Research Paper Volume 12, Issue 8 pp 6823—6851

Figure 6. IGFBP2 interacts with p21 and protects it from ubiquitin-mediated proteasome degradation. (A) IGFBP2, p53, p21, p-p21, p27 and p16 protein expression was detected by WB analysis in pso KC cultured at passage 4 (P4, senescent cells) and silenced (si-IGFBP2) or not for IGFBP2 (si-NC) for different time points (right panels). Similarly, cyclin D, p-Rb and PCNA protein expression was detected in pso KC cultured at passage 1 (P1, pre-senescent cells) and silenced or not for IGFBP2 (left panels). In A and B, graphs show the mean ± SD of densitometric intensity (D.I.) of three independent experiments. *p ≤ 0.05, **p ≤ 0.01 as calculated by paired Student’s t test comparing si-IGFBP2 with si-NC. (B) Co-immunoprecipitation experiments were performed on protein lysates obtained from pso KC left untreated or treated by M4 for 6 hours and then immunoprecipitated with antibodies against IGFBP2 or goat IgG as negative control (IP: IGFBP2, left panel), and with p21 or mouse IgG (IP: p21, right panel). The immunoprecipitates were probed with anti-IGFBP2, -p21 or –p16 antibodies, as shown in left and right panels. WB analysis was also performed on cell lysates (Input) to detect IGFBP2 and p21 levels. Figures are representative of three independent experiments. (C) Protein extracts of three distinct pso KC strains, transfected with si-IGFBP2 or si-NC for 24 h and then treated or not with 20 μM of the proteasome inhibitor MG132 for 6 h, were subjected to WB for the detection of IGFBP2, p21 and p16 expression. WB panels are representative of three independent experiments and D.I. indicates values of densitometric intensity. (D) Endogenous p21 was immunoprecipitated by protein lysates obtained from pso KC cultures silenced or not for IGFBP2 for 24 h and treated with 20 μM of MG132 for 6 hours. WB analysis was performed for the detection of p21 ubiquitination by using anti-ubiquitin antibody. WB was also performed on cell lysates (Input) to detect IGFBP2 and p21 levels, as well as β-actin as loading control.