Ptm 802

The western blotting assay was performed to detect the expression of the Khib proteins in PC tissues and cell lines. Briefly, cells or tissues were lysed by pre-formulated lysis buffer (RIPA, 1% Protease Inhibitor Cocktail, 3 μM TSA, and 50 mM NAM). The protein concentrations were measured by BCA kit (P0012S; Beyotime Biotechnology, China), and then we analyzed 50μg protein of every sample on 10% SDS-PAGE gels. All blots were visualized using ECL Kit (RM00020P; ABclonal, Wuhan, China) and the intensity of the bands was assessed by Image Lab (Bio-Rad, California, USA). The antibodies used in this study were: pan-lysine 2-hydroxyisobutyrylation antibody (1:1000; PTM-802; PTM Bio, Hangzhou, China), FITC antibody (1:50; AS011; ABclonal, Wuhan, China), β-actin antibody (1:1000; AC026; ABclonal, Wuhan, China). β-actin was used as a loading control and the intensity of the entire bands was assessed with ImageJ2 (National Institute of Mental Health, Bethesda, MD, USA). All experiments were performed three times following the same procedure.

Cells were spread evenly on coverslips and incubated for 24 hours, then fixed in 4% paraformaldehyde at room temperature for 15 min and permeabilized with 0.1% TX-100 for 10 min at room temperature. Then, the cells were blocked with 5% BSA in PBS for 1 hour and incubated overnight at 4°C with pan-Khib antibody (PTM-802; PTM Bio, Hangzhou, China). After washing three times with PBS, cells were incubated with the appropriate secondary antibody for 1 hour at 4°C. Coverslips were stained with DAPI and mounted. Immunofluorescence images were then observed and captured under a fluorescent microscope.

Tryptic peptides dissolved in NETN buffer were incubated overnight with prewashed antibody beads(PTM-802, PTM Bio), followed by washing with NETN buffer and H₂O. 0.1% trifluoroacetic acid was used to elute the bound peptides and then combined and vacuum-dried. Finally, all samples were desalted for LC-MS/MS analysis.