Tnt quick coupled translation system

In vitro translation was performed using TnT Quick Coupled Translation System (Promega, #L1170) according to the manufacture’s instruction. Briefly, pCS4-HA-RUNX3, pCS4-HA-G9a, pCS4-HA-G9a-ΔSET plasmids were translated with TnT rabbit reticulocyte lysate using a TnT SP6 RNA polymerase. In vitro translated proteins were electrophoresed in SDS-PAGE gel and western blot analysis was performed with anti-HA antibody (SC-7392, Santa Cruz) to confirm the protein synthesis. Proteins (400 µg) were immunoprecipitated with anti-RUNX3 antibody (ab40278, Abcam) and immunoblotted with anti-HA antibody to check their interaction.

Purified mitochondria were resuspended in the mitochondria isolation buffer (MIB, 220 mM mannitol, 140 mM Sucrose, 10 mM HEPES/KOH pH 7.4, 1 mM EGTA/KOH).
For in vitro import assay WT and MLS-TFEB cDNA were cloned into the pGEM-4 vector and transcribed in vitro using the TNT Quick coupled translation system (L1170, Promega), by incubating 200 ng of DNA with 7 μL of TNT-T7 reticulocyte lysate mix and 0.5 µL of 35S-labeled methionine (NEG709A500UC, PerkinElmer) for 1 h at 30 °C at 600 rpm. Next, the mitochondrial pellets were resuspended into mitochondrial import buffer (500 mM Sucrose, 10 mM MgAc, 160 mM KAc, 20 mM sodium succinate and 40 mM Hepes-KOH pH 7.4, 0.3 M ATP, and 1 M fresh DTT). Then, mitochondria were incubated with the lysate containing TFEB transcribed in vitro and the membrane potential was blocked by adding 1 µL of 20 mM CCCP at different time points. Mitochondria were also treated with Trypsin (T1426, Sigma Aldrich) (25 μg/mL for 10 min) followed by Trypsin inhibitor soya bean (Merck, 10109886001) (250 μg/mL for 15 min). 1% Triton X-100 was eventually added to permeabilize the mitochondrial membranes and allow the Trypsin to reach the mitochondrial matrix. The mitochondrial pellet was lysed in denaturing conditions and proteins were detected by autoradiography.