Odyssey infrared gel imager

After stimulating the cells under the indicated conditions, they were washed twice using PBS. We extracted nuclear proteins using the Nucleoprotein Protein Extraction kit according to the instructions of the manufacturer. The extracted nucleoproteins were stored at −80°C for the subsequent experiments. The nuclear proteins were mixed with 5μl DNA binding buffer and incubated at room temperature for 60 min. After that, we added the fluorescent molecule‐labelled dsDNA probe (5’CATTTCCCGTAAATC3’ and 5’GATTTACGGGAAATG3’¹⁰), specific competition dsDNA or non‐specific competition dsDNA and incubated at room temperature for another 60 min. Then, the mixture was subjected to non‐denaturing electrophoresis, and the Odyssey infrared gel imager (Li‐Cor) was used to scan gel.

The cells were stimulated with PRL or a mixture of PRL and different concentrations of H53, GH or a mixture of GH and different concentrations of H53. The cells were then washed with ice cold PBS, and the nuclear protein of 3 × 10⁶ cells were extracted using a nuclear protein extraction kit. The nuclear protein concentration was the determined using a BCA assay kit. Fluorescently labeled DNA probe sequence was mixed with nucleoprotein from different treatments. The mixture was then subject to EMSA (electrophoresis mobility shift assay) (Martens et al., 2005 (link)). The gels were imaged on an ODYSSEY infrared gel imager (Li-Cor). For supershift analysis, nuclear protein extracts were incubated on ice for 40 min with 1.5 mg of monoclonal anti-Stat5.