Anti p21 antibody

Frozen brains were serially sectioned in a coronal plane covering the entire length of the dentate gyrus (bregma point −2.3 to −6.3 mm) according to the coordinate Atlas [35 ] using a cryostat (Cryostat Series HM550 Microm international, A.S. Science Co., Ltd., Walldorf, Germany). p21 immunofluorescence staining was performed to investigate the cell cycle arrest as described previously [6 (link),21 (link)]. Forty micron sections were preserved in a cryoprotective buffer and stored in 24-well plates at 4 °C. Sections were incubated with an anti-p21 antibody (1:100, Santa Cruz Biotechnology, Texas, USA) at 4 °C overnight. Sections were incubated with an Alexa Fluor 488 rabbit anti-mouse IgG secondary antibody (1:300 Invitrogen, San Diego, CA, USA) at room temperature for 60 min and counterstained with propidium iodide (PI) (1:6000, Sigma-Aldrich, St. Louis, MO, USA). A systematic random sampling method was employed to choose every 8th section throughout the length of the dentate gyrus [36 (link)]. Nine sections were used for staining using a free floating method. p21-positive cells within three cells of the inner edge in the upper and lower blade of the DG were counted [10 (link),11 (link),37 (link)]. Sections were viewed at 10 × and 40 × by a Nikon ECLIPSE 80i fluorescence microscope (Melville, NY, USA).

CWR22Rv1 cells were seeded on cover slips in RPMI 1640 media supplemented with 10% FBS under an atmosphere of 5% CO₂ at 37°C overnight. Before the treatment, CWR22Rv1 cells were maintained in RPMI 1640 media with 0.5% FBS. For the observation of p21 and its nuclear localization, the cells were pretreated with Zyflamend (200 μg/mL) for 24 hr. After the treatment, the cells were fixed using 2% paraformaldehyde for 15 min, followed by blocking with 10% goat serum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 hr, and anti-p21 antibody (10 μg/mL) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C. After washing with PBS, coverslips were incubated with secondary antibody (10 μg/mL of Alexa Fluor 488 goat anti-rabbit IgG) for one hour at room temperature. Coverslips were mounted on glass slides with Prolong Gold w/ DAPI Antifade reagent (Invitrogen Corporation, Carlsbad, CA) and analyzed by epifluorescence microscopy. Four dual-channel images were captured from each sample using a 60x objective lens. Image analysis was performed using NIS-Elements software v3.1 (Nikon Instruments, Melville, NY). Mean fluorescence intensity per cell was calculated by the following: [(total p21 fluorescence)/(nuclei count)]. To assess p21 nuclear accumulation, p21 fluorescence was also measured within discrete nuclear regions as defined using a DAPI intensity threshold.

Human fibroblasts at day 10 and 27 (PDL of 10–11 and 21–22) were used for these experiments (S1A Fig). Cells were washed twice with PBS and lysed in lysis buffer containing 50 mM Tris HCl pH 7.5, 30 mM KCl, 5 mM EDTA, 1% NP-40, 1 mM dithiothreitol, and 0.1% sodium dodecyl sulfate (SDS), protease inhibitor (Nacalai Tesque, Kyoto, Japan), and phosphatase inhibitor (Pierce Thermo Fisher Scientific, IL, USA). Twenty μg of protein per lane was subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and then transferred to a polyvinylidene fluoride membrane. The membrane was incubated with primary antibody overnight at 4°C. Antibodies for total p53 and phosphorylated p53 (Ser 15) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-p21 antibody and anti-β-actin antibody were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and Sigma-Aldrich (St Louis, MO, USA), respectively. After incubation with horseradish peroxidase-conjugated secondary antibody for 1 hr at room temperature, signals were visualized with ImmunoStar LD (WAKO, Tokyo, Japan) or Chemi-Lumi One L solution (Nacalai Tesque, Kyoto, Japan).

Proteins from kidney samples were isolated using lysis buffer followed by centrifugation. Western blot analysis of the proteins in mouse kidneys was performed according to standard protocols. Immunoblotting was performed using anti-SIRT1 antibody (1:5000; Abcam, Cambridge, UK), anti-cleaved caspase-3 antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-pro-caspase-3 antibody (1:1000; Zen BioScience, Chengdu, China), anti-PCNA antibody (1:2000; Santa Cruz, Dallas, TX, USA), anti-p53 antibody (1:1000; Santa Cruz, Dallas, TX, USA), anti-acetyl-p53 antibody (K381) (1:1000; Abcam, Cambridge, UK), and anti-p21 antibody (1:2000; Santa Cruz, Dallas, TX, USA). The blots were detected using the Immobilon Western Chemiluminescent HRP Substrate kit (Millipore, Danvers, MA, USA) followed by exposure to Kodak-X-Omat film (Shanghai, China).

Reagents were purchased or obtained from the following sources: anti-myosin 1b antibody (ab194356, Abcam); anti-VCAM-1 antibody (sc-13160, Santa Cruz Technology); anti-λH2AX antibody (sc-517348, Santa Cruz Technology); ICAM-1 antibody (sc-8439, Santa Cruz Technology); anti-p21antibody (sc-6246, Santa Cruz Technology); anti-LC3A/B antibody (#4108S, Cell Signaling Technology); anti-LAMP1 antibody (#9091S, Cell Signaling Technology); anti-p62 antibody (18420-1-AP, Proteintech); anti-LRRK2 antibody (#5559S, Cell Signaling Technology); and anti-tubulin antibody (SAB4500087, Sigma-Aldrich). Duolink® In Situ Detection Reagents Red (DUO92008) was from Sigma. IRDye 800-conjugated affinity purified goat anti-rabbit IgG F(c) was purchased from LI-COR Biosciences (Lincoln, Nebraska, USA); goat anti-mouse IgG (H+L) secondary antibody Alexa Fluor® 680 conjugate, goat anti-mouse IgG (H+L) secondary antibody Alexa Fluor® 488 conjugate, goat anti-rabbit IgG (H+L) secondary Antibody Alexa Fluor® 488 conjugate, and goat anti-rabbit IgG (H+L) secondary antibody Alexa Fluor® 594 conjugate were from Invitrogen/Thermo Fisher Scientific (Waltham, MA, USA). Insulin-transferrin-selenite sodium and dexamethasone were from Sigma (St. Louis, Missouri, USA). All cell culture media and materials were purchased from Gibco/Thermo Fisher Scientific (Waltham, Massachusetts, USA).