Ab189955

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).

Immunohistochemical analysis was conducted according to a standard protocol. Briefly, samples were incubated with anti-SCUBE3 (ab189955; Abcam) and anti-mitochondria (ab92824; Abcam) as primary antibodies, followed by washing and incubation with horseradish peroxidase-conjugated secondary antibodies. Histometric observations were performed as described for the histological assessment.

Total cell proteins were lysed using radioimmunoprecipitation assay (Beyotime) according to the manufacturer’s protocol. Proteins in the conditioned medium were extracted using a liquid sample total protein extraction kit (Solarbio, Beijing, China). Western blotting was performed as previously described [10 (link)]. The primary antibodies used were anti-SCUBE3 (ab189955; Abcam), anti-AMBN (orb155652; Biorbyt, Cambridge, UK), TGFβR1 (ab31013; Abcam), anti-DSPP (sc-73632; Santa Cruz), anti-DMP1 (ab103203; Abcam), anti-OPN (ab8448; Abcam), anti-OSX (ab13418; Abcam), anti-BMP2 (ab14933; Abcam), anti-BMPR1A (ab264043; Abcam), anti-p-SMAD1/5 (9516; Cell Signalling Technology, Boston, MA, USA), and anti-SMAD1 (D59D7; Cell Signalling Technology). Anti-GAPDH (Rayantibody, Beijing, China) was used as an internal control. Antibody binding was detected using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA). The intensity of the bands was quantified using the ImageJ software (NIH, USA).