The E3 ubiquitin ligase DESYNAPSIS1 regulates synapsis and recombination in rice meiosis-Lijun Ren
This file provides original photos of western blotting analysis in the paper.
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Ubiquitination Assay in E. coli The in vitro ubiquitination assay was performed according to the procedure of Han et al. (2017) with some modifications. To conduct co-transformation assays in Transetta (DE3), the coding sequence of chloramphenicol in the pACYCDuet plasmid was replaced with the coding sequence of ampicillin, which was amplified from pGEX4T-2. Transetta (DE3) strains containing the expression cassettes of E1, E2, E3, ubiquitin, and substrate proteins, or strains lacking some of these components, were cultured at 37 ℃ until an ODq600 of 0.4-0.6 was reached. Protein expression was subsequently induced by addition of 0.5 mM IPTG. The strains were further cultured at 28 ℃ for 12 h. Crude protein extracts were prepared from 1 mL of cultured bacteria and separated by SDS-PAGE electrophoresis, followed by western blotting analysis with corresponding antibodies. Western Blotting The cultured bacteria were collected and resuspended in 1×PBS buffer. 5× Protein loading buffer was added to the suspension before boiling. Bacterial lysate was centrifuged at 13000 rpm for 10 min. The supernatants were used for western blotting analysis. SDS-PAGE was performed to separate the proteins in supernatants. And the proteins were transferred to a Polyvinylidene-Fuoride (PVDF) membrane (GE Healthcare) at 4 ℃ for 2 h. Subsequently, immunoblotting analysis was conducted by adding HRP-coupled anti-Flag or anti-Myc antibody and then was incubated at 37 ℃ for 1h. After washing off nonspecific binding antibodies, target proteins on the PVDF membrane were visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). The image was captured by Tanon-5200 Chemiluminescent Imaging System (Tanon, China).