Western Blot Analysis of IP6K2 in Glioma

We conducted the Western blot assay according to the previous study [29 (link)]. Brifely, treated glioma cells were collected and lysed using a lysis buffer containing the protease inhibitor PMSF (Beyotime, Nantong, China), according to the BCA protein quantification kit (Beyotime, Nantong, China) to measure the protein concentrations. SDS-PAGE protein loading buffer was added, and the mixture was heated at 100°C to denature the protein. After loading and running, the separated proteins were transferred to a PVDF membrane, which was then cut according to the molecular weight. Antigen blocking was performed with 5% skimmed milk powder blocking solution. Antibodies against IP6K2 (ab179921; 1/1000) and GAPDH (ab8245; 1/1000) were purchased from Abcam (Cambridge, MA, USA). HRP-conjugated secondary goat anti-mouse and goat anti-rabbit antibodies (Proteintech, USA) were then added to the membrane and incubated. The membranes were then immersed in ECL Plus (Millipore, USA) at room temperature. Protein expression levels were detected using a Bio-Imaging System (Bio-Rad, USA).