Ab178695

Protein expression was measured using Western blot analysis as previously described [18 (link)]. Briefly, proteins were extracted by homogenizing thoracic aorta tissues in 500 µL 1× RIPA buffer enriched with protease inhibitors (1 µg/mL phenylmethylsulfonyl fluoride and 1 µg/mL leupeptin). Protein separation was performed by SDS-PAGE (10% TRIS-HCl–SDS gels) using a Mini-PROTEAN Handcast system (BioRad, Hercules, CA, USA). The proteins were resolved and transferred onto a nitrocellulose membrane (0.45 μm, Millipore, Billerica, MA, USA) using standard procedures. The membranes were incubated for 1 h with blocking buffer and then overnight with primary antibodies against glucose-6-phosphate dehydrogenase (G6PD) (Cell Signaling Technology 12,263, Danvers, MA, USA, 1:1000), cysteine- and glycine-rich protein 2 (CSRP2) (Abcam ab178695, Cambridge, UK; 1:1000), tubulin alpha-4 A (TUBA4A) (Sangon D110022, Shanghai, China, 1:1000) and β-Actin (Santa Cruz Biotechnology sc1616, Santa Cruz, CA, 1:1000) at 4 °C overnight. Finally, the membranes were probed with secondary antibody (1:5000) at room temperature for 1 h. The membranes were washed three times, and then the protein band intensities were determined using the Odyssey® Imager system (LI-COR, Lincoln, NE, USA).