P1050

The relationship between Fascin-1 and TAZ in 7-day SCI tissues and myelin-treated microglia was assessed by coimmunoprecipitation (Co-IP) assay. The 7-day SCI tissues and myelin-treated microglia were lysed using Co-IP lysis buffer containing protease/phosphate inhibitors (P1050, Beyotime Biotechnology, Shanghai, China) for 30 min. The supernatants were extracted after centrifugation at 12,000 g for 30 min at 4°C. The supernatants were incubated with the primary antibodies, an anti-Fascin-1 antibody or IgG overnight at 4°C. After the immunoprecipitation reaction, the immunoprecipitated protein was mixed with protein A/G beads, and the mixture was incubated for 3 h at 4°C. The beads were washed three times with ice-cold washing buffer, and then eluted by heating in 2 × SDS loading buffer for 10 min. The proteins were collected for subsequent immunoblotting of Fascin-1 and TAZ.

For whole cell lysates, HAECs (2–3 × 10⁶ cells) were lysed with 100 μL lysis buffer (P10013, Beyotime) supplemented with 2% protease inhibitors and 2% phosphatase inhibitors (P1050, Beyotime) for 10 min on ice. The lysates were centrifuged at 13,000× g for 10 min at 4 °C. Protein concentration of the collected supernatants was measured by BCA Protein Assay Kit (23225, Thermo Fisher Scientific). Nuclear and cytoplasmic lysates were prepared by Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime). Equal amounts of protein samples (10 μg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked with 5% nonfat milk in TBST buffer at room temperature for 1–2 h, and analyzed by immunoblotting. Membranes were incubated with specific primary antibodies (for details, see Table S1) overnight at 4 °C and washed three times in TBST buffer before incubation in HRP-conjugated secondary antibody (111-035-003/115-035-003, Jackson Immunoresearch, West Grove, PA, USA). Membranes were washed three times again in TBST buffer before visualization with ECL substrate (1705061, Bio-Rad, Hercules, CA, USA). Images were acquired with the Clinx Chemi Analysis system (Clinx, Shanghai, China) and quantified with Image J software (National Institutes of Health, RRID: SCR_003070).

Protein extracts were prepared from mid-log phase cells. Briefly, mid-log phase cells were washed with ice-cold Phosphate-buffered saline (PBS, pH = 7.4), then scraped the cells and lysed on ice for 30 min in RIPA-lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) supplemented with a complete protease (P1050, Beyotime) and phosphatase (P1051, Beyotime) inhibitor cocktail. Extracts were clarified by centrifugation (12,000r for 20 min at 4 °C) and were stored at − 80 °C until analysis. The BCA assay kit (P0011, Beyotime) was used to determine the protein concentration in each sample according to the manufacturer's instructions.

Lung tissues and RAW264.7 cells were lysed in RIPA lysis buffer (P0013B,Beyotime) supplemented with protease inhibitor (ST505, Beyotime) and phosphatase inhibitor (P1050, Beyotime), and the protein concentration in each sample was determined using the BCA assay. Samples were denatured, fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The non-specific membrane binding sites were blocked with 5% bovine serum albumin (BSA) for 1 h. Membranes were then incubated at 4°C overnight with primary antibodies (all diluted 1:1000) against the following proteins: IL-17 (ab79056, Abcam), MCP-1 (catalog no. 2029, CST),p38 MAPK (catalog no. 9212, CST), and phosphorylated (p)-p38 MAPK (catalog no. 4511, CST). Blots were washed several times with PBS buffer, then incubated for 1 h with goat anti-rabbit IgG secondary antibody (diluted 1:15000; ab96899, Abcam). The bands were visualized using a fluorescent scanner. As an internal reference, GAPDH was immunostained using an antibody (1:1000; catalog no. 5174, CST).

Mock- and MO-infected MH-S cells plated on 12-well plates were collected at 24 hpi and lysed on ice with a lysis buffer (Beyotime, P0013) containing protease and phosphatase inhibitors (Beyotime, P1050) for 30 min. After centrifugation at 12,000 r/min for 5 min at 4°C, the supernatants were collected for protein quantification with a bicinchoninic acid (BCA) assay kit (Beyotime, P0012), and then mixed with appropriate amounts of SDS-PAGE loading buffer prior to boiling for 10 min. Samples were then subject to Western blot analysis as previously described [36 (link)], exploiting p53, caspase-8, caspase-12, Bax, Bcl-2, and β-actin specific primary antibodies. β-actin was regarded as a loading control. Grayscale value analyzed with ImageJ software was calculated for semi-quantisation of the target proteins.

For Co-IP assay, cells were collected 48 h after transfection and lysed in RIPA Lysis Buffer supplemented with protease/phosphates inhibitors (P1050, Beyotime Biotechnology, Shanghai, China). After centrifugation for 15 min at 14,000×g, supernatants were collected and incubated with the indicated antibodies followed by the addition of protein A/G beads (sc-2003, Santa Cruz, America). After incubation overnight at 4 °C, beads were washed four times with lysis buffer. Immunoprecipitated proteins were eluted by boiling with 4×SDS loading buffer.