Western ip lysis buffer

The IP assay was performed as previously described [48 (link)] to assess the interactions between proteins. In detail, Huh-7 cells were collected and lysed in Western/IP lysis buffer (Beyotime, Jiangsu, China) according to the manufacturer’s description. After preclearing with 50 μl of protein A/G-Sepharose (Novex, Oslo, Norway) for 1 hour, the supernatants were incubated overnight at 4 °C with anti-O-GlcNAc (no. 9875, Cell Signaling Technology), anti-ACSL4 antibody (no. ab110007, Abcam) or IgG for crosslinking and then incubated with protein A/G-Sepharose beads and washed with the Western/IP lysis buffer (Beyotime). Then, the beads were subjected to western blotting assay with the indicated antibodies.

Glomerular endothelial cells (GEnCs) lysates were prepared using Western-IP lysis buffer (Beyotime, Beijing, China) supplemented with proteinase inhibitor cocktail (Roche, Basel, Switzerland). Total protein extracts were subjected to targeting proteins separating, transferred to a PVDF membrane, and blotted with antibodies against targeting proteins (collagen I, collagen III, fibronectin, CD31, α-SMA, VE-cadherin, vimentin, NOX-4, TGF-β1, p-Smad2, t-Smad2, p-Smad3, t-Smad3, GAPDH). The secondary antibody was a goat HRP-conjugated secondary antibody (1:20,000 dilution). The images were captured using a ChemiDoc XRS (Bio-Rad Laboratories, CA, United States) with the SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., IL, United States) and further analyzed with Gel-Pro analyzer (Ye et al., 2016 (link)).

According to the manufacturer’s instructions, proteins were extracted from cells or tissues using Western-IP Lysis Buffer (Beyotime, Shanghai, China). The protein concentration was determined using a bicinchoninic acid (BCA) protein concentration determination assay kit (Beyotime, Shanghai, China). Prepared samples were electrophoresed in a 10% SDS-PAGE gel and blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) using the Tetra Handcast system (Bio-Rad, USA). The membranes were blocked for 3 h at room temperature and incubated overnight at 4°C with an appropriate primary antibody in Tris-buffered saline with 0.05% Tween (TBST) containing 5% non-fat milk. After washing in TBST, the membranes were incubated with secondary antibody, washed and visualized using a supersensitive enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s protocol. The protein bands were detected and quantified using the Gene Gnome Syngene Bio Imaging System (SYNGENE, UK).
Primary antibodies used for Western blotting included a mouse monoclonal anti-SK4 antibody (1:100; Alomone Labs, Israel), a rabbit polyclonal ER antibody (1:1,000; a gift from Dr. Yibing Hu), a rabbit monoclonal anti-EMT antibody sampler kit (1:1,000; Cell Signaling Technology, USA) and a mouse monoclonal anti-tubulin antibody (1:500; abcam, USA).