Anti pfkp

Expression of the enzymes involved in glucose metabolism and in cytosolic and ER PPP was determined by Western blot, using standard procedures. Anterior and posterior brain regions were homogenized in PBS in the presence of a protease inhibitor cocktail for mammalian cells, and total protein was measured by Bradford assay. After SDS-PAGE, performed according to the standard method on 10% precast gels (BioRad), proteins were transferred to a nitrocellulose membrane. The membrane was blocked for 1 h with Tris Buffered Saline (TBS) plus 0.15% Tween 20 (TBSt) containing 5% nonfat dry milk and incubated overnight at 4 °C with the following rabbit polyclonal antibodies: anti-HK II (1:1000, Cell Signalling #2867), anti-G6Pase (1:1000, Abcam, ab93857), anti-PFKP (1:200, Cell Signalling #8164), anti-G6PD (1:1000, Abcam, ab210702), anti_H6PD (1:1000, Abcam, ab170895), or anti-actin (1:10,000, Thermo-Fisher MA5-11869). After washing with TBSt, the membrane was incubated with an anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) (BioRad) and developed with Clarity Western ECL Substrate (BioRad). Bands were detected and analyzed for density using an enhanced chemiluminescence system (Alliance 6.7 WL 20 M, UVITEC, Cambridge, UK) and UV1D software (UVITEC). Bands of interest were normalized for actin levels in the same membrane.

Western blot was performed as previously described (Lam et al., 2020 (link)). Specific primary antibodies [mouse monoclonal anti-human β-actin (Sigma-Aldrich); anti-PCNA, anti-PARP (Santa Cruz Biotechnology, Inc., Santa Cruz, California, United States of America); anti-PFKP, anti-LDHB, anti-Bcl-2, anti-survivin, anti-XIAP, anti-AKT, anti-CDK2, anti-CDK4, anti-CDK7, anti-Cyclin D2, H, anti-CDK4, anti-N-cadherin, anti-β-catenin (Cell Signaling Technology, Danvers, Massachusetts, United States of America); and corresponding horseradish peroxidase (HRP)-conjugated secondary (Cell Signaling Technology)] were purchased. An enhanced chemiluminescence (ECL) kit (GE Healthcare) was used to detect protein expression. Beta-actin was selected as a reference protein (Lam et al., 2020 (link)).

Recombinant human TNF-α, IL-1β, and IL-17α were purchased from R&D Systems (Bio-Techne), and LPS was obtained from MilliporeSigma. DMEM, FBS, antibiotics, trypsin EDTA, PBS, and other products for cell culture were purchased from Invitrogen, Thermo Fisher Scientific. The primary antibodies used were as follows: anti-SAE1 (ab38434, Abcam), anti-UBA2 (ab189289, Abcam), anti–SUMO-1 (ab11672, Abcam), and anti-STAT5A (ab32043, Abcam) were purchased from Abcam. Anti-HK2 (2867, Cell Signaling Technology), anti-PFKP (8164, Cell Signaling Technology), anti-PKM2 (4053, Cell Signaling Technology), and anti–p-PKM2 (3827, Cell Signaling Technology) were purchased from Cell Signaling Technology. Anti–β-actin (A5441) was purchased from MilliporeSigma. GA (15:1) was purchased from MilliporeSigma. SKN was purchased from Selleckchem.