*4.6. Microscale Thermophoresis*

RNA-protein binding assays were carried out on a Microscale Thermophoresis system (Monolith NT. 115 from NanoTemper Technologies, München, Germany). Standard treated capillaries (Cat. Number: MO-K002) were used for measurements. Instrument settings are presented in Table 2.


**Table 2. Instrument settings for MST**.

Normalized fluorescence values after 1.25 s after turning on the IR laser were used as T-jump values.

RNA concentrations were set to give an initial raw fluorescence between 300 and 1000 counts and varied between 30 and 100 nM. All experiments were done at room temperature. DEPC-treated PBS buffer containing 0.05% NP-40 was used as assay buffer.

#### *4.7. Electrophoretic Mobility Shift Assay (EMSA)*

LightShift® Chemiluminescent RNA EMSA Kit (Thermo Scientific, Cat. No. 20158, Thermo Fisher Scientific, Waltham, UK) was used for the EMSA experiments. Assay control was performed according to the instructions of the manufacturer with the control reaction provided with the kit. In short, 6.25 nM biotin-labeled IRE RNA was incubated with 2 μg of cytosolic liver extract with or without 1 μM of unlabeled IRE RNA. The result of the assay control is presented on Supplementary Figure S6. Binding, electrophoresis and detection of the tested RNAs with the proteins were carried out following the protocol of the kit. Briefly, proteins of varying concentrations were incubated with 1 or 2 nM of RNAs for 30 min at room temperature, then loaded on 4 or 6% native polyacrylamide gels. RNA was transferred to nitrocellulose membranes using Trans-Blot® Turbo™ Transfer System (Bio-Rad, Hercules, CA, USA) and crosslinked to the membrane by UV-light crosslinking. After proper washing and blocking, biotin labeled RNA was detected by chemiluminescence using Streptavidin-Horseradish Peroxidase Conjugate.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/19/11/3478/ s1.

**Author Contributions:** Conceptualization, A.T.; Data curation, B.S. (Beáta Szabó), E.S. and B.S. (Bálint Szeder); Formal analysis, J.K.; Funding acquisition, J.K., L.B. and A.T.; Investigation, B.S. (Beáta Szabó), N.M., R.A. and J.K.; Methodology, B.S. (Beáta Szabó) and A.T.; Project administration, B.S. (Beáta Szabó) and A.T.; Resources, B.S. (Beáta Szabó) and N.M.; Software, E.S.; Supervision, L.B. and A.T.; Validation, J.K. and L.B.; Visualization, A.T.; Writing—original draft, A.T.; Writing—review & editing, B.S. (Beáta Szabó), N.M., R.A., E.S., J.K., B.S. (Bálint Szeder) and L.B.

**Funding:** This research was funded by the National Research, Development and Innovation Office, Hungary (grants: K-125340 (A.T.), K-120391 (J.K.), KH-125597 (J.K. and A.T.), TÉT\_16-1-2016-0134 (J.K.), 2017-1.2.1-NKP-2017-00002 (J.K.), Medinprot synergy grant (A.T. and J.K.)) and a Korea-Pan-European Research Agreement (A.T.).

**Conflicts of Interest:** The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
