Immortalized RPE Cells for IGF-1 Studies

RPE cells isolated from WT or immunoproteasome KO mice (L2 and L7M1) were immortalized as previously described [37 (link)]. RPE cells were cultured in Dulbecco’s Modified Eagle Medium as described previously [15 (link)], with 5% heat-inactivated fetal bovine serum (Atlanta Biologicals). For IGF-1 treatments, cells were serum starved for 24 hours, then treated with recombinant mouse IGF-1 (100 ng/mL, R&D Systems) and harvested at times indicated in the figures. Concentration of IGF-1 utilized in the study was determined by assessing time- and concentration-dependent phosphorylated-Akt (p-Akt) activation by Western blot in WT-RPE cells from 0–3 hours and 0–300 ng/mL IGF-1, respectively (Data not shown).