Engagement of FcγRs on immune cells with trastuzumab Fc is required for HER2 downregulation. (A) WB detection of HER2 in BT474 breast cancer cells with or without co-culture with PBMCs in the presence of trastuzumab, scIgG-T, N297A-T, or F(ab’)2-T for 48 h as labeled on the top of each panel. The same amount of protein lysates was loaded on each lane and β-actin was used as a loading control. (B) WB detection of HER2 in BT474 cancer cells after co-culture with NK cells, monocytes, T cells and PBMCs as labeled on each panel in the presence and absence of trastuzumab for 48 h. (C) PBMCs were pretreated with isotype IgG (10 μg/ml) for 1 h to block FcγRs before conducting co-culture with BT474 cancer cells in the presence or absence of trastuzumab (5 μg/ml) for 48 h. HER2 expression was detected by WB. (D) WB detection of HER2 downregulation in cancer cells after co-culture in transwell chambers with PBMC (the right two lanes) in the presence and absence of trastuzumab. Cancer cells co-cultured with PBMCs without the transwell chamber (the left two lanes) were used as controls. Fab, fragment, antigen binding; Fc, fragment, crystallizable; FcγR, Fc gamma receptor; HER2, human epidermal growth factor receptor 2; NK cell, natural killer cell; PBMC, peripheral blood mononuclear cell; scIgG-T, single hinge cleaved trastuzumab; WB, Western blotting.