Phenyl methyl sulfonyl fluoride (pmsf)

For the rats, the brains were dissected to separate the substantia nigra and striatum for homogenization in RIPA Lysis Buffer 10 μL/per mg tissues (Beyotime Biotechnology, Shanghai, China, 50 mM Tris-HCl, pH7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) with protease inhibitor PMSF (Beyotime, RIPA: PMSF = 100:1). For Drosophila, 5 heads of each experimental group were homogenized in RIPA Lysis Buffer with protease inhibitor. After 12.5% SDS–PAGE gel (Epizyme Biotech, Shanghai, China) electrophoresis, the proteins were transferred onto a PVDF membrane (Millipore). The membrane was then blocked with 5% nonfat milk in TBST and then incubated with primary antibodies overnight at 4 °C and, subsequently, with secondary antibody for 2h at room temperature. The primary antibodies used for the Western blot included anti-Nrf2 (1:1000, Assaybiotech), anti-GCLC (1:1000, BD), anti-HO-1 and anti-LC3I/II (Sigma), anti-Ref(2)P (1:1000, BD Pharmingen, San Jose, CA, USA), and anti-β-tubulin (1:5000, Sigma-Aldrich). The bands were detected by using chemiluminescence substrates (Beyotime Biotechnology) and analyzed with the NIH ImageJ software. The experiments of WB were repeated at least three times and we performed the statistical analysis of the results using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA).

Total protein was extracted from rapidly frozen retinas by using RIPA buffer (Beyotime) and 1% protease inhibitor (PMSF, Beyotime) (RIPA:PMSF ratio of 1:100). Twenty-five micrograms of protein lysate from each sample was electrophoretically separated with a 10% SDS–PAGE gel and then transferred to a polyvinylidene fluoride membrane. Anti-CXCR2 (Invitrogen, PA5-102662, 1:200) and anti-GAPDH (Cell Signalling Technology, CST, D16H11, 1:1000) were used to incubate the membranes at 4 °C overnight and then with secondary antibodies. We developed all the blots with the ECL-Plus chemiluminescence system and used ImageJ software for densitometry analysis. GAPDH was used as an internal control.

Cells for the experimental assay were collected and washed twice with PBS, then the cells were lysed with a mixture of Ripa cell lysate (Biyuntian, Shanghai) and PMSF (Biyuntian, Shanghai) to prepare a lysis solution to extract total cellular protein. Equal amounts of cell lysate were separated on duplicate 8%–10% SDS-PAGE gels (Solebro, China) and transferred to PVDF membranes (Weiao, China). The membranes were blocked with 5% skimmed dry milk at the end of the transfer and incubated with the primary antibody: β-actin antibody (CST, USA), RUNX2 antibody (CST, USA), ALP antibody (Abcam, UK), Wnt6 antibody (Abcam, UK), p38 MAPK antibody (CST, USA), and Phospho-p38 MAPK antibody (CST, USA). The average expression levels of target proteins are relative to β-actin. All original WBs images are shown in Supplementary 4.

RIPA lysate (Beijing Kangwei Century Company Biotechnology, Beijing, China), containing PMSF and protease inhibitors (Shanghai Biyuntian Biotechnology, Shanghai, China) was used to extract the total proteins of the 20 mg lung lobe tissue cells of each group. Total protein concentration of about 3 μg/μL by BCA method and 10 μL total proteins were taken from each lane and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE). Subsequently, the proteins were wet-transferred to a polyvinylidene fluoride membrane and blocked at 37 °C for 1 h with 5% skimmed milk powder. Primary antibodies, including anti-VEGFA (1:500; Abcam, Cambridge, UK), anti-Ang-1 (1:2,000; Abcam, Cambridge, UK), anti-FOXP3 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-IRF4 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), and β-actin (1:1,000; Cell Signaling Technology, Danvers, MA, USA) were added respectively, and incubated at 4 °C overnight. After washing the membrane, secondary antibody IgG (1:5000; Immunoway, Plano, Texas, USA) was added, and the membrane was incubated at 37 °C for 1 h. Enhanced chemiluminescence was used for visualisation. The relative level of the target protein was determined as the ratio of the grey value of the target protein to the β-actin band.