Fig 1: Knockdown of ASC1 down-regulates expression of erythroid transcription factors but does not activate the UPR. a. Xbp-1 mRNA splicing in ASC1-depleted K562 cells. mRNA from DTT (dithiothreitol)-treated HeLa cells was used as a positive control. b. Expression of Grp78 in K562 cells with knockdown of ASC1, Uba5 and UFBP1. The data are presented as the relative level to K562 cells treated with scrambled shRNA. * p < 0.01 (n = 3). c. Expression of GATA-1 and Klf1 in ASC1-knockdown K562 cells. The data are presented as the relative level to the cells with scrambled shRNA. * p < 0.01 (n = 3). d. GATA-1 and KLF1 protein level in ASC1 knockdown cells. e. Subcellular localization of ASC1 in UFBP1 and Uba5 knockdown K562 cells. Subcellular fractionation of K562 cells was performed according to [6]. Lamin B1 was used as the nuclear marker, while GAPDH was used as the cytosolic marker. f. ChIP analysis of ASC1 association to the promoters of GATA-1 and Klf1 genes in BM cells. BM and spleen cells were isolated from phenylhydrazine-treated control and UFBP1 CKO mice, and subjected to ChIP assays. c-Myc and CCND1 promoters were used as positive ASC1 targets while actin promoter was the negative control. Data are presented as means ± SD (n = 3). g. A working model for the role of ufmylation in regulation of hematopoiesis.