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. 2022 Nov 8;13:6732. doi: 10.1038/s41467-022-34612-y

Fig. 4. The UBX domains of IARS1 are responsible for BRCA1 stability.

Fig. 4

a Protein extracts from HeLa cells transfected with control siRNA or two siRNAIARS1 were immunoblotted with anti-IARS1, anti-BRCA1, and anti-Actin (loading control) antibodies. b SV40 NLS fused wild-type IARS1 or -IARS1 UNE-I (residues 942-1262) was ectopically expressed following depletion of IARS1 using 3’UTR-specific siRNAIARS1. a, b The data were representative of three independent experiments. Source data are provided as a Source Data file. c The stability of BRCA1 in cells with or without IARS1 depletion was assessed by cycloheximide-chase analysis. HEK293T cells were transfected with control siRNA or 3’UTR-specific siRNAIARS1 and treated with 100 µg/mL cycloheximide. Lysates prepared at the indicated time points were immunoblotted with an anti-BRCA1 antibody. The graph shows relative levels of BRCA1 expression. Error bars indicate standard deviation (SD) of the mean (n = 3, independent cell cultures). d In vivo ubiquitylation assay of BRCA1 in transfected HEK293T cells with or without IARS1 depletion. The blot with short exposure is shown. HEK293T cells co-transfected with control siRNA or 3’UTR-specific siRNAIARS1 and Ub were treated with 10 µM MG132 for 4 hr and analyzed by immunoblotting with the indicated antibodies. BRCA1 immunoprecipitated with an anti-BRCA1 antibody was analyzed by immunoblotting with an anti-Ub antibody. Quantification of ubiquitylated BRCA1 and the blot with long exposure are shown in Supplementary Fig. 10a and c. e In vivo ubiquitylation assay of BRCA1 in HEK293T cells transfected with full-length IARS1, IARS1ΔC, and IARS1 UNE-I alone. The blot with short exposure is shown. HEK293T cells co-transfected with indicated cDNAs and Ub were treated with 10 µM MG132 for 4 h and analyzed by immunoblotting with the indicated antibodies. BRCA1 immunoprecipitated with an anti-BRCA1 antibody was analyzed by immunoblotting with an anti-Ub antibody. Quantification of ubiquitylated BRCA1 and the blot with long exposure are shown in Supplementary Fig. 10b and d. d, e The data are representative of three independent experiments. Source data are provided as a Source Data file.