Torin 2

Zfp418 was amplified from mouse ventricular RNA extractions by PCR, 5ʹ-tagged with Flag and cloned into pShuttle-CMV with SalI and HindIII restriction enzymes. For overexpression of Flag-mZfp418, GFP-CryAB^R120G or GFPu [60 (link),61 (link)], NRVMs were transduced with the respective adenoviruses for 2 h. For proteasome inhibition, NRVMs were treated for 24 h prior to harvest or fixation with 250 nM epoxomicin (Calbiochem, 324,801). For the GFPu chase assay, cells were incubated in culture medium with 100 µM cycloheximide (Sigma-Aldrich, c4859) for indicated times. For ALP activation, NRVMs were treated with 1 μM torin 1 (Tocris Bioscience, 4247) or 50 nM torin 2 (LC laboratories, T8448) for 3 h prior to harvest or fixation. For ALP inhibition, NRVMs were treated with 100 μM leupeptin (Sigma-Aldrich, L8511) for 24 h or 30 nM bafilomycin A₁ for 3 h prior to harvest or fixation. Medium was changed with every compound treatment.