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. 2023 Jan 18;120(4):e2217255120. doi: 10.1073/pnas.2217255120

Fig. 3.

Fig. 3.

Desi3a deSUMOylates BRI1 upon changes in ambient temperature. A. In vivo SUMOylation analyses of BRI1 at 21 °C or 26 °C. Immunoprecipitation was carried out using anti-GFP antibodies on solubilized protein extracts from mono-insertional homozygous BRI1-mCitrine plants grown at 21 °C or 26 °C. Detection of immunoprecipitated proteins used the anti-SUMO1 and anti-GFP antibodies. B. Quantification of BRI1 SUMO at 21 °C and 26 °C relative to immunoprecipitated BRI1-mCit levels. Error bars represent SD (n = 3). The asterisks indicate a statistically significant difference between 21 °C and 26 °C (Mann–Whitney). C. Confocal microscopy analyses of BRI1 and Desi3a at 21 °C and 26 °C. BRI1-mCit and Desi3a-mch were transiently expressed in N. benthamiana leaves and incubated at 21 °C or 26 °C for 2 d. Similar confocal detection settings were used to compare the effect of temperature on BRI1 and Desi3a proteins levels. Several independent experiments were carried out and multiple regions of interest analyzed to overcome the variability in transformation efficiency. (Scale bars, 20 μm.) D. Quantification of BRI1 and Desi3a fluorescence levels in experiments carried out as in C. Multiple regions of interest analyzed for to overcome the variability in transformation efficiency. Error bars represent SD (n = 15). The asterisk indicates a statistically significant difference between BRI1-mCit or Desi3a-mCh at 21 °C and 26 °C (Mann–Whitney). E. Western blot analyses monitoring the accumulation of Desi3a-HA protein in Arabidopsis plants grown at 21 °C or 26 °C. Detection of Desi3a-HA was performed with anti-HA antibodies. The membrane was stripped and probed with anti-FBPase antibodies as loading control. F. Quantification of Desi3a protein at 21 °C and 26 °C relative to FBPase levels. Error bars represent SD (n = 3). The asterisks indicate a statistically significant difference in Desi3a protein level at 21 °C and 26 °C (Mann–Whitney). G. In vivo interaction between BRI1 and Desi3a at 21 °C and 26 °C. BRI1-mCit and Desi3a-HA were transiently expressed in N. benthamiana and incubated at 21 °C or 26 °C for 2 d prior to immunoprecipitation using anti-GFP antibodies on solubilized protein extracts. GFP alone was used as negative control. Detection of immunoprecipitated proteins used the anti-HA and anti-GFP antibodies.