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. 2014 Mar 13;289(18):12680–12692. doi: 10.1074/jbc.M114.558981

FIGURE 3.

FIGURE 3.

A, interaction of C18ORF1 with Smads. COS7 cells were transfected with the indicated plasmids and harvested for coimmunoprecipitation (co-IP) experiments. The interaction between C18ORF1 and Smads is shown in the upper panel. The middle and lower panels indicate the total expression of FLAG-Smads and C18ORF1α1, respectively. B, endogenous interaction between Smad3 and C18ORF1. HaCaT cells were fixed, followed by addition of rabbit anti-Smad3 and/or mouse anti-C18ORF1 antibodies. Then PLA was performed. Upper panel, only Smad3 antibody was added to the section. Lower panel, both Smad3 and C18ORF1 antibodies were added. The red dots indicate colocalization. Nuclear staining with DAPI is indicated in blue. C, no detectable dots in HaCaT cells when rabbit anti-Smad3 and mouse anti-Myc9E10 antibodies were used for PLA assay. HaCaT cells were fixed, followed by addition of rabbit anti-Smad3 and mouse anti-Myc9E10 antibodies. Then, PLA was performed. Red dots were not visualized because mouse anti-Myc9E10 antibody was used as a control antibody. Left panels, interaction between two molecules that are recognized by anti-Smad3 and anti-Myc9E10 antibodies. Middle panels, nuclear staining with DAPI is indicated in blue. Right panels, merge image. D, C18ORF1 equally binds to nonphosphorylated and phosphorylated AR-Smads. Left panel, illustration of how cell lysates were prepared from each dish in which indicated plasmids were transfected. Middle panel, each cell lysate was mixed and subjected to co-IP experiments. Upper panel, interaction of Smad2 with C18ORF1α1; 2nd panel, total expression of Smad2; 3rd panel, Smad2 phosphorylation; lower panel, total expression of C18ORF1α1. Right panel, each cell lysate was mixed and subjected to co-IP experiments. Upper panel, interaction of Smad3 with C18ORF1α1; 2nd panel, total expression of Smad3; 3rd panel, total expression of C18ORF1α1; lower panel, total expression of ALK5. E, C18ORF1 inhibits interaction between Smad2 and Smad4. COS7 cells were transfected with the indicated plasmids and harvested for co-IP experiments. The interaction between Smad2 and Smad4 is shown in the upper panel. The 2nd, 3rd, 4th, and lower panels indicate the total expression of FLAG-Smad4, 6×Myc-Smad2, ALK5ca/HA, and C18ORF1α1/V5, respectively. F, C18ORF1 interferes with endogenous AR-Smad·Smad4 complex formation. NMuMG cells were infected with indicated adenoviruses, stimulated with 5 ng/ml TGF-β for 90 min, and harvested for co-IP experiments. Upper panel, interaction between AR-Smads and Smad4; 2nd panel, expression of total Smad2/3; 3rd panel, expression of total Smad4; lower panel, expression of C18ORF1.