Skip to main content
. 2023 Dec 22;120(52):e2310542120. doi: 10.1073/pnas.2310542120

Fig. 4.

Fig. 4.

HEI10 promotes RPA1a ubiquitination. (A) Measurement of ubiquitination levels of RPA1a immunoprecipitated from tobacco cell protein extracts by anti-Flag and anti-UBQ11 antibodies. GFP-Flag is used as the negative control. (B) Tube2 IP tests the enrichment of ubiquitin-modified RPA1a-Flag proteins in tobacco cells. GFP-Flag is used as the negative control. (C) Detection of the ubiquitination levels in protein extracts from tobacco cells expressing RPA1a alone or with HEI10. The RPA1a protein level is determined with anti-Myc antibody; the ubiquitination level is determined with anti-UBQ11 antibody. (D) In vitro ubiquitination assay, the recombinant His-Sumo-RPA1a proteins are incubated with the extracts from central inflorescences of Col-0 and HEI10 overexpressing plants (HEI10-OE) at room temperature for 2 h by adding 10 mM ATP plus 50 μM MG132. The samples are analyzed by western blot using anti-His and anti-UBQ11 antibodies. (E) Investigation of the stability of RPA1a proteins treated by 26S proteasome inhibitor MG132 in tobacco cells. The samples are collected at 48 h and 60 h after agrobacterium injection. 50 μM MG132 is injected into the leaves for 12 h before sampling. The protein level of RPA1a-Myc is determined with anti-Myc antibody. GFP is coexpressed with RPA1a as the expression control.