Ab72543

Saponin detergent was from Sigma Life Sciences (St. Louis, MO; http://www.sigmaaldrich.com/life-science.html?) and Live/Dead Viability/Cytotoxicity Kit and fluorescent-labeled zymosan particles for phagocytosis assay were from Molecular Probes/Invitrogen, (Eugene, OR; http://www.lifetechnologies.com/us/en/home/brands/molecular-probes.html). Aqueous humor was collected from fresh postmortem porcine eyes by inserting a 27 gauge needle through the cornea into the anterior chamber and slowly removing 100–150 µl/eye. This was stored at −20°C and centrifuged at 15,000g for 10 minutes before use. Antibodies used were: CD44 (352-020, Ancell (Bayport, MN; http://www.ancell.com/) and ab65829, Abcam; Cambridge, UK; http://www.abcam.com/); CHI3L1 (ab88847; Abcam); α3 integrin (NBP1-19724, Novus Biologicals; Littleton, CO; http://www.novusbio.com/); KLF4 (ab72543, Abcam); LAMP1 (ab25630, Abcam); Wnt1 (ab15251, Abcam); AQP1 (sc-20810, Santa Cruz; Santa Cruz, CA; http://www.scbt.com/); NANOG (sc-33759, Santa Cruz); OCT3/4 (sc-5279, Santa Cruz); SOX2 (sc-20088, Santa Cruz); and α-tubulin (04-1117, Millipore; Darmstadt, Germany; http://www.emdmillipore.com).

Radioimmunoprecipitation assay (RIPA) buffer (CW2333; Cwbiotech, China) supplemented with a protease inhibitor cocktail (PI003; BOCAI Technology, China) and phenylmethylsulfonyl fluoride (PMSF; Dingguo Changshen Biotech, China) was used to isolate cellular proteins. Equivalent amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with specific primary antibodies against Oct4 (2840; Cell Signaling Technology, USA), Glis1 (SAB2700289; Sigma, USA), Klf4 (ab72543; Abcam, UK), Sox2 (3579; Cell Signaling Technology, USA), L-Myc (sc-790; Santa Cruz Biotechnology, USA), and GAPDH (KC-5G4; KangChen Biotech, China), followed by horseradish peroxidase-conjugated secondary antibodies: goat anti-Rabbit IgG (ZB-2301; ZsBio, China) and anti-mouse IgG-HRP (IH-0031; Dingguo Changshen Biotech, China). Bands were visualized using enhanced chemiluminescence (ECL) (34087; Thermo, USA).

Homogenates of the rat cerebral cortex and hippocampus or HT-22 cell lysates were prepared for Western-Blot analysis. Proteins were incubated overnight at 4 °C with a primary antibody against HCP1 (1:1000, ab25134, Abcam, USA), ALAS1 (1:1000, AB21560, Sangon, China), HO-1 (1:500, ab13248 Abcam, USA), KLF4 (1:250, ab72543, Abcam, USA), and GAPDH (1: 2000, Cell signal technology, USA). The blots were developed by incubation in ECL chemiluminescence reagent (Amersham Life Science, Arlington Heights, IL, USA) and subsequently exposed to BioMax Light Film (Eastman Kodak Co., USA).