Ab210702

Immunoblots were conducted on hippocampal protein extracts according to our previously described procedure^{28 (link),35 (link)}. The primary antibodies used included: Esd (1:10,000; ab133631, Abcam), Idh2 (1:1,000; ab131263, Abcam), Dld (1:10,000; ab133551, Abcam), Sdha (1:1,000; ab137040, Abcam), G6pdx (1:1,000; ab210702, Abcam), Dlat (1:1,000; ab172617, Abcam), Pkm (1:1,000; C103A3, CST), Aldh2 (1:1,000; ab108306, Abcam), Glo1 (1:1,000; ab137098, Abcam), Ogdhl (1:500; 17110–1-AP, Proteintech), Anxal (1:2,000; ab214486, Abcam), Por (1:10,000; ab180597, Abcam), Prdx6 (1:1,000; ab133348, Abcam), Prnp (1:5,000; ab52604, Abcam), and Tpp2 (1:1,000; ab180177, Abcam). Anti-mouse or anti-rabbit horseradish peroxidase-conjugated IgG (1:10,000; Bio-Rad) was used as a secondary antibody. After gel electrophoresis and immunodetection, the protein band intensities were analyzed using Quantity One (Bio-Rad) software. Non-target protein bands in Coomassie blue-stained gels before being transferred to a PVDF membrane were used as the loading control^{28 (link),36 (link)}.

Cells were lysed using the RIPA buffer. The BCA method was used to detect the thrombospondin-1 (THBS1) protein concentration. Next, 25 μg of protein was loaded onto 4–20% Express PLUSTMPAGE gels (GenScript, USA) for separation, followed by transfer onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with a primary antibody overnight. Subsequently, the membranes were incubated with an HRP-conjugated secondary antibody (1:5000). The proteins were detected using the EZ-ECL kit (Biological Industries, Israel). The following antibodies were obtained from Abcam (Cambridge, UK): anti-SDC1 (ab128936), anti-SDC4 (ab74139), anti-vWF (ab134193), anti-THBS1 (ab1823), and anti-PKC-A (ab32326), anti-uPA (ab3218106), anti-VEGFA (ab1316), anti-EGFR (ab52894), anti-G6PD (ab210702), anti-GAPDH (ab8245).

MM cells were harvested, washed, and lysed using RIPA lysis buffer (#FMS-WB035, Fcmacs Biotech Co., Ltd., Nanjing, China). Protein concentration were detected by the BCA protein assay kit (#P0011, Beyotime Biotechnology, Shanghai, China). Total protein samples (20–40 μg) were heated in SDS/β-mercaptoethanol buffer and loaded on 10–15% SDS-gels. The proteins were resolved using SDS-PAGE, and then transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk at room temperature for 2 h and incubated with primary antibodies against G6PD (1:1,000 dilution, #ab210702, Abcam, Shanghai, China), β-catenin (1:1 000 dilution, #51,067-2-AP, Proteintech Group, Wuhan, China) and β-actin (1:1000 dilution, #4970S, Cell Signaling Technology, Mass, USA) overnight at 4 °C. Blots were incubated with secondary antibodies using horseradish peroxidase-conjugated rabbit anti-mouse (1:10,000 dilution, #S0002, Affinity, OH, USA) or goat anti-rabbit IgG (1:10,000 dilution, #FMS-Rb01, Fcmacs, Nanjing, China) at room temperature for 1 h. Finally, blots were developed using a chemiluminescence ECL kit (#180-5001, Tanon, Shanghai, China).