Figure 5. Direct interaction between p14/ARF-progerin can be blocked by pVHL. (A) p14/ARF (p14) directly and specifically interacts with progerin. GST-progerin (bead; PG) and GST-Lamin A (LMA) were incubated with p14- or p14-/NBS-transfected 293 lysates for 2 h at RT. Precipitated proteins with GST-beads were subjected to SDS-PAGE and WB analysis. (B and C) Interaction of p14/ARF and progerin in C2 and HGPS (HS) cells. Each cell lysate was incubated with p14 Ab or Progerin Ab. After IP, associated progerin, or p14, was detected by SDS-PAGE and WB. Actin was used as a control. (D) pVHL blocks the interaction between p14-progerin. Using GST-PG, a pull-down assay was performed. Associated p14 was diminished by the addition of pVHL-lysate. Instead, pVHL was associated with progerin (GST-PG). (E) GST pull-down assay using GST-p14. The association of p14-progerin was confirmed by GST-p14 and GFP-PG lysate. In addition, other types of Lamin proteins did not show an interaction with GST-p14. Moreover, pVHL could block the interaction between GST-p14 and GFP-PG. (F) Progerin (PG) blocked the interaction between p53-p14. The interaction between GST-p53 and p14 was reduced by the addition of PG-lysate. (G) Progerin (PG) regulates the half-life of p14. To address the effect of progerin on p14 half-life, we performed a pulse-chase analysis using CHX. The half-life of p14 (approximately 2 h) was obviously reduced by GFP-progerin (PG) transfection; whereas si-PG extended the half-life to 6 h. (G) pVHL extends the half-life of p14. Using a similar procedure, we also measured the effect of pVHL on the half-life of p14.