Quantification of Rapid Myosin Regulatory Light Chain Phosphorylation Using High-Throughput In-Cell Western Assays: Comparison to Western Immunoblots

Quantification of phospho-proteins (PPs) is crucial when studying cellular signaling pathways. Western immunoblotting (WB) is commonly used for the measurement of relative levels of signaling intermediates in experimental samples. However, WB is in general a labour-intensive and low-throughput technique. Because of variability in protein yield and phospho-signal preservation during protein harvesting, and potential loss of antigen during protein transfer, WB provides only semi-quantitative data. By comparison, the "in-cell western" (ICW) technique has high-throughput capacity and requires less extensive sample preparation. Thus, we compared the ICW technique to WB for measuring phosphorylated myosin regulatory light chain (PMLC(20)) in primary cultures of uterine myocytes to assess their relative specificity, sensitivity, precision, and quantification of biologically relevant responses.ICWs are cell-based microplate assays for quantification of protein targets in their cellular context. ICWs utilize a two-channel infrared (IR) scanner (Odyssey(R)) to quantify signals arising from near-infrared (NIR) fluorophores conjugated to secondary antibodies. One channel is dedicated to measuring the protein of interest and the second is used for data normalization of the signal in each well of the microplate. Using uterine myocytes, we assessed oxytocin (OT)-stimulated MLC(20) phosphorylation measured by ICW and WB, both using NIR fluorescence. ICW and WB data were comparable regarding signal linearity, signal specificity, and time course of phosphorylation response to OT.ICW and WB yield comparable biological data. The advantages of ICW over WB are its high-throughput capacity, improved precision, and reduced sample preparation requirements. ICW might provide better sensitivity and precision with low-quantity samples or for protocols requiring large numbers of samples. These features make the ICW technique an excellent tool for the study of phosphorylation endpoints. However, the drawbacks of ICW include the need for a cell culture format and the lack of utility where protein purification, concentration or stoichiometric analyses are required

Myxoma of the maxilla: A case with extremely aggressive biologic behavior

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A systematic review of transfusion-associated graft-versus-host disease

Accepted April 14, 2015.Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare complication of blood transfusion. The clinicolaboratory features of TA-GVHD, and the relative contributions of recipient and component factors remain poorly understood. We conducted a systematic review of TA-GVHD reports. The Human Leukocyte Antigen (HLA) relationship between donor and recipient was classified as D=0 when no donor antigens were foreign to the recipient, versus D≥1 when ≥1 donor antigen disparity occurred. We identified 348 unique cases. Criteria for component irradiation were met in 48.9% cases(34.5% immune-compromised, 14.4% related-donor), though non-irradiated components were transfused in the vast majority of these(97.6%). Components were typically whole blood and red cells. When reported, component storage duration was ≤10 days in 94%, and 23(6.6%) were leukoreduced (10 bedside, 2 pre-storage, 11 unknown). Among 84 cases with HLA data available, the category of D=0 was present in 60(71%) patients at either HLA class I or II loci, and was more common among recipients without traditional indications for component irradiation. These data challenge the historic emphasis on host immune defects in the pathogenesis of TA-GVHD. The dominant mechanism of TA-GVHD in both immunocompetent and compromised hosts is exposure to viable donor lymphocytes not recognized as foreign by, but able to respond against, the recipient.Ilana Kopolovic, Jackie Ostro, Hideki Tsubota, Yulia Lin, Christine M. Cserti-Gazdewich, Hans A. Messner, Amy K. Keir, Neal DenHollander, Walter S. Dzik, and Jeannie Callu