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. Author manuscript; available in PMC: 2020 Sep 19.
Published in final edited form as: Cell Chem Biol. 2019 Jul 18;26(9):1283–1294.e5. doi: 10.1016/j.chembiol.2019.07.002

Figure 4: TCH-165 stimulates proteasomal degradation of SNAP29 and STX17:

Figure 4:

(A) Percent disorder in autophagy fusion proteins were predicted using predictor of natural disorder region (PONDR) software(Xue et al., 2010). (B) HeLa cells were treated with either vehicle, torin1 (200 nM), TCH-165 (10 μM) or combinations for 16h. Cell lysates were immunoblotted for STX17, SNAP29, VAMP8 and GAPDH. (C) HEK293T or RPMI-8226 cells were treated with either vehicle or TCH-165 (10 μM) for 16h and whole cell lysates immunoblotted for STX17, SNAP29, VAMP8 and GAPDH. (D) U-87 MG cells were treated with cycloheximide (100 μg/mL) plus vehicle, TCH-165 (10 μM), bortezomib (BTZ; 5 μΜ) or combinations for 8h. Whole cell lysates were immunoblotted for STX17, SNAP29, p62, LC3B and GAPDH. The degradation of SNAP29 (E) and STX17 (F) were quantified with image J software. Statistical analyses were performed on four independent experiments (n=4) using unpaired t-test on GraphPad Prism7. (G) U-87 MG cells were treated with vehicle or TCH-165 (10 μM) for 8h or 24h and mRNA quantified by RT-qPCR. Data are presented as mean ± SD of two independent experiments (n=2), each ran in triplicate. See also supplemental table S1