Figure 6
Downregulation of Sp1 expression in highly invasive lung adenocarcinoma cells is caused by instability of Sp1 protein. (Aa) Whole-cell extracts of cells were collected for western blotting with antibodies against Sp1, pSp1 (T739) and tubulin. Reverse transcriptase–PCR (RT–PCR) was used to determine the mRNA level of Sp1. (Ab) Sumoylation and ubiquitination in an equal Sp1 expression level of CL1-0 and CL1-5 cells. (Ac) The quantitated result for the ratio of pSp1 (T739) with Sp1 in CL1-0 and 1-5 cells. Data are representative of three independent experiments and are presented as mean±s.e.m. (**P<0.01). (B) After MG132 treatment (50 μM) for 6 and 12 h, cells were harvested as whole-cell extracts for western blotting. (C) The association of Sp1 with RNF4. The whole-cell extracts of cells were immunoprecipitated with an anti-RNF4 antibody or IgG, and immunoblotted with an anti-Sp1 antibody. (D) Sp1 sumoylation in CL1-5 cells. Whole-cell extracts of cells were immunoprecipitated with an anti-Sp1 antibody or IgG, and immunoblotted with the antibody against SUMO-1 or Sp1. SUMO-Sp1 is indicated. (E) Sp1 ubiquitination in CL1-5 cells. After overexpression of myc-ubiquitin in the presence of MG132 for 12 h, whole-cell extracts were immunoprecipitated with an anti-Sp1 antibody, and immunoblotted with the antibody against myc or Sp1. Ubi-Sp1 is indicated. pSp1 represents phosphorylated Sp1. (F) Proposed model of Sp1 downregulation caused by Sp1 protein instability in highly invasive lung adenocarcinoma cells.
