Ascl1

The antibodies used were: ASCL1 (Abcam, #ab211327), INSM1 (Abcam, #ab170876), E2F7 (Abcam, ab245655), RB1 (Abcam, ab181616), SYP (Santa Cruz, #sc-17750), RCOR1 (Santa Cruz, #sc-376567), CCN1 (Santa Cruz, #sc-374129), CCN2 (Santa Cruz, #sc-365970), YAP/TAZ (Santa Cruz, #sc-101199), P53 (Santa Cruz, #sc-126), E2F1 (Santa Cruz, #sc-251), GAPDH (Santa Cruz, #sc-47724), LATS1 (Cell signaling, #3477), LATS2 (Cell signaling, #5888), p-LATS (Cell signaling, #8654), H3 (Cell signaling, #4499), H3K27ac (Cell signaling, #8173), H3K4me3 (Cell signaling, #9751), VIN (Cell signaling, #13901), Flag (Sigma, #1804),

Tissues (male hippocampus) were homogenized in RIPA buffer (ATTO, Tokyo, Japan) with proteinase and phosphatase inhibitors (ATTO, Tokyo, Japan). Antibodies against β-actin (Cell Signaling, Danvers, MA USA), FOXO3 (Cell Signaling, Danvers, MA, USA), ASCL1 (Abcam, Cambridge, UK), and TFAM (Millipore, Burlington, MA, USA) were used as primary antibodies. After conventional Western blotting, bands were visualized using the Pierce^TM ECL western blotting substrate (Thermo Scientific, Waltham, MA, USA). Protein expression levels were calculated using ImageJ software (NIH, Bethesda, MD, USA).

Lysates of kidney tissue or HK-2 cells were prepared. All the antibodies in western blot were purchased from Cell Signaling Technology Inc. (Beverly, MA) and the dilute concentration is based on antibody instruction. After centrifugation, the protein level was determined in supernatants using a Micro BCA protein assay kit with BSA as a standard (Pierce, Thermo). In brief, 30 μg protein was used for electrophoresis on SDS-PAGE and transferred to a nitrocellulose fibrous membrane. The blots were blocked in 5% nonfat dry milk for 1 h, followed by overnight incubation at 4 °C with the following primary antibodies, respectively, Nrf2: (rabbit anti-human, 1:1000 Abcam, USA), ASCL1 (rabbit anti-Human, 1:1000; Abcam). Rabbit anti-human β-actin-specific antibody (1:1 000; Abcam, Cambridge, UK) was used for loading controls on stripped membranes. After being washed with TBS, blots were incubated with an HRP-conjugated secondary antibody (goat anti-rabbit, 1:1 000 Vectastain Elite; Vector Laboratories, Peterborough, UK) at room temperature for 1 h, and then enhanced chemiluminescence (Thermo, Rockford, IL) was used to visualize the bands.