A6235

Cells were lysed with ice-cold RIPA buffer and centrifuged at 13,000 g for 15 min at 4 °C. 20 ug of protein lysate (concentration determined by bicinchoninic acid assay) was run on a 10% SDS-PAGE gel at 120 V for 1.5 h and then transferred to nitrocellulose filter membranes (Millipore, Bedford, MA). After blocking with 5% BSA at room temperature (RT) for 1 h, the membranes were incubated with the following primary antibodies: α-SMA (#19245, 1:1000, Cell Signaling Technology, Danvers, MA), fibronectin (ab2413, 1:1000, Abcam, Cambridge, UK), arginase-1 (Arg1) (16001-1-AP, 1: 5000, Proteintech, Wuhan, China), ornithine δ-aminotransferase (OAT) (A6235, 1:1000, ABclonal Technology, Wuhan, China), PYCR1 (13108-1-AP, 1:4000, Proteintech), proline dehydrogenase (oxidase) 1 (PRODH) (22980-1-AP, 1:2000, Proteintech) and β-actin (20536-1-AP, 1:5000, Proteintech) at 4 °C overnight. The next day, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG(H + L) (SA00001-2, 1:10000, Proteintech) for 1 h at room temperature and then washed three times for 10 min with tris-buffered saline containing 0.1% Tween 20. Quantitative analysis was performed on the immunoreactive bands using ImageJ software.

Samples were fixed with 4% paraformaldehyde overnight, then embedded in paraffin. 5 mm sections were processed and stained with hematoxylin and eosin (H&E) and Masson’s Trichrome Stain Kit following protocol (Solarbio, Beijing, China).
For immunohistochemistry and immunofluorescence, 5 mm sections were incubated with primary antibodies against Piezo1 (ab128245, 1:1000, Abcam), α-SMA (ab7817, 1:200, Abcam), Collagen I (ab6308, 1:200, Abcam), GFP (ab290, 1:500, Abcam), Arg1 (16001-1-AP, 1:400, Proteintech), OAT (A6235, 1:100, ABclonal Technology), PYCR1 (13108-1-AP, 1:200, Proteintech), PRODH (22980-1-AP, 1:400, Proteintech) overnight at 4 °C. Sections were then incubated with Alexa Fluor 594 goat anti-rabbit secondary antibody (125369, 1:200, Jackson lab), and Alexa Fluor 488 goat anti-mouse secondary antibody (133384, 1:200, Jackson lab) for immunofluorescence or an HRP-conjugated goat anti-rabbit secondary antibody (138729, 1:500, Jackson lab) for immunohistochemistry. Images were captured using a Nikon Eclipse E800 microscope (Nikon, Melville, NY, USA).