Anti acsl4 antibody

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.

Immunohistochemistry assays were carried out to detect ACSL4 expression patterns in HCC tissues and paired normal tissues according to the manufacture protocol. Briefly, after dewaxing, antigen repair, and serum sealing with 5% goat serum (Solarbio, Beijing, China), the 4-μm sections were incubated with anti-ACSL4 antibody (1: 100 dilution, no. ab110007, Abcam, MA, USA), anti-O-GlcNAc antibody (1:200 dilution; no. MA1-072, Thermo Fisher Scientific, MA, USA) or anti-GLUT1 (GLUT) antibody (1:200 dilution; no. PA5-16793, Thermo Fisher Technology) at 4 °C overnight. The next day, the sections were incubated with the corresponding second antibody (Zhongshanjinqiao, Beijing, China) at room temperature for 1 hour. The staining results were evaluated by using a microscope, and the immunohistochemical scores were calculated by two researchers who were blinded to the grouping information. For staining extent, positive cells <5% were scored as 0, positive cells 5%-25% were scored as score 1, positive cells 25%-50% were scored as score 2, and positive cells >75% were scored as score 3. For staining intensity, 0, 1, 2 and 3 represent no, weak, intermediate and strong staining, respectively. The staining extent score and intensity score were multiplied to obtain the final score.

Animal tumor tissues were sectioned and stained with a 1:1000 dilution of anti-ACSL4 antibody (Abcam), 1:1000 dilution of anti-A20 antibody (Abcam), 1:500 dilution of anti-CD34 antibody (Abcam). Five regions were selected randomly for each specimen.