Ab176560

Cell lines or tumor tissues were harvested and lysed in RIPA buffer (1% NP 40, 0.5% sodium deoxycholate, 0.1% SDS, 10 ng/ml PMSF, 0.03% aprotinin, and 1 μM sodium orthovanadate) on ice for 30 min. After centrifugation for 15 min at 14,000 rpm, the supernatants were collected, and the protein concentration was quantified by using Bradford assay. The proteins were separated on 12% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and incubated with primary antibodies, such as MLX (12042-1-AP, Proteintech), β-actin (81115-1-RR, Proteintech), TRC/CD71 (66180-1-Ig, Proteintech), SLC40A1/FPN1 (26601-1-AP, Proteintech), Transferrin (66171-1-Ig, Proteintech), FTH1 (3998S, Cell Signaling Technology), cleaved Capase-3 (9661S, Cell Signaling Technology), SLC7A11(12691S, Cell Signaling Technology), Tubulin (ab176560, Abcam) and 4-Hydroxynonenal (ab46545, Abcam), Caspase-3 (ab184787, Abcam), and corresponding horseradish peroxidase-conjugated secondary antibodies were used against each primary antibody. Proteins were detected and filmed by using chemiluminescent detection reagents.

To analyze the macrophage phenotypic shape markers arginase-1 and iNOS, the cells cultured on each nanofiber membrane sample were separated by 0.25 % trypsin and transferred to the lysis buffer containing 1 % benzenesulfonyl fluoride; after 30 min of complete lysis at 4 °C, protein samples were collected by centrifugation at 25,000 rpm for 15 min, and the total protein concentration was measured by commercial kits. For Western blotting, the main antibodies used were anti-arginase-1 (1:1000, PA5-85267, Gibco), anti iNOS (1:1000, PA3-030a, Gibco), and anti-α-tubulin (1:1000, ab176560, Abcam), the subsequent procedures were performed as described previously [26 (link)–28 (link)].

Whole cell extracts (40-50 µg) from cells or tissues were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (PVDF; Bio-Rad, Hercules, CA, USA). The membranes were blocked and then probed with antibodies against TWIST1 (ab50887) and anti-alpha tubulin (ab176560) as a loading control (Abcam, Cambridge, MA, USA). After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies.

The transfected J82 and T24 cells were lysed using protein inhibitor modified RIPA lysate (Thermo Fisher) on ice, and the total cell protein was extracted. After accurate loading, electrophoresis separation was performed on a 10% SDS-PAGE gel, and then transferred to a superior PVDF membrane. The PVDF membrane was blocked with skim milk (BD) for 2 h. The following primary antibodies were added and incubated overnight at 4 °C, anti-SHMT2(1:1,000, ab224428; Abcam, Cambridge, UK), anti-Tubulin(1:10,000, ab176560; Abcam, Cambridge, UK), anti-MMP7(1:1,000, ab205525; Abcam, Cambridge, UK), anti-Integrin β-4(1:1,000, ab182120; Abcam, Cambridge, UK), anti-Laminin β-4(1:500, Sc-130540; Santa Cruz, CA, USA), anti-SHMT1(1:800, Cat.14149–1-AP; Proteintech, Wuhan, China), anti-E-cadherin (1:2,000, Cat.60335–1-Ig; Proteintech, Wuhan, China), anti-N-cadherin (1:2,000, Cat.66219–1-Ig; Proteintech, Wuhan, China), anti-MMP2 (1:2,000, Cat.66366–1-Ig; Proteintech, Wuhan, China), anti-GAPDH (1:10,000, Cat.60004–1-Ig; Proteintech, Wuhan, China). The membrane was washed the next day and secondary antibodies were added and incubated for 30 min. After washing, the membrane was treated with ECL chemiluminescence reagent for 1 min and finally imaged using a fluorescence imaging system. The gray value of protein bands was analyzed using Image J software and the experimental data were recorded.

Total protein of cells and heart tissues were extracted and quantified with BCA Protein Assay kit (Beyotime, Shanghai, China), separated by SDS-PAGE, and then transferred to a PVDF membrane. Each membrane was blocked for 1.5 h at room temperature in 5% non-fat milk, followed by incubation overnight at 4°C with the primary antibodies against NLRP3 (1:1000, abcam, ab263899, United Kingdom), apoptosis associated speck like protein containing a CARD (ASC) (1:500, abcam, ab180799, United Kingdom), Caspase-1 (1:1000, Immunoway,YT5743, United States), cleaved-Caspase-1 (1:1000, abcam, ab179515 United States), YAP1 (1:2000, proteintech, 66900-1-Ig, China), phosphor-YAP1 (ser127) (1:1000, cell signaling technology, 13008s, United States), YAP1 (1:2000, proteintech, 66900-1-Ig, China), phosphor-YAP1 (ser127), LATS (1:1000, proteintech, 17049-1-AP, China), phosphor-LATS1 (Thr1079/1041) (1:1000, immunoway, YP1222, United States), and α-tubulin (1:2000, abcam, ab176560, United Kingdom). The membrence then incubated with the horseradish peroxidase (HRP)-labeled goat anti-rabbit and goat anti-mouse secondary antibodies for 1 h at room temperature. And the expression of protiens was visualized using enhanced chemilumi-nescence reagents (biosharp, BL520A, China).