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. 2012 Feb;32(3):704–716. doi: 10.1128/MCB.06448-11

Fig 3.

Fig 3

SIRT1-Δ2/9 protein is downregulated by CUGBP2. (A) Exogenous SIRT1-Δ2/9 protein is not detectable in ARPE19 cells. MH-SIRT1-Δ2/9 was exogenously expressed in HCT116 p53+/+ and ARPE19 cells. Cells were harvested after 24 h for RNA and protein. RT-PCR results for exogenous SIRT1-Δ2/9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (upper panel) and Western blot analysis results for SIRT1-Δ2/9 (lower panel) are shown. ∗, a nonspecific band; arrowhead, SIRT1-Δ2/9 protein. (B) Downregulation of CUGBP2 by siRNA allows the expression of exogenous SIRT1-Δ2/9 protein in ARPE19 cells. CUGBP2 was silenced by siRNA prior to the exogenous expression of MH-SIRT1-Δ2/9 in ARPE19 cells. Protein samples were blotted for SIRT1-Δ2/9 and phosphorylated SIRT1 serine 47. (C) Detection of endogenous and exogenous SIRT1-Δ2/9. Total cell lysates from HCT116 p53+/+ cells treated with vector alone or MH-SIRT1-Δ2/9 plasmid were analyzed. The blots were probed with anti-SIRT1 residues 1 to 131 (α Sir2; catalog number 07-131; Upstate Biotechnology). (D) RT-PCR results showing mRNA expression of CUGBP2 (upper panel) and qRT-PCR results (lower panel) for the indicated cell lines. (E) CUGBP2 does not influence SIRT1-Δ2/9 or SIRT1-FL splicing. Expression levels of SIRT1-Δ2/9, SIRT1-FL, and CUGBP1 mRNAs (RT-PCR results [lower panel]) in CUGBP2 silenced ARPE19 cells (qRT-PCR results [upper panel]) are shown.