Immunofluorescence Analysis of SCUBE3 and TGFβIIR

Bel7404 and HepG2 cells were plated in 35 mm confocal culture dishes for 24 h. On the following day, the cells were treated as previously described [24 (link)]. Primary antibodies against SCUBE3 (ab189955, Abcam,1:50) and TGFβIIR (sc-17799, Santa cruz,1:50) were used at 1:50 dilution. After washing, the cells were then incubated with fluorescence-labelled secondary antibodies Alexa-Fluor 555 anti-mouse IgG and anti-rabbit IgG (1:200 dilution, Cell Signaling Technology, Danvers, MA, USA). Then, cells were washed and stained with DAPI. Finally, images of cells positive for SCUBE3 and TGFβIIR were captured using a confocal microscope (Nikon Corporation, Tokyo, Japan).