Skip to main content
. 2012 May 31;8(5):761–777. doi: 10.7150/ijbs.4036

Figure 5.

Figure 5

Knockdown of ZFP36L1 affects Mkp-1 mRNA stability in preadipocytes. (A) 3T3-L1 preadipocytes were infected with lentivirus carrying shRNA against ZFP36L1 (shL1), ZFP36L2 (shL2), or luciferase (shLuc, negative control) mRNA. WCEs from ZFP36L1 or ZFP36L2 knockdown cells were immunoblotted with anti-ZFP36L1, anti-ZFP36L2, anti-MKP-1, anti-p-ERK, anti-ERK, and anti-β-tubulin (upper panel). The bracket indicates the multiple forms of ZFP36L2. The arrow indicates the increased MKP-1 protein under knockdown of ZFP36L1. RNA was isolated for quantitative PCR to analyze the expression of Zfp36l1 and Zfp36l2 (lower panel). **P < 0.01; ***P < 0.001; ns, not significant. (B) ZFP36L1 knockdown by shL1 increases the basal level of Mkp-1 mRNA. RNA was isolated from knockdown cells for quantitative PCR with primers to Mkp-1 and β-actin. The relative amounts of Mkp-1 mRNA are presented after normalizing to β-actin mRNA values as the mean ± SD from three independent samples. *P < 0.05. (C) Mkp-1 mRNA half-life is increased in ZFP36L1 knockdown cells. 3T3-L1 cells were treated with shLuc, shL1, or shL2, and actinomycin D was added for the indicated times to stop RNA synthesis. RNA was isolated for quantitative PCR. Each experiment was performed three to five times independently. The mRNA half-life (t1/2) was calculated and indicated. *P < 0.05. (D) Determination of Mkp-1 mRNA half-life at 1 h of differentiation in control and knockdown cells. Two-day post-confluent 3T3-L1 cells knocked down with control shLuc, shL1, or shL2 were induced to differentiate for 1 h followed by actinomycin D treatment for 0 min (untreated), 10 min, 20 min, and 30 min. RNA was isolated for quantitative PCR with primers directed to Mkp-1 and β-actin. The mRNA half-life (t1/2) was calculated and indicated. **P < 0.01.