Fig. 2

In vitro CAV1 depletion increases HER2 stability at the cell membrane. a Western blot of biotinylated cell surface-associated HER2 along with HER2 and CAV1 in total lysates of NCIN87 cells transfected with CAV1 siRNA or scrambled control siRNA constructs or b transfected with CAV1 CRISPR activation plasmid or control CRISPR activation plasmid. HER2 protein levels increase at the cell surface after CAV1 depletion (1.4 ± 0.2, mean ± S.E.M, n = 3). BT biotinylation. c Western blot of biotinylated cell surface-associated HER2 in NCIN87 cells transfected with CAV1 siRNA or scrambled control siRNA constructs and blocking protein synthesis with 80 μg mL⁻¹ CHX. CHX cyclohexamide. d Half-lives of cell surface-associated HER2 (mean ± S.E.M, *P < 0.05 based on a Student’s t-test, n = 5). e, f Western blot analysis of internalized HER2 (e) or cell surface–associated HER2 (f) on NCIN87 cells transfected with either control or CAV1 siRNA and treated with 1 μM trastuzumab. g Membrane-bound and internalized [⁸⁹Zr]Zr-DFO-trastuzumab before and after CAV1 knockdown or CAV1 depletion with methyl-β-cyclodextrin (MβCD) or filipin in NCIN87 cancer cells (bars, n = 5, mean ± S.E.M, ***P < 0.001 based on a Student’s t-test). h NCIN87 cells transfected with CAV1 siRNA or scrambled control siRNA constructs were incubated with [⁸⁹Zr]Zr-DFO-trastuzumab (0 to 125 μM) for 3 h at 4 °C (upper panel). Specific binding of [⁸⁹Zr]Zr-DFO-trastuzumab (black circles, red squares) and non-linear regression curve fit (dotted lines). Data are presented as mean ± S.E.M, n = 3. Binding parameters (lower panel) of [⁸⁹Zr]Zr-DFO-trastuzumab to NCIN87 cells scrambled control, treated with CAV1 siRNA or with lovastatin (*P < 0.05 based on a Student’s t-test, n = 5). i Western blot of CAV1 and phosphorylated HER2 proteins from total extracts of NCIN87 cells incubated with 25 μM of lovastatin for 4 h. β-actin was used as a loading control. j Confocal images of immunofluorescence of HER2 in NCIN87 cells incubated with 1 μM trastuzumab for 90 min in the presence and absence of lovastatin. Scale bars: 20 μm