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Fig. 4 | Cell & Bioscience

Fig. 4

From: p52 signaling promotes cellular senescence

Fig. 4

Extracellular CYPA and S100A4 induce p52. a Immunoblot (IB) with anti-p52 and anti-GAPDH using lysate from WI-38 cells incubated in regular media (RM) or conditioned media (CM) pre-treated with proteinase K (PK). b Schematic of CM fractions partitioned based on molecular weight. c IB with anti-p52 using lysate from MEFs incubated for 24 h in either whole RM (Whole) or the specific CM fraction indicated. d IB using the 10–30 kDa fraction isolated from RM or CM probed with the indicated antibody. e IB using lysate from WI-38 cells at D0 and D7 following prolonged culture, probed with indicated antibodies. f IB probed with the indicated antibody using lysate from MEFs 24 h following treatment with recombinant CYPA (10 ng/mL) and/or S100A4 (50 ng/mL). g IB probed with indicated antibodies using the 10–30 kDa fraction of CM following immunodepletion (ID) with anti-S100A4 (upper) or anti-CYPA (lower); ID with anti-IgG used as control. h IB using lysate from WI-38 cells incubated for 24 h with RM or CM that has undergone ID with the indicated antibody. IB probed with indicated antibodies. i Quantification of β-gal positive WI-38 cells 3 days following treatment with CYPA (10 ng/mL) and S100A4 (50 ng/mL). Data represent mean value of three biological samples ± SEM. *P < 0.05 (two-tailed t test). Blots are representative of at least two biologically independent experiments. Analysis of fold-change normalized to control lane shown below IB where indicated

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