Anti gs

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.

The procedures were performed according to the previous report (Cao et al., 2018a (link)). Briefly, zebrafish embryos were fixed in 4% PFA at 4°C overnight. They were then dehydrated in 30% sucrose until they sank, embedded in OCT tissue freezing medium, and cryosectioned at 10-μm thickness using a Leica Cryostat Microtome. Slides were subjected to antigen retrieval for 30 min at 98°C, except for those analyzed with anti-Zn5 antibodies. They were then incubated with primary antibodies overnight at 4°C, including anti-Zn5 (1:500, Zebrafish International Resource Center), anti-Pax6 (1:500, Covance, PRB-278P-100), anti-αPKC (1:500, Santa Cruz Biotechnology, sc-208), anti- GS (1:500, BD Biosciences, 610518), anti-GFAP (1:500, ThermoFisher Scientific, PA5-16291), anti-Recoverin (1:500, Millipore, AB5585), and anti-Rho 1D4 (1:500, Abcam, ab5417). Then, the slides were washed with PBST three times and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit antibody (1:500, Invitrogen, A-10042) and Alexa Fluor 568-conjugated donkey anti-mouse antibody (1:500, Invitrogen, A-21202) at room temperature for 2 h. Images were obtained using a confocal microscope (Ism 710; Zeiss).