FIG. 4.

CSIG expression regulates PTEN in the translational level without influencing PTEN mRNA levels. (A and B) Ectopic intervention of CSIG expression was achieved by transiently transfecting HEK 293 cells either with pIRES-CSIG along with an empty vector (A) or with siRNA targeting CSIG (20 nM [lane 3] and 50 nM [lane 4]) along with a Ctrl siRNA (20 nM [lane 1] and 50 nM [lane 2]) for 48 h. (B) CSIG and PTEN expression were detected by Western blot analysis; β-actin was served as a loading Ctrl. Relative PTEN abundances were estimated by densitometry and are expressed as increases relative to PTEN levels in control cells. (C and D) Results of Northern blot analysis of PTEN mRNA levels after CSIG overexpression (C) or CSIG knockdown (D); GADPH served as a loading Ctrl. The PTEN mRNA abundance is expressed as the increase relative to PTEN mRNA levels in Ctrl cells. (E) Analysis of PTEN translation in CSIG-silenced HEK 293 cells. Newly translated PTEN was measured by incubating cells with l-[³⁵S]methionine and l-[³⁵S]cysteine for 20 min, followed by IP using either anti-PTEN antibody, anti-GAPDH antibody, or IgG; resolving immunoprecipitated samples by SDS-PAGE; and transferring for visualization of signals by using a PhosphorImager. (F) Results of Western blot analysis of PTEN and p27^Kip1 (left panel), and results of Northern blot analysis (panel right) of PTEN in young (∼28 pdl) and senescent (∼58 pdl) 2BS cells. β-Actin and GADPH served as loading Ctrls for Western or Northern blot analysis, respectively.