Fig 6. Comparison in western blotting for ferritin and TfR1 proteins between heated and unheated samples from Caco-2 cells treated with iron.

Caco-2 cells were untreated or treated with 50uM hemin, 250 and 500uM FAC, or 25uM DFO for 24hr. 25ug of Caco-2 WCLs along with 10ug of WCL from HEK293 cells transfected with pCMV or pCVMFpn1 were heated at 95°C for 5min or not heated (at room temperature). They were subjected to successive western blotting with A) anti-Fpn1 (SLC40A1), B) anti-DMT1 (SLC11A2), C) anti-ferritin H, D) anti-TfR1, and E) anti-GAPDH antibodies. The larger area of Fpn1 western blot for HEK293 cells transfected with pCMV or pCVMFpn1 is shown right next to the panel A. The arrow indicates the transfected Fpn1 protein stuck on the top of the separation gel. The asterisk in D) may be a TfR1 dimer only seen in unheated samples. The experiments were repeated three times and the representative western blots are shown. The same experiment repeated in HepG2 cells are shown in S2 Fig.