Figure 2.
Depletion of KSRP enhances iNOS 3′-UTR RNA stability in in vitro degradation assays. DLD-1 cells were preincubated for 18 h in medium without FCS and phenol red. Then the cells were incubated with the CM for 6 h and cytoplasmic proteins were isolated. Parts of these extracts were immunodepleted of KSRP by using a polyclonal anti-KSRP antibody (KSRP-depl). The extracts were incubated with radiolabeled 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNA with (iNOS 3′-UTR) or without (non-AU) the AREs for different time periods from 0 to 30 min. Then the degradation of the RNA in the different samples was stopped by adding SDS. To normalize the subsequent purification steps in vitro transcribed radiolabeled β-actin RNA fragments were added. After phenol extraction and ethanol precipitation the material was separated on denaturing urea polyacrylamide gels. (A) Scheme of the human 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs used for in vitro decay experiments. The positions of AU-repeats are indicated by arrowheads. (B) The degree of KSRP depletion of two different immunodepletion reactions (IP α-KSRP) were analyzed by western blotting using specific anti-KSRP- and anti-β-tubulin antibodies. For comparison the corresponding untreated samples were analyzed. The position of KSRP and β-tubulin is indicated. (C) Summary of densitometric analyses of eight different in vitro decay assays using 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs containing the AREs (iNOS 3′-UTR). Data points (means ± SEM) represent relative iNOS 3′-UTR RNA levels (100% = 0 min incubation; circles: undepleted extracts from CM-treated cells; squares: KSRP-depleted extracts from CM-treated cells; **P < 0.01; ***P < 0.001; ns = not significant versus undepleted extract). (D) Summary of densitometric analyses of five different in vitro decay assays using 5′-capped and 3′-polyadenylated iNOS 3′-UTR RNAs without the AREs (non-AU). Data points (means ± SEM) represent relative non-AU iNOS 3′-UTR RNA levels (100% = 0 min incubation; diamonds: undepleted extracts from CM-treated cells; squares: KSRP-depleted extracts from CM-treated cells; ns = not significant versus undepleted extract).