Fig. 5.

TRIM72 salvages stress-induced p53 activation in vitro. a Upper panels: immunostaining of total p53 (red) on stretch-injured RLE cells treated with bovine serum albumin (BSA) or rhT72; cells are counter-stained with DAPI (blue); lower panels: representative images of repaired cells labeled with FITC-dextran (green), non-repaired cells labeled with fixable cell vitality dye eFluor450 (blue) and post-fixation immunostaining of p53 (red) in these cells; blue arrows = p53+ FITC+ or p53 + eFluor+ cells; scale bar = 20 μm; b the number of p53 positive cells per 20 × field and quantification of p53+ cells among non-stretched and membrane injured cells (FITC+ plus eFluor+); c Western blot of Ser15 phosphorylated p53 (P-p53), total p53, and TRIM72 in CTRL or T72OE RLE cells with or without treatment of 50 μg/ml bleo; b Western blot detection of ubiquitin and total p53 in T72OE or CTRL, in the presence and absence of bleo and with or without MG132 to inhibit proteasome degradation of ubiquitinated substrates. Stars: Bleo+MG132-treated CTRL and T72OE RLE cells; brackets: bleo-induced total p53 with or without MG132 treatment; c and d quantification of P-p53 and p53 (in the absence and presence of MG132) from bleo-treated RLE cells. Relative protein expression of P-p53 or p53 was normalized to β-actin. n = 4, *P < 0.05, or **P < 0.01 based on two-tailed student t-test (P-p53) or one-way ANOVA with post hoc analysis (p53)