Fig. 4.

Spontaneous, post-confluent differentiation is associated with increased IR-B mRNA and protein expression, and signaling. Caco-2 cells were grown on Transwell filters or plastic for 2 days (subconfluent) or Transwell filters for 21 days post confluence (differentiated). qRT-PCR and western blot to measure (A) differentiation biomarker and brush border enzyme sucrase isomaltase (SI) mRNA and protein, (B) total insulin receptor (IR) mRNA and protein. (C) RT-PCR evaluated the ratio of IR-B∶IR-A mRNA and IR-B protein levels were measured by western blot. All qRT-PCR data were normalized to invariant control, ribosomal phosphoprotein P0. Data represent mean ± s.e.m. (n = 5). Immunoprecipitation and western blot to measure tyrosine phosphorylation of (D) IR-B and (E) downstream mediator AKT in serum-deprived cells at 5 minutes after treatment with insulin (Ins; 200 ng/ml) or serum-free medium (SFM) alone. Data represent mean ± s.e.m. (n = 2 in duplicate). All western blots use β-actin as the loading control. β-actin image shown corresponds to p-AKT blot. Loading was identical for total AKT. *P<0.05 differentiated versus undifferentiated Caco-2 cells, unpaired t-test.