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. 2018 Jul 31;7:e36362. doi: 10.7554/eLife.36362

Figure 3. Biophysical properties of G2-PQSs from PSPs: Gel migration, Thioflavin T fluorescence, UV (295 nm), CD.

All biophysical studies were performed in 100 mM K+. PAGE migration and Thioflavin T staining were performed at different migration times on distinct gels. Melting temps (TM 295) are shown in the plots; a K+-effect (1 mM and 100 mM K+) on TM 295 was found for ARG25Q1 (+18.8°C), OAZ25Q2 (+20.9°C) and SMS5Q1 (+18.4°C); mutated controls ARG25Q1M, OAZ25Q1M, SMS5Q1M, AZIN15QM1, AZIN15QM2, AZIN15QM3, did not show any (TM 295) melting transitions (data not shown and Figure 3—figure supplement 1; Figure 5—figure supplement 3). CD measurements were not performed for ARG23Q1 due to the presence of multiple RNA species. Error bars represent the standard error (SE) from two independent replicates. (Gels in this figure were cropped: full length gels are in Figure 3—figure supplement 3 and 4.

Figure 3.

Figure 3—figure supplement 1. UV melt-anneal profiles for mutant G2-PQS's.

Figure 3—figure supplement 1.

UV melt-anneal profiles for NRASM, ARG25Q1M, ODC5Q2M, OAZ25Q1M, OAZ25Q2M, SMS5Q1M and ARG23Q1M at 295 nm (performed in the presence of 100 mM K+, UV performed in 1 mM K+ for NRAS). Two replicates of each biophysical experiment were performed. NRAS: UGUGGGAGGGGCGGGUCUGGG. NRASM: UGUAAAAGGGGCGGGUCUGGG.
Figure 3—figure supplement 2. Additional biophysical data.

Figure 3—figure supplement 2.

(a) Biophysical properties of the G2-PQS from SMS3Q1 and SAT5Q1 and mutants where relevant. Gel migration, Thioflavin T fluorescence, UV (295 nm), CD. All biophysical studies were performed in 100 mM K+. For the Thioflavin-T assay, the error bars represent the standard error (SE). (b) UV melt profile (295 nm) of AZIN15Q1 and CD trace of AZIN15Q1 and AZIN15Q1M. Performed in the presence of 100 mM K+. Two replicates of each biophysical experiment were performed.
Figure 3—figure supplement 3. Full size migration gels from cropped images.

Figure 3—figure supplement 3.

Full-length gels (shown as cropped images in Figure 3 and Figure 3—figure supplement 2).
Figure 3—figure supplement 4. Full size thioflavin gels from cropped images.

Figure 3—figure supplement 4.

Full-length gels (shown as cropped images in Figure 3 and Figure 3—figure supplement 2).