Quantitative Analysis of STAT3 and pSTAT3 in MDSC Subtypes

The whole cell lysates from MO- and PMN-MDSCs were prepared as previously described [38 (link)], and total protein contents were measured using the Nanodrop (Thermo Scientific). Equal protein amounts (30 μg/lane) were loaded per lane for the MO- and PMN-MDSC samples. Protein was separated by electrophoresis on precast gel (Bio-Rad Laboratories), and transferred onto PVDF membranes (Millipore Corp.). The membranes were incubated overnight at 4 °C with rabbit anti-mouse monoclonal antibody targeting STAT3, anti-rabbit monoclonal antibody targeting pSTAT3, or an anti-rabbit anti-GAPDH (Cell Signaling) and then with the secondary anti-rabbit or anti-mouse antibodies HRP for 1 h. Signal was developed with a chemi-luminescent substrate (Thermo Fisher Scientific), and Kodak M35 X-OMAT Automatic Processors was used for the detection of signals. Bands were quantitated in densitometry units after the processing with the ImageJ software (NIH).

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Cantoni C., Cignarella F., Ghezzi L., Mikesell B., Bollman B., Berrien-Elliott M.M., Ireland A.R., Fehniger T.A., Wu G.F, & Piccio L. (2016). Mir-223 regulates the number and function of myeloid-derived suppressor cells in multiple sclerosis and experimental autoimmune encephalomyelitis. Acta neuropathologica, 133(1), 61-77.

Publication 2016

Nanodrop precast gel PVDF membranes chemi-luminescent substrate

Anti antibodies Anti antibody Densitometry Electrophoresis Gapdh Luminescent Mdsc Membranes Monoclonal antibody Mouse Protein Pvdf Rabbit Signals detection Stat3

Corresponding Organization :

Other organizations : Washington University in St. Louis, Hope Center for Neurological Disorders

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Variable analysis

independent variables

Preparation of whole cell lysates from MO-MDSCs and PMN-MDSCs

dependent variables

Protein content
Levels of STAT3 and phosphorylated STAT3 (pSTAT3)

control variables

Equal protein amounts (30 μg/lane) loaded per lane for the MO- and PMN-MDSC samples
Antibodies used for detection of STAT3, pSTAT3, and GAPDH

controls

Anti-rabbit anti-GAPDH antibody used as a loading control

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