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. 2022 Aug 3;12:13362. doi: 10.1038/s41598-022-17559-4

Figure 2.

Figure 2

Increased phosphorylation and nuclear localization of the NF-κB p65 subunit upon ADAR3 expression. (a) Control (ADAR3−) and ADAR3-expressing (ADAR3+) U87 cells were lysed and subjected to immunoblotting with antibodies against ADAR3, p65, phosphorylated S356 p65 (p-p65), and β-actin. As phosphorylated and unphosphorylated p65 migrate at the same position on SDS-PAGE, expression of p-p65 and p65 was examined on independent immunoblots, with control detection of β-actin performed for each immunoblot. Blot is a representative image (replicate 2) of three biological replicates of the p65 and p-p65 immunoblots and one replicate of the ADAR3 immunoblot. Uncropped blot images are included in the supplementary information file. (b) Quantification of phosphorylated p65 protein (p-p65) to total p65 protein levels was determined relative to β-actin controls for each immunoblot. The relative p-p65/p65 ratios were normalized to the control cell line. Error bars represent the standard error of the mean (SEM) for three biological replicates. Statistical significance was determined using a two-tailed unpaired t-test. *p ≤ 0.05 (c) Cell equivalent amounts of nuclear and cytoplasmic fractions of control and ADAR3-expressing U87 cells were subjected to immunoblotting using antibodies against p65, tubulin and Histone H3. To determine the accuracy of the subcellular fractionation, the entire immunoblot was probed with Tubulin and Histone H3 antibodies. As cytoplasmic levels of p65 are significantly higher than nuclear p65 levels, to allow for exposure in the dynamic range and proper quantification, the entire p65 blot was incubated with primary antibody and exposed to capture the cytoplasmic p65 levels. Subsequently, the nuclear p65 immunoblot was cropped away from the cytoplasmic p65 samples and re-subjected to enhanced chemiluminescence detection for detection within the dynamic range. Blot is a representative image (replicate 1) of three biological replicates and uncropped blot images are included in the supplementary information file. (d,e) Quantification of the p65 level relative to Histone H3 in nuclear (d) and tubulin in cytoplasmic (e) fractions were normalized to the control cell line for three biological replicates. Error bars represent SEM. Statistical significance was determined by a two-tailed unpaired t-test. *p ≤ 0.05, ***p ≤ 0.0005.