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. 2023 Jul 17;133(14):e164428. doi: 10.1172/JCI164428

Figure 4. Trastuzumab induces retrograde transport of DPAGT1.

Figure 4

(A) Representative IF staining images of DPAGT1 and DAPI in the indicated cells. Red arrows indicate the location of the cell membrane. Scale bar: 10 μm (B and C) IB analysis of DPAGT1 expression in the extracted plasma membrane (PM) and whole lysate (WL) from SK-BR-3, BT-474, MDA-MB-231, and MCF-7 cells (B), or from Vector- or DPAGT1-silenced SK-BR-3 and BT-474 cells (C). PMCA1 was used as a PM marker. α-Tubulin was used as a loading control. (D) Representative IF staining images of DPAGT1 in SK-BR-3 cells treated with IgG, trastuzumab, or nimotuzumab (NIMO, 20 μg/mL). The percentage of DPAGT1 PM+ cells was quantified in 10 random fields. Scale bar: 10 μm (E) IB analysis of expression of DPAGT1, HER2, EGFR, and HER3 in the extracted PM, extracted ER, and WL of SK-BR-3 cells treated with IgG, trastuzumab, or nimotuzumab. Calnexin was used as an ER marker. PMCA1 was used as a PM marker. α-Tubulin was used as a loading control. (F) Representative IF staining image (left) and quantification (right) of DPAGT1 PM+ cells in the indicated cells. Scale bar: 10 μm (G) IB analysis of DPAGT1 expression in the extracted PM, extracted ER, and WL of indicated SK-BR-3 cells. Calnexin was used as an ER marker. PMCA1 was used as a PM marker. α-Tubulin was used as a loading control. (H) IB analysis of expression p-CAV1Y14, CAV1, p-c-SrcY416, and c-Src in IgG- or trastuzumab-treated SK-BR-3 cells. α-Tubulin was used as a loading control. (I) IB analysis of expression of DPAGT1, c-Src, and CAV1 in the lipid rafts isolated from IgG- or trastuzumab-treated SK-BR-3 cells. (J) IB analysis of DPAGT1 expression in the extracted PM and ER fractions from SK-BR-3 cells treated with IgG, trastuzumab, or trastuzumab + c-Src inhibitor Dasatinib (4 μM). Data in D and F were plotted as the mean ± SD of biological triplicates and analyzed with an unpaired 2-sided Student’s t test. The arrows in panels D and F indicate plasma membrane expression. Relative protein expression was quantified by Image J. *P < 0.05, **P < 0.01, ***P < 0.001.