FIGURE 2.

Inhibition of GSK increases transport of IGF-I. A, MDCK cells transfected with either GFP or mini-megalin and exposed to a GSK inhibitor (200 μm, GSK inhibitor I; Calbiochem) or NP12 (100 μm; Noscira) showed increased internalization of bIGF-I. A representative blot is shown. Membranes were reblotted with anti-β-actin antibody as a loading control (n = 4). Lower blots, note the increased presence of megalin, but not IGF-IR in the surface of the cell (membrane fraction) after treatment with NP12. B, inhibition of GSK3 involves the GSK3 site located at the SH3 region of the C terminus of megalin. Mini-megalin mutants lacking either the entire SH3 region (SH3) or just the GSK3 site (GSK3) did not show increased bIGF-I in response to GSK3 inhibition with NP12. Representative blots and quantitation histograms are shown (n = 6; *, p < 0.05 versus respective controls). C, transcytosis of IGF-I across the choroid plexus monolayer is markedly increased by NP12. Significantly greater amounts of bIGF-I were detected in the CSF side (upper chamber) of the double-chamber system (for details, see “Experimental Procedures”). Representative blots of upper-chamber supernatants and quantitation histograms are shown (n = 5; **, p < 0.01). D, transcytosis of digoxigenin-labeled albumin (Dig-albumin) is not affected by NP12. No digoxigenin-labeled albumin was detected in the upper chamber, whereas it was readily detected in the lower chamber. IGF-I did modulate transcytosis of albumin, as already published (12). Error bars, S.E.