Anti gls

Total proteins were extracted through a conventional method using RIPA lysis buffer (Beyotime Biotechnology, China) with protease inhibitor cocktail (Beyotime Biotechnology, China) at 4 °C. Protein concentrations were determined by BCA protein assay (Beyotime Biotechnology, China), and equal amounts of total proteins were separated by 10% SDS-PAGE (Thermo Fisher Scientific, USA), and then transferred onto PVDF membranes (Millipore, USA). Next, the membranes were blocked with 5% nonfat milk in 0.1% Tween washing buffer for 1 h at room temperature, and then incubated with primary antibodies including anti-GLS (Abcam, USA), anti-GRIA3 (Abcam, USA), anti-GRIN1 (Novus, USA), anti-EAAT1 (Abcam, USA) and anti-GAPDH (Abcam, USA) overnight at 4 °C. On the next day, the membranes were incubated with the conjugated secondary antibody (Invitrogen, USA) for 1 h at room temperature and the bands were visualized using ChemiDoc Touch Gel Imaging System (Bio-Rad, USA). The blots were quantified by densitometry using ImageJ software (NIH).

After the treatments indicated, the cells were harvested and the proteins extracted with lysis buffer (1.5 mmol/L EDTA, 50 mmol/L Hepes pH 7.4, 150 mmol/L NaCl, 10% (v/v) glycerol, and 1% (v/v) NP40) containing proteases and phosphatase inhibitors (Thermofisher scientific). The lysates were centrifuged at 12, 000 g for 20 minutes at 4°C and the supernatants were collected. Equal amounts of proteins (40 µg) were separated onto a 4% to 20% SDS-PAGE gel (Bio-Rad), transferred onto a nitrocellulose membrane (Protran, Whatman) at 70 V, 35 minutes at 4°C and blocked with 5% w/v skimmed milk or BSA for 1 hour. The antibodies used in immunoblotting were as follows: homemade anti-UCP2 (UCP2–605) (29 (link)), anti-Notch1^ICD valine1744 (Cell signaling, 4147); anti-cMyc (Cell Signaling, 5605); anti-HK2 (Cell signaling, 2867); anti-PDH (Cell signaling, 2784); anti-GLS (Abcam, ab93434); anti-GS (BD, 610517); anti-CS (Abcam, ab85669); anti-GADPH (Cell Signaling, 2118); anti-ASNS (proteintech, 14681-1-AP) and anti-ß-Actin (Sigma-Aldrich, A5441), anti-α-Tubulin (Sigma-Aldrich, T5168). A direct recording of the chemiluminescence and a quantification (Fusion^® software) were carried out. Western blot analyzes were performed using independent samples.