Fig. 5: Expansion of LYMTAC platform to other LMPs. | Nature Communications

Fig. 5: Expansion of LYMTAC platform to other LMPs.

From: LYMTACs:chimeric small molecules repurpose lysosomal membrane proteins for target protein relocalization and degradation

Fig. 5

A Schematic of Lysosome Membrane Proteins (LMPs). B CHX assay in FLAG-MTH1-LAPTM4a-stably expressing HCT116 cells. Cells were pre-treated with DMSO, 200 nM Bafilomycin A1 (BafA1), or 1 µM E1 inhibitor (TAK-243) for 30 min, followed by co-treatment with CHX for 4 h. Cells were harvested at 0 h to include as a control. Cells were lysed and subjected to immunoblotting with MTH1 and vinculin antibodies. Quantified data are representative of two independent experiments. C CHX assay in FLAG-MTH1-LAPTM5-stably expressing HCT116 cells. Quantified data are representative of two independent experiments. D HiBiT cellular degradation assay in FLAG-MTH1-LAPTM4a- and FLAG-MTH1-LAPTM5-stably expressing HCT116 (knock-in-HiBiT- FKBP12F36V-KRASG12D: (HF-KRAS)) cells. Cells were treated with increasing concentrations of LYMTAC-2 for 24 h, and HiBiT levels were measured using Nano-Glo® HiBiT lytic reagent. Data are representative of three independent experiments and reported as the mean ± S.E. E HCT116 (HF-KRAS) cells stably expressing FLAG-MTH1-LAPTM5 were pre-treated with BafA1, 2 BTZ, or TAK-243 for 30 min, followed by co-treatment with DMSO or 500 nM LYMTAC-2 for 6 h and subjected to immunoblotting with KRAS and tubulin antibodies. Quantified data are representative of two independent experiments. F KRAS ubiquitylation assay. HA-ubiquitin was transfected into HCT116 expressing FLAG-MTH1-LAPTM5, which were pre-treated with 200 nM BafA1 for 30 min, followed by co-treatment with DMSO or 1 µM LYMTAC-2 treatment for 4 h. Next, cells were lysed and subjected to immunoprecipitation with anti-HiBiT antibody. Whole cell lysate was used as the input. The respective blots were probed with HA, MTH1, HiBiT, and vinculin antibodies. Data are representative of two independent experiments. G AsPC-1 cells stably expressing FLAG-MTH1-LAPTM4a were pre-treated with either pan-KRAS inhibitor or MTH1 ligand, followed by DMSO or 100 nM LYMTAC-4 treatment for 6 h. Cells were lysed and subjected to immunoblotting with KRAS, pERK, and tubulin antibodies. Quantified data are representative of two independent experiments.

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