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. 2016 May 6;48(5):e231. doi: 10.1038/emm.2016.17

Figure 5.

Figure 5

Unphosphorylated STAT3 binds to NFκB in starved cancer cells. (a) Nuclear and cytoplasmic fractions were prepared from HeLa cells expressing STAT3WT (ST3W)-V5 or STAT3 Y705F (ST3F)-V5 incubated in HBSS for the indicated time periods and western blotted with pY705–STAT3 Ab, V5 Ab, p65 Ab, p50 Ab, histone H3 Ab and tubulin Ab. Arrows indicate pY705-STAT3 (left), p105 (right, upper) and p50 (right, lower), respectively. (b) HeLa cells stably expressing control vector, ST3W-V5 or ST3F-V5 were incubated in HBSS for the indicated time periods, and NFκB reporter activity was measured by real-time PCR. Data are presented as relative quantitation (RQ)±s.d. (c) HeLa cells expressing ST3WT-V5 and p65-HA, or ST3F-V5 and p65-HA, were western blotted with V5 Ab, HA Ab and tubulin Ab (left). The nuclear and cytoplasmic fraction were prepared from HBSS-incubated HeLa cells expressing ST3WT-V5 and p65-HA or ST3F-V5 and p65-HA, immunoprecipitated with anti-p65 Ab, and western blotted with STAT3 Ab and p65 Ab (right). *P<0.05 and **P<0.01 compared with the respective control. Ab, antibody; HBSS, Hank's balanced salt solution; IP, immunoprecipitate; NFκB, nuclear factor κB; STAT3, signal transducer and activator of transcription 3.