Phenyl methyl sulfonyl fluoride (pmsf)

Retina protein was collected in 300 μl cell lysis buffer (Cell Signaling) with protease inhibitors (Roche Complete, FUT-175, phosphatase inhibitor cocktail II and PMSF [Cell Signaling]) in 500 μl screw cap tube (Sarstedt). Tissue was homogenized using two 3 mm tungsten beads with a Qiagen Tissuelyser II, 3 minutes at 30 Hz and centrifuged for 15 minutes at 4 °C at 18,000 × g. Lysate was aliquoted and stored at −80 °C. ELISAs for mouse C5a (RayBiotech) and CCL2 (DuoSet, R&D Systems) were performed according to manufacturers instructions and read on Spectramax Plus (Molecular Devices). Luminex analysis on 5 retina lysates was performed using Milliplex 32-plex panel (EMD Millipore). We only reported chemokines that were detected in retina samples.

The 5 × 10⁶ human NSCLC cells were suspended in hypotonic buffer (20 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) Cell Signaling Technology) and incubated on ice for 15 min. Nonionic detergent NP-40 (10%; Sigma-Aldrich) was then added to the cell suspension, which was mixed vigorously. Next, the cell homogenate was centrifuged at 5000 rpm for 10 min at 4 °C. The supernatant was collected as the cytoplasmic fraction, and the pellet was suspended in cell extraction buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Roche) and 1 mM PMSF. The suspension was incubated on ice for 30 min with intermittent vortexing. Finally, the sample was centrifuged at 14,000 × g for 30 min at 4 °C, and the supernatant was collected as nuclear extract.

Protein was isolated from cells by lysis (BestBio, Shanghai, China) with phenylmethanesulfonyl fluoride (PMSF, cat #: 8553, Cell Signaling Technology, Danvers, MA, United States) for western blotting. Cells were harvested and resuspended in PBS, and cell protein extracts were obtained by centrifugation at 12,000 rpm for 10 min. Protein levels were determined using the BCA assay kit (Tiangen, Beijing, China). Protein extracts (10–20 μL) were separated using 6–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidene fluoride membranes. After blocking with 5% nonfat dried milk in Tris-buffered saline, the PVDF membranes were incubated overnight at 4°C with the rabbit anti-NOX4 (1:1000), anti-RhoA (1:1000), anti-ROCK1 (1:1000), anti-α-SMA (1:200), anti-collagen-I (1:1000), anti-MMP1 (1:2000), anti-TIMP1 (1:2000), anti-F-actin (1:2000), and anti-GAPDH (1:1000) antibodies. A secondary HRP-conjugated anti-rabbit or anti-mouse IgG antibody (Zhongshan Golden Bridge Biotechnology Co., Beijing, China) was then added, and specific bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific Inc., Waltham, MA, United States).

After being anesthetized through isoflurane inhalation, mice were perfused with 1X PBS through the left ventricle to remove blood. Brain samples were collected and frozen in dry ice. Peri-infarct brain regions were dissected out and homogenized in 1X cell lysis buffer (Cell Signaling, Danvers, MA, USA), supplemented with 1 mM PMSF (Cell signaling). The lysates were centrifuged at 13,000 rpm for 25 minutes, and the supernatants, collected. Protein concentrations were measured using the Bradford Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). IL-1ß (R&D systems, Minneapolis, MN, USA) and IL-6 (R&D systems) levels were measured (using 312 μg protein from each animal) through ELISA assays following the manufacturer’s instructions.

Protein from lymphoma cell lines from Lck-Dlx5 mice was extracted with cell lysis buffer supplemented with 2 mM PMSF (Cell Signaling); 50 μg/well proteins were loaded into Tris-Glycine SDS-PAGE gels (Invitrogen) and transferred onto PVDF membranes (Millipore). Protein blots were incubated with antibodies overnight at 4°C. Signals were developed after incubation with HRP-conjugated secondary antibody.