Table 1.
All chemicals and reagents used in this study.
Fig 1.
Heated samples for Fpn1 (SLC40A1) western blotting.
A) 25ug of whole cell lysates (WCLs) prepared from indicated cells (*two different preparations of SW480 WCLs) were subjected to Fpn1 western blotting as detailed in the Materials and Methods. This experiment used sample loading buffer containing 5% β-mercaptoethanol, heated at 95°C for 5 min, anti-Fpn1 antibody (Novus, rabbit polyclonal, 1,000-fold diluted with 1% BSA in T/TBS) at 4°C overnight followed by anti-rabbit IgG-HRP conjugated (8,000-fold diluted with 1% BSA in T/TBS) at room temperature for 1.5hr. Unless otherwise noted, the rest of western blots in this study were performed in the same procedures with or without sample heating prior to gel loading. B) SW480 cells were treated with 0.1–0.5mM FAC (ferric ammonium citrate), 2-100ug/ml cisplatin, or FAC 0.25mM plus cisplatin 10ug/ml for 18hr. 20ug of WCLs were subjected to Western blotting with anti-Fpn1 antibody (top). The membrane was soaked in the stripping solution at room temperature for 1hr, washed with T/TBS, and incubated with anti-ferritin H mouse monoclonal antibody (sc-135667, Santa Cruz Biotechnology). In A) and B), the asterisks indicate the predicted position (63 kDa) of the endogenous Fpn1. The experiments were repeated three times and the representative western blots are shown.
Fig 2.
Comparison in Fpn1 (SLC40A1) western blotting between heated and unheated protein samples from Fpn1 transfected cells.
A) WCL in RIPA buffer was prepared from HEK293 cells transfected with pCMV or pCVMFpn1. 25ug of the WCL mixed with 2X SDSPAGE sample loading buffer was heated at 95°C for 5min and subjected to Fpn1 western blotting. The arrow indicates the Fpn1 protein stuck on the top of the separation gel (also in B and C). B) WCL in RIPA buffer, in RIPA plus sonication for 10 seconds three times in ice-water, or in transmembrane protein extraction reagent (Five Photon Biochemicals) was prepared from HEK293 cells transfected with pCMV or pCVMFpn1(6ug, *Fpn1: 12ug DNA). 15ug of the WCLs mixed with 2X SDSPAGE sample loading buffer were heated at 95°C for 5min or not heated (at room temperature), and subjected to Fpn1 western blotting. The arrowhead indicates the transfected Fpn1 band. C) WCLs in RIPA buffer from HEK293 cells transfected with pCMV or pCVMFpn1 were mixed with 2X SDSPAGE sample loading buffer containing 5% β-mercaptoethanol (regular), 4M urea, or both 5% β-mercaptoethanol and 4M urea. One sample set was heated at 95°C 5min, another set was not heated (at room temperature), and subjected to Fpn1 western blotting. Positions of molecular weight protein size marker are indicated on the left. The experiments were repeated three times and the representative western blots are shown.
Fig 3.
Comparison in DMT1 (SLC11A2) western blotting between heated and unheated protein samples.
A) WCL in RIPA buffer, or cytoplasmic (Cyto) and nuclear (Nuc) fractions were prepared from HEK293 cells transfected with pCMVFlag, pCMVDMT1 or pCVMFlagDMT1. 30ug of the WCL, Cyto, and Nuc fractions mixed with 2X SDSPAGE sample loading buffer were heated at 95°C for 5min or not heated (at room temperature), and subjected to western blotting with anti-Flag antibody (top) or anti-DMT1 antibody (bottom). B) WCLs in RIPA buffer were prepared from SW480 cells treated with 0.1–0.5mM FAC (ferric ammonium citrate), 2-100ug/ml cisplatin, or FAC 0.25mM plus cisplatin 10ug/ml for 18hr. 20ug of WCLs mixed with 2X SDSPAGE sample loading buffer without heating were subjected to Western blotting with anti-DMT1 antibody (top: longer exposure, bottom: shorter exposure). As a control, heated or unheated WCL from pCMVDMT1-transfected HEK293 cells was loaded. The experiments were repeated four times and the representative western blots are shown.
Fig 4.
Comparison in DMT1 and Fpn1 western blotting between heated and unheated protein samples from 12 human cell lines.
25ug of WCLs in RIPA buffer from indicated 12 human cell lines were mixed with 2X SDSPAGE sample loading buffer, either heated at 95°C for 5min or not heated (at room temperature), and subjected to A) DMT1 and B) Fpn1 western blotting. As a control, 10ug of non-heated WCL from HEK293 cells transfected with pCMV, pCMVDMT1, or pCMVFpn1 was loaded. The arrowheads indicate the positions of transfected DMT1 and Fpn1 bands. In A), the arrow indicates the DMT1 protein stuck on the top of the separation gel. The experiments were repeated four times and the representative western blots are shown.
Fig 5.
Verification of the band specific to endogenous Fpn1 protein.
A) Caco-2 cells were transfected with Control- or Fpn1-siRNA using Lipofectamine RNAiMax for 18hr, followed by treatment with FAC at the final 250 and 500uM for additional 8hr in the presence of siRNA. Unheated Caco-2 WCLs in RIPA buffer along with unheated WCL from HEK293 cells transfected with pCMV or pCVMFpn1 were analyzed by western blotting for Fpn1 (top) followed by incubation with anti-GAPDH antibody. B) Caco-2 cells were untreated or treated with 500nM hepcidin (3 plates each) for 22h. 20ug of three-independent hepcidin (-) and hepcidin (+) WCLs in RIPA buffer were mixed with 2X SDSPAGE sample loading buffer, unheated, and subjected to Western blotting with the anti-Fpn1 antibody. Unheated WCL from HEK293 cells transfected with pCMV or pCVMFpn1 was loaded as a control. The membrane was incubated with the anti-GAPDH antibody to assess an equal amount of sample loading. The experiments were repeated five times and the representative western blots are shown.
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.