Fig. 3.

IGF2BP expression in adult mice and tumor-derived cells. a Semi-quantitative RT-PCR analysis of IGF2BP expression (40 PCR cycles) in adult mouse tissues. Total RNA was analyzed from tissues isolated from either a 16- or 80-week-old male mouse. 28S RNA served as a loading control (20 PCR cycles). Total RNA isolated from E17 mouse embryonic fibroblasts (MEF) was used as positive control. Total lung RNA without reverse transcription (−RT) and water served as negative controls. b IGF2BP protein expression in indicated tumor-derived cells was analyzed by western blotting using mouse monoclonal antibodies directed against each of the three paralogues. Recombinant IGF2BP proteins (20 ng; including IGF2BP2-a and IGF2BP2-b) served as controls. Note, the IGF2BP3-directed antibody shows a significant cross-reactivity with IGF2BP1 (see also supplemental Fig. S4), presumably reflecting the high sequence similarity of both proteins. The cross-reactivity of both anti-IGF2BP1 (6A9) and anti-IGF2BP3 (6G8) with IGF2BP2 is low and presumably negligible for most studies (see also supplemental Fig. S4). Notably, one or two IGF2BP paralogues are expressed at very low levels in some tumor-derived cells, whereas all three paralogues are expressed in other cancer-derived cells. Additional controls for paralogue specificity of used monoclonal antibodies are shown in Fig. S4