PCAF Accelerates Vascular Senescence via the Hippo Signaling Pathway

Figure 3

PA upregulated PCAF in HUVECs depended on decreased MDM2 mediated ubiquitination. (a) HUVECs were treated with cycloheximide (35 μmol/L) to block nascent protein synthesis for indicated time points in the presence of PA or not. Stability of PCAF protein was assessed by western blotting. (b) The stability of PCAF mRNA was assessed in HUVECs treated with actinomycin D (100 μg/ml) by RT-PCR in the presence of PA or not. (c) Western blotting was used to assess the expression of PCAF in HUVECs treated with MG132 (10 μM) for indicated times points in the presence of PA or not. (d) His-tagged ubiquitin plasmid and flag-tagged PCAF plasmid were cotransfected into HUVECs, PBS, or PA were then stimulated for 48 h. 12 h before cells were harvested, MG132 was added into the culture dishes. Flag-tagged magnetic beads were used to bind to PCAF, total Ub level was detected by western blotting. (e) and (f) His-tagged MDM2 plasmid or Flag-tagged PCAF plasmid was transfected into HUVECs before stimulated with PBS or PA for 48 h, Co-IP were performed by using corresponding magnetic beads. Data are expressed as ( for each experiment. , , , n.s. ).

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