Fig. 3: LTβR modulates p53 and MDMX protein expression.
From: Reduction of lymphotoxin beta receptor induces cellular senescence via the MDMX-p53 pathway
A Relative p53 mRNA levels in A375 cells with LTβR knockdown or LTβR-overexpressing cells determined by real-time PCR. B, C Western blot analysis of p53 and p21 protein levels in A375 cells after 20 μM of MG-132 treatment for 4 h, following siRNA (B) or LTβR plasmid (C) transfection. p53 and p21 relative band intensities were quantified using ImageJ software, and normalized to β-actin. D Western blot analysis of MDMX, p53, MDM2, and p21 protein expression in A375 cells after 12 h or 24 h of siControl or siLTβR transfection. E, F Cycloheximide (CHX, 100 μg/ml) chase assays were conducted to determine MDMX protein stability in LTβR-overexpressing and LTβR knockdown cells for the indicated time and quantified using ImageJ. G–J A375 cells were treated with 80 nM of BTZ for 4 h, and whole cell lysate was subjected to immunoprecipitation to confirm MDMX ubiquitination and p53 ubiquitination. Relative expression levels of ubiquitinated MDMX and p53 were measured using ImageJ software, normalized, and presented as mean ± SD from three independent experiments. Graphical data are presented as means ± SD from three independent experiments (n = 3). n.s not significant, using an unpaired Student’s t-test (A). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, using Fisher’s LSD post hoc test (B, C) or Šidák’s multiple comparison test (E, F). n.s not significant.
