Ab44986

NP tissues from three patients in each group were extracted and immediately placed on ice. The tissues were washed with pre-cooled PBS and homogenized using RIPA buffer (Aspen, China) supplemented with phenylmethanesulfonyl fluoride and protease and phosphatase inhibitors (Aspen, China). Tissue lysates were sonicated on ice, and protein concentrations were determined using a BCA protein assay kit (Sigma). Total protein was separated via SDS-PAGE on 10% gels and subsequently transferred onto PVDF membranes (EMD Millipore Corporation, USA) at 250 mA. After blocking with 5% non-fat dried milk in TBST at room temperature for 1 h, the membranes were incubated with anti-Notch1 (Abcam, ab44986), anti-NICD (Abcam, ab83232), anti-Notch2 (CST, 5732), anti-Notch3 (CST, 5276), anti-Notch4 (CST, 2423), anti-HIF-1α (Novus, NB100–105), anti-HES1 (Abcam, ab49170), and anti-GAPDH (ProteinTech, 60,004–1-Ig) primary antibodies overnight at 4 °C. After washing with TBST three times, the protein bands were incubated using HRP-conjugated secondary antibodies (ProteinTech) at room temperature for 1 h. The protein bands were detected using enhanced chemiluminescence detection reagents.

For immunocytochemistry, the cells were incubated in 200 nM Rapamycin (rap), 400 nM Bafilomycin A1 (baf), 100 mM Trehalose (treh) for 8 h or starved with HBSS for 2 h (1 × wash with HBSS before incubation for 2 h).
The cells were fixed for 3 min with ice cold methanol or for 10 min with 4% paraformaldehyde (PFA). Concentration of the primary and secondary antibodies are described below. The mounting solution was from Molecular Probes.
Dilution of primary antibodies. 1:50: Notch1 ms (ab44986, Abcam)
1:100: Notch1 rb (ab52627, Abcam), NICD (ab8925, Abcam), Hes1 (ab71559, Abcam), Pax6 (ab5790, Abcam), EEA1 (ab70521, Abcam), Dll1(ab76655, Abcam), ATG16L1 (Cell Signalling)
1:200: ATG9 (ab108338, Abcam)
1:300: LC3 (clone 5F10, Nanotools)
1:500: Tbr1 (ab31940, Abcam), Tbr2 (ab23345, Abcam), Nestin (ab6142. Abcam), 3β-tubulin (ab7751, Abcam)
1:800: AP2 (ab52222, Abcam)
The secondary antibodies Alexa 488, 568, 594 or 647 goat anti-mouse or goat anti-rabbit were obtained from Molecular Probes and used at 1:500.
Imaging was conducted with LSM710 Zeiss confocal with 63 × oil-immersion lense. The colocalization was measured using Volocity software for Mander's coefficient.