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4 protocols using ab72543

1

Aqueous Humor-Derived Cell Characterization

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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).
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2

Western Blot for Pluripotency Factors

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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).
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3

Western Blot Analysis of Oxidative Stress Markers

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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).
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Western Blot Analysis of KLF4 in Mice

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Adult mice of indicated genotypes were killed, and retinas were dissected and lysed with lysis buffer (Cell Signaling Technology) containing 0.5 mm phenylmethylsulfonyl fluoride (Sigma-Aldrich). Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific). Samples (25 μg) were separated by SDS-PAGE in 4-20% gradient Tris-glycine precast gels (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated for 1 h in blocking solution containing 5% nonfat milk powder and 0.1% Tween-20, pH 7.6. This was followed by overnight incubation at 4°C in the blocking buffer containing rabbit primary antibodies against KLF4 (1:50; Ab72543, Abcam). Subsequently, the labeled proteins were visualized by incubation with a horseradish peroxidase (HRP)-conjugated anti-goat or rabbit IgG (1:2000; Santa Cruz Biotechnology) followed by development with a chemiluminescence substrate for HRP (Thermo Fisher Scientific). The images of Western blots were captured by GE ImageQuant. Relative band intensities were analyzed using ImageJ software and normalized to GAPDH.
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