Tnt t7 insect cell extract protein expression system

ABCG2-HA was transcribed and translated using the TNT T7 Insect Cell Extract Protein Expression System (Promega). After affinity purification of ABCG2-HA from extracts of SF21 insect cells and ENPP1-MF or GFP-F from extracts of 293T cells, co-immunoprecipitation was performed as described above.

We tested three different commercially available cell-free expression systems: the PURExpress In Vitro Protein Synthesis System (New England Biolabs, Ipswich, MA, USA), the S30 Extract System (Promega, Madison, WI, USA), and the TnT T7 Insect Cell Extract Protein Expression System (Promega) based on the S. frugiperda cell line Sf21. We followed the manufacturer’s recommendations for each kit, using 1.5 mL Eppendorf tubes for each reaction. The S30 Extract System lacks methionine, so we added this amino acid to match the concentrations in the other systems. All tubes were stored at −20 °C after the reaction. Protein synthesis was confirmed by 1D SDS-PAGE. We mixed 1 µL of the reaction with 1 µL of Tricine sample buffer and incubated it for 5 min at 95 °C. The sample was then loaded onto a 16.5% Mini-PROTEAN Tris-Tricine Gel (Bio-Rad, Hercules, CA, USA) and placed in a Mini-PROTEAN Tetra System chamber (Bio-Rad) filled with 10x Tris/Tricine/SDS running buffer. After electrophoresis at 100 V for 100 min, the gel was stained overnight with Roti-Blue quick solution (Carl Roth, Karlsruhe, Germany).

The smORF sequences cloned into the pF25A ICE T7 Flexi vector (Promega) were translated in vitro using TnT^® T7 Insect Cell Extract Protein Expression System (Promega). After addition of Laemmli buffer, translation products were separated on 4–20% SDS-PAGE gels (BioRad) and transferred to 0.1 µm nitrocellulose membrane (Amersham GE Healthcare, UK) according to Lauressergues et al. 2015 [72 (link)]. The membrane was blotted overnight at 4 °C with rabbit anti-HA (C29F4) from Cell Signalling Technology (Beverly, MA, USA), washed then incubated at room temperature with HRP-conjugated secondary antibodies (sc-516102, Santa Cruz Biotechnology, CA, USA). After washes, detection was achieved with chemiluminescence detection reagent (Clarity Western ECL substrate, BioRad). Image acquisitions of immunoblots were performed with a ChemiDoc Touch imaging system (BioRad).