human heart liver tissue lysates  (Proteintech)

Journal: PLoS ONE

Article Title: Common Minor Histocompatibility Antigen Discovery Based upon Patient Clinical Outcomes and Genomic Data

doi: 10.1371/journal.pone.0023217

Figure Lengend Snippet: The iTopia epitope binding assay was used to measure T4A and the alternately encoded T4E peptide (GLYTYWSAGE) binding to HLA-A2 compared to the percentage of maximal binding of the iTopia control peptide (FLPSDFFPSV). T4A binding to HLA-A0201 is 95% of that compared to the positive control; however, T4E binds HLA-A0201 at 24% that of the positive control. According to the manufacturer, peptides binding of >30% that of the control peptide are candidate HLA-A0201 epitopes (4A). The iTopia assay was also used to measure the ED 50 and dissociation t 1/2 of T4A to HLA-A2 relative to the iTopia positive control (FLPSDFFPSV) labeled “POS”. T4A binds with a 50% effective dose of ED 50 = 1.3 µM and a dissociation half time of t 1/2 = 1.00 hr (4B, 4C). Western blots on whole cell lysates of normal human tissue (colon, heart, liver, skin, testis and PBMC), 4 human AML samples and Jurkat cells (positive control) are shown (4D). Western blotting of Jurkat cells reveals 2 bands of roughly 100 kDa (full length TRIM42) and 50 kDa (unknown protein product). The TRIM42 band is seen at various levels in all AML samples and faintly in PBMC. No TRIM42 protein is detected in the other human tissues. GAPDH expression (40 kDa) was used as a loading control. Subcellular fractionation was performed on AML blasts and isolated healthy donor granulocytes (4E). The fractions are labeled C, cytosolic; N, nuclear; M, membrane; Sk, cytoskeletal. No TRIM42 protein is observed in granulocytes, but the same bands seen in the Jurkat cell positive control are detected in the N and Sk fractions of the AML blasts. The allele frequencies and genotypes for rs9876490, the T4A associated cSNP, in the original ethnic populations used by the International HapMap Project (CEU, HCB, JPT, YRI) are shown (4F). The T4A recipient phenotypes (and frequencies in the CEU population) are AC (0.450) or CC (0.133), and the donor genotype is AA (0.417). The predicted gIR+ in an unrelated transplant scenario can be calculated: P(AA) × P(AC + CC) and applied over a range of allele frequencies yielding the MUD SCT curve (4G). When parental genotypes are considered in the MRD SCT setting (Supplement 1) a similar curve with generally lower gIR+ frequency at each donor allele frequency is generated (4F). In the case of rs9876490 the T4A gIR+ frequency observed in our cohort (•) fits the predicted MRD SCT curve well.

Article Snippet: Normal human tissue lysates (heart, testis, colon, skin, liver) prepared using RIPA buffer and SDS sample buffer were purchased from Prosci (Poway, CA).

Techniques: Binding Assay, Control, Positive Control, Itopia Assay, Labeling, Western Blot, Expressing, Fractionation, Isolation, Membrane, Generated