Locali z ation of exocyst subunits to LAMP1Δ-RUSH post-Golgi carriers, a C-terminal tag negatively affects EXOC6 function and validation of gene abrogation. (A) TIRF imaging of LAMP1Δ-RUSH (gray) cells expressing heterologous EXOC1-HALO (purple) after ∼35 min in biotin. From Video 11. Scale bar: 10 μm (B) TIRF imaging of LAMP1Δ-RUSH (gray) cells expressing heterologous HALO-EXOC6 (purple) after ∼35 min in biotin. From Video 12. Scale bar: 10 μm. (C) Immunoblot confirming loss of EXOC1 in a transient LAMP1Δ-RUSH EXOC1 KO cell population. (D) Recovery of EXOC6-HALO to EXOC6+6B KO cells, demonstrating a less efficient recovery than N-terminally tagged EXOC6 (Fig. 3 A). (E) Overexpression of EXOC6-HALO has a moderate but significant effect on cell-surface delivery of LAMP1Δ-RUSH. (F) Validation of guide RNA KO efficiency by qRT-PCR. Data from all conditions was internally normalized to GAPDH expression and is represented as fold change of control LAMP1Δ-RUSH Cas9 cells. ELKS2/ERC2 and UNC13C were not detectable by qPCR in control conditions. Error bar = SD of at least three independent experimental repeats. Tukey’s multiple comparisons test (HSD, FWER = 0.05) was performed on data in D and E. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.