Legumain

The detailed procedures of immunohistochemical and immunofluorescence analyses have been described previously34 (link). Protein expression was detected in OCI-Ly3 xenograft tissue sections with antibodies against F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA), CD206 (eBioscience, San Diego, CA), CD31 (BD Biosciences), collagen I (Abcam), and legumain (Santa Cruz Biotechnology). Specimens from DLBCL patients were fixed and immunohistochemically stained for CD163 (BD Biosciences). Images were captured by an IX51 research microscope. (Olympus)

Kidney tissue samples and cell pellets were homogenized in RIPA buffer that contains protease and phosphatase inhibitor cocktails (Sigma‐Aldrich). The protein concentration was determined by the Bradford Assay (Thermo Fisher Scientific). Equal amounts of proteins were separated on a 10%–15% SDS‐PAGE gel and were then transferred to a PVDF membrane (EMD Millipore, Billerica, MA). After blocking with 5% dry skim milk, the membranes were incubated overnight at 4°C with primary antibodies. The following primary antibodies were used: legumain, fibronectin, α‐SMA, CTGF, PAI‐1, p21, LAMP1, and β‐actin (Santa Cruz Biotechnology, Dallas, Texas, USA); collagen I, TGFβ1, ATP6V0D1, ATP6V1G1, p62/SQSTM1, prohibitin, and Pink1 (Abcam, Cambridge, MA).
p16^Ink4a, Tomm20, and Parkin (Proteintech, Rosemont, IL); LC3 (MBL Life Science, Tokyo, Japan), and TFEB (Cell Signaling Technology, Danvers, MA). After washing with TBST buffer three times, the membranes were probed with respective HRP‐conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature and then visualized using an ECL kit (EMD Millipore).