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. 2017 Feb 1;37(4):e00521-16. doi: 10.1128/MCB.00521-16

FIG 2.

FIG 2

NF-κB p65 is necessary for full induction of iNOS and other mRNAs in response to hyperosmotic treatment. (A) RT-qPCR analysis of mRNA isolated from wild-type and p65-deficient MEFs treated with 600 mosM sucrose. (B) Western blot analysis of wild type MEFs treated with 600 mosM sucrose. (C) RT-qPCR analysis of mRNA isolated from MEFs cotreated with the IKKε inhibitor (100 nM) and hyperosmotic medium. (D) MEFs treated with shIKKε before treatment with hyperosmotic medium for the indicated durations. IKKε depletion was verified by Western blotting. (E) RT-qPCR analysis of mRNA isolated from wild-type and p65-deficient MEFs treated with 600 mosM sucrose. (F) Control and shPKR MEFs were treated with DSS medium, and wild type MEFs were treated with Tg for the indicated durations. Cell lysates were analyzed by immunoblotting. IKKε protein was detected at ∼75 kDa, tubulin was detected at 55 kDa, iNOS was detected at 130 kDa, ATF4 was detected at ∼48 kDa, GADD34 was detected at ∼110 kDa, Xbp1s was detected at 54 kDa, CHOP was detected at 27 kDa, BiP was detected at 75 kDa, and PERK was detected at ~140 to 160 kDa. mRNA levels in panels A to E were normalized to the level of GAPDH. Error bars represent standard errors of the means. *, P < 0.05; ns, not significant (Student's t test).