Jetpei transfection reagent

Tsc2+/+Tp53−/− and Tsc2−/−Tp53−/− mouse embryonic fibroblasts (MEFs) were a kind gift from Prof. D. Kwiatkowski (Harvard University, Boston, MA, USA) and were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal calf serum and 100 μ/mL penicillin streptomycin (Life Technologies/ThermoFisher Scientific, Waltham, MA, USA). Human TSC2-deficient angiomyolipoma AML 621-101 cells [17 (link)] were grown as above but were supplemented with 20% (v/v) fetal calf serum. Transfections were carried out using a JETPei transfection reagent (VWR International, Leicestershire, UK) as directed by the manufacturer’s protocol. Drugs were treated as indicated. Hypoxia experiments were set up in a Binder CB150 hypoxic chamber set at 1% O₂ overnight (18 h). Cells were lysed directly in sample buffer (62.5 mM Tris–HCl (pH 7.6), 50 mM dithiothreitol, 2% (w/v) sodium dodecyl sulfate, 10% (w/v) glycerol, and 0.1% (w/v) bromophenol blue), sonicated for three 20 s cycles on full power (30 amplitude microns), heated at 95 °C for 10 min, and centrifuged at 13,000 rpm for 8 min. Protein quantification was carried out using the Pierce 660 nm protein reagent (with an ionic detergent compatibility reagent bought from ThermoFisher Scientific, Waltham, MA, USA) before being subjected to Western blotting.

The FLAG-tagged FGF23, Myc-tagged ANGPTL3 with the T₂₂₅G modification (Song et al., 2014 (link)), and untagged ppGalNAc-T3 (from [Kato et al., 2006 (link)] and cloned into PCDNA 3.0 using BamH1 sites) were transfected into HEK cells using the JetPEI transfection reagent (VWR International, Radnor, PA, Cat#101–40N) according to the manufacturer’s instructions. After 24 hr, the medium was replaced with serum-free MEM containing the compound for 6 hr. The medium and cells were then collected and, after trichloroacetic acid precipitation of the medium, analyzed by immunoblot using anti-FLAG antibody at 1:1000 or anti-Myc antibody at 1:2000 and then the peroxidase-coupled secondary antibodies. Emission was captured and quantified using a ChemiDoc Touch Imaging System with Image Lab Software (BioRad, SCR_014210). For ppGalNAc-T3 determinations in different cell lines, cells were collected and lysed with 100 µl buffer (10 mM Tris-HCl (pH8.0), 1 mM EDTA (ACROS ORGANICS, Cat#446085000), 1% Triton X-100, 0.1% sodium deoxycholate (Fisher Scientific, Cat#BP349-100), 0.1% SDS (Fisher Scientific, Cat#BP166-500), 140 mM NaCl (Fisher Scientific, Cat#S271-3), 1 mM PMSF (Sigma-Aldrich, Cat#PMSF-RO). Then 15 µl of each lysate was analyzed by immunoblotting using the purchased anti-ppGalNAc-T3 and anti-α-tubulin antibodies.