Fig. 1
From: REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice
Abrogation of rXCI in Rnf12−/− differentiating ESCs is rescued by knockout of Rex1. a Scatter plot showing the correlation of the H:L log2 ratios for the quantitatively identified proteins in the SILAC experiment in ESCs between two biological replicates (heavy-Rnf12−/−:light-WT and heavy-WT:light-Rnf12−/−) showing RNF12-dependent changes in REX1 stability. Upregulated proteins are indicated in blue (log2 ratios >0.585 and log2 ratios <−0.585 on the x and y axes, respectively). Depleted proteins are indicated in green (log2 ratios <−0.585 and log2 ratios >0.585 on the x and y axes, respectively). b Genotyping analysis of Rnf12 and Rex1 deletions in WT, Rnf12CR−/CR−, Rnf12CR−/CR−:RexCR−/CR− (clones 36 and 27) and Rnf12CR−/CR−:Rex+/CR− (clone 17) ESCs. Bottom panel shows a Pf1M1 restriction fragment length polymorphism (RFLP) analysis to detect the presence of two X chromosomes in the same ESC lines. Location of green, purple and red genotyping primers is depicted in Supplementary Fig. 2b. c Nuclear extracts of WT, Rnf12CR−/CR−, Rnf12CR−/CR−:RexCR−/CR− (clones 36 and 27) and Rnf12CR−/CR−:Rex+/CR− (clone 17) ESCs were immunoblotted with RNF12 and REX1 antibodies. ACTIN was used as a loading control. Uncropped WB images are found in Supplementary Fig. 10a. d Immunohistochemistry of REX1 (grey), RNF12 (red) and DNA (DAPI, blue) of WT, Rnf12−/− and Rnf12CR−/CR− ESCs. Note the recognition of the remaining 333 aa in RNF12−/− ESCs by the RNF12 antibody. Scale bar: 20 μm. e Xist RNA-FISH (FITC) analysis on WT, Rnf12CR−/CR−, Rnf12CR−/CR−:RexCR−/CR− (clones 36 and 27) and Rnf12CR−/CR−:Rex+/CR− (clone 17) ESCs at day 6 of differentiation. DNA was stained with DAPI (blue). Scale bar: 20 μm. f Quantification of cells with Xist clouds in WT, Rnf12CR−/CR−, Rnf12CR−/CR−:RexCR−/CR− (clones 36 and 27) and Rnf12CR−/CR−:Rex+/CR− (clone 17) ESCs at day 3 and day 6 of differentiation. g QPCR analysis of Xist expression in undifferentiated, day 3 and day 6 differentiated WT, Rnf12CR−/CR−, Rnf12CR−/CR−:RexCR−/CR− (clones 36 and 27) and Rnf12CR−/CR−:Rex+/CR− (clone 17) ESCs (average expression ± s.d., n = 3 biological replicates)
