Dear colleagues, I really struggle to find a good antibody for human NTCP (gene symbol SLC10A1) for Western Blot and will really appreciate your help.

In our lab, we developed an excellent positive control - adenovirus for overexpression of NTCP. The CDS of this AdNTCP is sequenced and 100% identical to the RefSeq RNA for NTCP.

We know that this AdNTCP is functional because when we infect HepG2 cells (which do not express NTCP protein), we observe a clear dose-dependent uptake of fluorescently labelled taurocholic acid (fTCA) (which can not enter freely the cell and can be transported only by NTCP in presence of Na2+ in the uptake buffer). We do not observe any uptake in cells infected with empty adenovirus, and moreover, there is no uptake in AdNTCP infected cells, when fTCA was provided in a buffer without Na2+ .

Even though the predicted molecular weight of NTCP is about 40kDa, according to several papers, in HepG2 it is heavily glycosylated and the apparent Mw increases to 50-60kDA.

So far I have tried several antibodies: Abcam 131084, Abcam 97947, Sigma ANTI-SLC10A1 (C-TERM) SAB1303863, but none of these provided a specific band which is clearly visible for the AdNTCP infected HepG2 and not visible for the emptyAd -infected cells.

Even though NTCP is a membrane protein, we do not do membrane fractionation, and we work with total cell lysate because our qPCR results show that in AdNTCP infected cells we increase the expression to Cq16 (versus Cq40 for the emptyAd -infected cells).

Does anyone have experience with the immunodetection of NTCP by Western Blot? Please share your experience and recommend anti-NTCP antibodies and experimentally validated protocol.