA), anti-p300 (#86377, CST, USA), anti-integrinα6 (ab181551, Abcam, USA), anti-integrin β4 (ab236251, Abcam, USA), anti-ABCG1(ab218528, Abcam, USA), anti-HA (#3724, CST, USA), and anti-β-actin (#4970, CST, USA).

The protein was separated on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to PVDF membranes (Millipore, USA), and blocked with 5% non-fat dry milk in TBST. After washing three times with TBST, the following primary antibodies dissolved in antibody buffer (Keygentec, China) were used: anti-RNase L (Abcam, USA), anti-phosphor-H2A.X (Serl39) (Millipore, USA), anti-phosphor-H2B (Serl4) (Millipore, USA), anti-H2A.X (Abcam, USA), anti-H2B (Abcam, USA), anti-ROCK-1 (Abcam, USA), anti-Caspase-3 (Cell Signaling Technology, USA), ECLhCZIBPBHhf-iFeN8r/" target="_blank">anti-PARP (Cell Signaling Technology, USA), anti-p21 (Cell Signaling Technology, USA), and anti-β-actin (Cell Signaling Technology, USA). After the secondary antibody incubation, the membrane was washed three times with TBST and exposed with ECL (Millipore, USA). The corresponding semi-quantitative analysis was performed by measuring the optical density using ImageJ software.