Senescence-Associated p21 and p53 Expression

Total protein was extracted from young (P3) and senescent (P16) RPESCs (1 x 10⁶ cells/sample) as described previously [16 (link)], with some modifications. Membranes were incubated overnight with the primary p21 and p53 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using GAPDH as the endogenous control, followed by incubation with the secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology). Protein detection on the membrane was performed using the Clarity Western ECL Substrate Kit (Bio-Rad). The signals were captured with an Alliance Mini (UVITEC Cambridge, Cambridge, UK) system; the p21 and p53 bands were quantified with UVITEC software and their intensity was normalized by comparison to the housekeeping protein β-actin, used as a loading control. The intensity of each band was compared to the negative controls and any change was expressed as a percentage.

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Lazzarini R., Nicolai M., Pirani V., Mariotti C, & Di Primio R. (2018). Effects of senescent secretory phenotype acquisition on human retinal pigment epithelial stem cells. Aging (Albany NY), 10(11), 3173-3184.

Publication 2018

secondary antibody conjugated to horseradish peroxidase Clarity Western ECL Substrate Kit

Actin Antibody Cells Gapdh Horseradish peroxidase Membrane protein Membranes Protein

Corresponding Organization :

Other organizations : Marche Polytechnic University

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Variable analysis

independent variables

Age of the RPESCs (young vs. senescent)

dependent variables

Protein expression levels of p21 and p53

control variables

Total protein extracted from 1 x 10^6 cells per sample
GAPDH used as endogenous control
β-actin used as loading control

controls

Negative controls were used for comparison of p21 and p53 band intensity

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