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. 2023 Mar 15;222(5):e202205137. doi: 10.1083/jcb.202205137

Figure 5.

Figure 5.

Exocyst is essential for the secretion of various soluble cargoes. (A) Schematic of novel PiggyBac transposon RUSH backbone. Genes encoding proteins of interest were either cloned upstream (BamHI) or downstream (XbaI) of HALO. (B) Schematic representation of guide infection followed by RUSH and flow cytometry analysis/imaging of reporter protein of interest. (C) Flow cytometry–based analysis of protein secretion 12 h after biotin addition of indicated RUSH cargoes in WT and EXOC3 KO cells. Each condition is normalized to 0 h control resulting in a percent of cargo secreted per sample and condition. Minimum of 30,000 cells per biological repeat, three repeats per sample (blue dots). Statistical comparison = two-tailed t test. (D) Live lattice-SIM imaging of RUSH cargo proteins in EXOC3 KO cells 12 h after biotin addition. When compared to WT, EXOC3 KO cells show substantial accumulation of carriers containing cargo proteins of interest near the plasma membrane. Scale bar: 10 μm; insert: 2 μm. Error bar = SD of at least three independent experimental repeats. Student’s t test was performed on data in C. **P ≤ 0.01; ***P ≤ 0.001.