Article Figures & Data
Figures
- Figure 1
Ex vivo CD34+ human hematopoietic progenitor cell culture model of erythropoiesis. (A) Illustration of the morphological changes that occur during erythropoiesis. (B) Representative images of cells from days 1, 5, and 7 of erythroid differentiation. Cells were cytospun, fixed, and stained with the May-Grunwald and Giemsa stains. Bar, 15 nm. IL, interleukin; SCF, stem cell factor; EPO, erythropoietin.
- Figure 2
Ubiquitin-specific protease 50 (USP50) is increased during erythropoiesis. (A) Total RNA was extracted from human CD34+ hematopoietic progenitor cells (HPCs) on day 1 and day 3 of erythroid differentiation. Real-time quantitative PCR was used to compare ubiquitin-specific protease mRNA levels. Amplification of each sample was normalized to its glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript levels. Shown are the ratios of day 3/day 1 in a representative experiment from five independent experiments. (B) Total RNAs were extracted on days 1, 3, 5, and 9 from HPCs. USP50 mRNA levels were measured by real-time qPCR. Shown are data in a representative experiment from five independent experiments. (C) Whole cell lysates were made from HPCs on days 0, 1, 3, 5, and 9 of erythroid differentiation. USP50 and β-actin proteins were detected by immunoblot. Representative blots were shown from five independent experiments.
- Figure 3
Ubiquitin-specific protease 50 (USP50) is localized in the nuclei and its binding partners are identified. (A) Mouse lung epithelial (MLE12) cells were transfected with USP50-V5 plasmids and incubated for 24 hours. Cells were fixed and stained with a V5 antibody. USP50-V5: green; DAPI (4',6'-diamidino-2-phenylindole, nuclear DNA stain): blue. (B) HEK293T cells were transfected with USP50-V5 plasmid and incubated for 24 hours. Immunoprecipitation with a V5 antibody, followed by SDS-PAGE and LC-MS/MS analysis. Shown are potential USP50 binding partners separated by known intracellular location or function based on literature.
- Figure 4
Ubiquitin-specific protease 50 (USP50) reduces Ku70 protein stability. (A) Mouse lung epithelial (MLE12) cells were transfected with 0–4 µg USP50-V5 plasmids per 35 mm dish and incubated for 24 hours. Total RNA was extracted and Ku70 mRNA levels were compared by real-time qPCR. The expression of Ku70 transcripts was normalized to Gapdh. The results were also normalized to 0 µg of USP50 plasmid. (B) MLE12 cells were transfected with USP50-V5 plasmids (0–4 µg of DNA per 35 mm dish) and incubated for 24 hours. Protein expression in whole cell lysates was analyzed by immunoblotting with antibodies against Ku70, V5, and β-actin. (C) MLE12 cells were transfected with USP50-V5 plasmid and incubated for 24 hours. Cells were treated with cycloheximide (CHX) (40 µg/mL) for 0–8 hours. Expression of proteins in whole cell lysates was analyzed by immunoblotting with antibodies against Ku70, V5, and β-actin.
- Figure 5
Ubiquitin-specific protease 50 (USP50) is a not stable protein. (A) Mouse lung epithelial (MLE12) cells were transfected with USP50-V5 plasmids and incubated for 24 hours. Cells were treated with cycloheximide (CHX) (40 µg/mL) with or without MG132 (20 µg/mL) or leupeptin (20 µg/mL) for 0–2 hours. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin. (B) MLE12 cells were transfected with USP50-V5 and treated with 5 µg/mL lipopolysaccharide (LPS) for 5 hours. Total RNA was extracted and USP50 mRNA levels were determined by real-time qPCR. The expression of USP50 transcripts was normalized to Gapdh.
