Skip to main content
. 2016 Sep 21;113(40):11336–11341. doi: 10.1073/pnas.1605246113

Fig. S8.

Fig. S8.

Antibody specificity control experiments. (A) Western blot analysis of the (Upper) GR H-300 and (Lower) MR H-300 antibodies. From left, lanes 1 and 2, nuclear fractions of FS30 hippocampus tissue; lane 3, protein ladder under UV illumination; lane 4, ladder under normal light. The ladder contains markers at 75 and 100 kDa. The lanes containing the nuclear fractions show bands generated by the GR antibody and the MR antibody at ∼92 and 103 kDa, respectively, which correspond with the predicted protein molecular masses. There was no cross-over in the staining pattern, because the GR antibody did not visualize a protein band at 103 kDa and the MR antibody did not recognize a band at 92 kDa. (B) Preabsorption analysis and ChIP on hippocampal chromatin using MR N-17 and GR M-20 antibodies. Chromatin was prepared from rats killed under baseline AM conditions (BL) or at 30 min after FS (FS30). Aliquots of MR N-17 and GR M-20 antibody were preincubated with peptide preparations against which the antibodies had been raised for 3 h at 4 °C as indicated in B. Next, the antibody–peptide mixture was added to chromatin samples, and the ChIP procedure continued as described. qPCR analysis of bound DNA was conducted using primers for Fkbp5 GRE2. Data are expressed as B/I (mean ± SEM; n = 3). Statistical analysis: two-way ANOVA: MR (B, Left): effect of stress: F(1, 12) = 244.1, P < 0.0001; effect of antibody treatment: F(2, 12) = 88.63, P < 0.0001; interaction: F(2, 12) = 57.82, P < 0.0001; GR (B, Right): effect of stress: F(1, 12) = 119.8, P < 0.0001; effect of antibody treatment: F(2, 12) = 32.7, P < 0.0001; interaction: F(2, 12) = 29.64, P < 0.0001. Bonferroni posthoc test. *P < 0.05 compared with the respective BL control. (C) IgG binding to Fkbp5 GRE2 under baseline conditions and after stress. ChIP with normal IgG antibody was conducted with hippocampal chromatin prepared from rats killed under baseline AM or PM conditions or at 30 min after FS. The graph shows lack of IgG enrichment (B/I; mean ± SEM; n = 3) at Fkbp5 GRE2 under all conditions. Statistical analysis: one-way ANOVA: F(2, 8) = 0.7582, P = 0.5087.