Fig. 4: THEMIS2 regulated the interaction between p-MET and PTP1B in TNBC cells.

A MDA-MB-231 and Hs578T cells were used in the co-IP experiments. Cell lysates were blotted directly or subjected to IP with a PTP1B antibody; this was followed by blotting with the indicated antibodies. IgG served as the negative control. B Cells were treated with or without HGF (30 ng) for 30 min, after which the lysates were blotted directly or subjected to IP with a PTP1B antibody first. This was followed by blotting with the indicated antibodies. IgG served as the negative control. C Cells were treated with or without HGF (30 ng) for 30 min, after which the lysates were blotted directly or subjected to IP with a p-MET antibody first. This was followed by blotting with the indicated antibodies. IgG served as the negative control. D Hs578T cells were transfected with the negative control siRNA or THEMIS2 siRNA for 8 h, serum starved for 24 h, and then treated without or with 30 ng/mL HGF for 30 min (left and right panels, respectively). A Duolink PLA was then performed. Top: p-MET antibody only served as the negative control; middle: protein–protein interactions between p-MET and PTP1B or between MET and PTP1B was analyzed using both antibodies; bottom: protein–protein interactions between p-MET and PTP1B or MET and PTP1B in THEMIS2-depleted cells. The signals are represented by white arrows and the nonspecific signals by red arrows. The close proximity (<40 nm) association of the two proteins in each set is indicated by small, distinct red dots, which were detected using fluorescence microscopy. Nuclei were counterstained with DAPI (blue). E Quantification of signals by number of PLA puncta per cell. The histograms represent means ± SEs from three independent experiments (**P < 0.01).