Fig 1: Localization of exocyst subunits to LAMP1?-RUSH post-Golgi carriers, a C-terminal tag negatively affects EXOC6 function and validation of gene abrogation. (A) TIRF imaging of LAMP1?-RUSH (gray) cells expressing heterologous EXOC1-HALO (purple) after ~35 min in biotin. From Video 11. Scale bar: 10 µm (B) TIRF imaging of LAMP1?-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 LAMP1?-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 LAMP1?-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 LAMP1?-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.
Fig 2: ELKS and its associated proteins and/or homologs are not necessary for LAMP1?-RUSH post-Golgi tubule fusion. (A) TIRF imaging of LAMP1?-RUSH (gray) cells expressing heterologous HALO-ELKS (red) mostly localized to fusion sites (blue arrowheads). From Video 10. Scale bar: 10 µm. (B) Cell-surface ratio quantification (flow cytometry) of LAMP1?-RUSH at the plasma membrane after stable ELKS KO and 35 min of biotin exposure. (C) Immunoblot confirming loss of ELKS in a stable LAMP1?-RUSH ELKS KO clonal cell line. (D) Cell-surface ratio quantification (flow cytometry) of LAMP1?-RUSH at the plasma membrane after transient ELKS KO and 35 min of biotin exposure. (E) Cell-surface ratio quantification (flow cytometry) of LAMP1?-RUSH at the plasma membrane after transient KO of known ELKS interaction partners and 35 min of biotin exposure. This experiment was carried out in a stable clonal ELKS KO cell line. (F) Cell-surface ratio quantification (flow cytometry) of LAMP1?-RUSH at the plasma membrane after transient KO of EXOC1, ELKS, and combined. Error bar = SD of at least three independent experimental repeats. Two-tailed t test was performed on data in B and D, and Tukey’s multiple comparisons test (HSD, FWER = 0.05) was performed on data in F. *P = 0.05; ***P = 0.001.
Fig 3: Exocyst is essential for secretion of a broad array of endogenous soluble secreted proteins. (A) Volcano plot from the mass spectrometry data on soluble proteins secreted over a period of 6 h by WT HeLa cells demonstrates that EXOC3 KO causes a dramatic and significant decrease of protein secretion in these cells. Significantly different proteins are shown in red (P = 0.01). (B) Immunoblot showing intracellular accumulation of selected proteins with downregulated secretion in EXOC3 KO cells. (C) Immunoblot confirming downregulation of Exoc1 in a siRNA-treated population of mouse 3T3-L1 adipocytes. (D) Immunoblot confirming downregulation of Exoc3 in a siRNA-treated population of mouse 3T3-L1 adipocytes. (E) ELISA quantification of secreted leptin from 3T3-L1 adipocytes siRNA treated with non-targeting (NT) or Exoc1. Results normalized to non-targeting control. (F) ELISA quantification of secreted leptin from 3T3-L1 adipocytes treated with non-targeting (NT) or Exoc3 siRNA. Results normalized to non-targeting control. (G) Immunoblot showing that downregulation of EXOC3 in a population of human JK6L lymphocytes correlates with a significant decrease in IgG secretion over a period of 24 h. (H) Quantification of secreted heavy chain IgG over four independent experiments described in F. (I) Quantification of secreted light chain IgG over four independent experiments described in F. Error bar = SD of at least three independent experimental repeats. Two-tailed t test was performed on data in E, F, H, and I. **P = 0.01; ***P = 0.001.
Fig 4: Exocyst is recruited to post-Golgi carriers and multiple associated proteins are essential. (A) TIRF imaging of heterologous expression of EXOC3-HALO in EXOC3-KO LAMP1?-RUSH cells. EXOC3-HALO (magenta) specifically co-localizes with LAMP1?-RUSH (green) carriers near the plasma membrane. Orange arrowheads indicate co-localizing structures. From Video 9. Scale bar: 5 µm. (B) Percent of LAMP1?-RUSH carriers positive for EXOC3-HALO, untagged HALO as a control, three biological repeats (total carriers quantified within 3 µm of plasma membrane edge = 298), statistical analysis = two-tailed t test. (C) Percent of LAMP1?-RUSH carriers positive for EXOC1-HALO, untagged HALO as a control, three biological repeats (total carriers quantified within 3 µm of plasma membrane edge = 228), statistical analysis = two-tailed t test. (D) Percent of LAMP1?-RUSH carriers positive for HALO-EXOC6, untagged HALO as a control, three biological repeats (total carriers quantified within 3 µm of plasma membrane edge = 206), statistical analysis = two-tailed t test. (E) Schematic representation of RUSH carrierIP assay. (F) Gels containing resolved proteins from carrierIP assay denoting enrichment of exocyst subunits (HALO-tagged EXOC1, EXOC2, EXOC3, EXOC4, EXOC5, EXOC6, EXOC7, and EXOC8) in LAMP1?-RUSH post-Golgi carriers (LAMP1 immunoblot). Molecular weight markers are indicated in kD. (G) Cell-surface ratio quantification (flow cytometry) showing reduced amounts of LAMP1?-RUSH at the plasma membrane after transient KO of PIP5K homologs and 35 min of biotin exposure. (H) Widefield imaging of LAMP1?-RUSH reporter in triple PIP5K1A/B/C KO cells 1 h after biotin addition. When compared to WT, KO cells show accumulation of post-Golgi LAMP1?-RUSH carriers. Scale bar: 20 µm; insert: 4 µm. Nucleus stain = DAPI. Error bar = SD of at least three independent experimental repeats. Student’s t test was performed on data in B, C, and D, and Tukey’s multiple comparisons test (HSD, FWER = 0.05) was performed on data in G. ***P = 0.001.
Supplier Page from Abcam for Anti-EXOC1 antibody