Fig. 4.

Effects of high glucose, IBMX and DETAnonoate on the affinity of RNA binding proteins to oligonucleotides representing specific 3′- and 5′-UTR sequences of insulin mRNA in INS-1 cells. INS-1 cells were cultured in RPMI 1640 medium with 1.67 mM glucose (control; C), 16.7 mM glucose (high glucose; HG), 1 mM IBMX and 1 mM DETAnonoate (DNO) for 2 h. Cytoplasmix extracts from the cells were incubated with beads carrying both the long 3′- and the long 5′-UTR oligonucleotides, and the eluted proteins were analyzed by SDS-PAGE and immunoblotting. The affinity of hnRNP U, PTB and TIAR was first normalized against hnRNP L, and then normalized against the control. The lower panel in A shows a representative blot with all four proteins. The image is acquired after blotting for hnRNP U, hnRNP L, PTB and TIAR in sequence, thus showing the correlation from a single experiment. The lower panel shows representative blots for all four proteins. Results are means ± SEM for 3–4 independent experiments and * denotes p < 0.05 using Student’s t-test when comparing vs. corresponding control. Full images can be found in Supplemental Fig. 4.