Quantification of total and phosphorylated STAT3 by calibrated western blotting

Quantification of intracellular proteins is essential to understand signaling. Here, we describe quantification of the expression and phosphorylation of the transcription factor STAT3. We present isolation of total and phosphorylated STAT3 from cell lysates by immunoprecipitation, followed by SDS-PAGE and western blot together with known amounts of a calibrator protein that shares an epitope with the precipitated proteins. Finally, we explain how to relate the amount of precipitated protein to the amount of calibrator protein considering the efficiency of immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Dittrich et al. (2012)1 and Reeh et al. (2019).2.