AMZ30 is a selective inhibitor of PME-1 (IC50: 600 nM). It also reduces the demethylated form of PP2A in living cells.

PME-1:600 nM

将HEK 293T细胞与不同浓度的AMZ30孵育，得到了PME-1抑制的原位IC50值为3.5 μM。在稳定过表达PME-1的HEK 293T细胞中，AMZ30 (20 μM) 使去甲基化的PP2A水平降低了约80%。AMZ30处理还显著增加了PP2A的甲基化形式。同样，AMZ30 (20 μM) 也在未转染的HEK 293T细胞中降低了PP2A的去甲基化形式，这些细胞表达PME-1的基础水平。

Purified wild-type PME-1 (500 nM, 2.6 mL total volume in PBS) was incubated with DMSO or AMZ30 (50 μM) at 25 °C. After 30 min, 100 μL was removed from each reaction (Fraction A). The remaining 2.5 mL of each reaction was passaged over a Sephadex G-25M column and eluted in a volume of 3.5 mL PBS (Fraction B). 100 μL of both fractions A and B were labeled with FP-rhodamine (2 μM). After 30 min, the reactions were quenched with 2× SDS-PAGE loading buffer, separated by SDS-PAGE, and analyzed by in-gel fluorescence scanning. Gels were then subjected to Coomassie staining with InstantBlue to verify equivalent protein loading.

HEK 293T cells were grown in DMEM SILAC media supplemented with dialyzed fetal bovine serum and 12C14N-lysine and - arginine for "light" cells or 13C615N2-lysine and -arginine for "heavy" cells. Cells were treated as indicated with DMSO or 28 (1 h, 37 °C), washed, harvested, and soluble and membrane proteomes were isolated as described above. Light and heavy proteome fractions (0.5 mg each) were combined (1 mL total volume) and labeled with 5 μM of FP-biotin for 1 hr at 25 °C. After incubation, the membrane proteomes were solubilized with 1% Triton-X and rotated at 4 °C for 1 hr. Enrichment of FP-labeled proteins was achieved as previously described.34 The streptavidin-enriched proteome was washed two times for 3 min with (1) 1% SDS in PBS, (2) 6 M urea in PBS, (3) PBS (pH 7.5) and finally resuspended in 200 μL 8 M urea in 25 mM ammonium bicarbonate. Samples were then prepared for on-bead digestion by reduction with 10 mM TCEP for 30 min at 25 °C and alkylation with 12 mM iodoacetamide for 30 min at 25 °C in the dark. Samples were diluted to 2 M urea with PBS (pH 7.5) and digestions were performed for 12 hr at 37 °C with sequence-grade modified trypsin (4 μL of 0.5 μg/μL) in the presence of 2 mM CaCl2. Lastly, peptide samples were acidified with formic acid to a final concentration of 5%.