Research Paper Volume 12, Issue 20 pp 20702—20727

Stress-induced p53 drives BAG5 cochaperone expression to control α-synuclein aggregation in Parkinson's disease

Figure 2. p53 is responsible for BAG5 induction. (A) U2OS, HeLa, and SH-SY5Y cells were transfected with various amounts of a p53-expressing plasmid (pcDNA3.1-p53). Expression levels of p53 and BAG5 were detected by Western blotting. β-Actin served as a loading control. After 24 or 48 h of pretreatment with etoposide (10 μM) or H2O2 (250 μM), p53 was repressed in etoposide-treated (B) and H2O2-treated (C) U2OS, HeLa, and SH-SY5Y cells by transfection of shp53-3755, shp53-3756, or shLuc control for 48 h and selecting with puromycin (2 μg/ml) for 48 h. The protein expression levels of BAG5, p53, and β-actin were detected by Western blotting. BAG5 transcripts were detected in shLuc and two p53 shRNAs knockdown cells. The expression levels of BAG5 transcripts were normalized to those of GAPDH and standardized with those of shLuc cells. (D) Total RNAs and cell lysates were harvested from HCT116 p53 wild-type (p53+/+) and null (p53-/-) isogenic colorectal cancer cell lines. BAG5 transcripts were detected by RT-Q-PCR. The relative fold in BAG5 mRNA expression was normalized to the GAPDH control and the vector control (upper panel). The protein expression levels of BAG5, p53, and β-actin were detected by Western blotting (lower panel). The relative fold in protein expression was normalized to the internal control β-actin and standardized with the vector control. Error bars represent the SD of the means calculated using data from three independent experiments (Student’s t-test; *, p < 0.05, **, p < 0.01, ***, p < 0.001).