The CALM NES is necessary for aberrant H3K79 methylation and elevated Hoxa cluster expression. (A-B) Western blot (A) and confocal IF (B) of MEFs stably infected with empty vector, CALM-AF10, and CALM(mutNES1+2)-AF10. Data are representative of one set of MEFs (out of 3) stably infected with the respective constructs. (C) Representative western blot of di-me H3K79 and histone H3 levels in stably infected MEFs. Quantification data (bottom) represent the mean ± SEM of di-me H3K79 values normalized to actin from separately generated MEF lines for each construct (n = 3). Statistical analysis was performed by one-way ANOVA followed by Dunnett’s multiple comparison test using empty vector-transduced MEFs as the control; **P < .01 (D) Confocal IF of MEFs coexpressing DOT1L (red) and Flag-tagged CALM-AF10 (green, left), CALM(NESmut1+2)-AF10 (green, middle), or CALM-AF10^ΔOMLZ (green, right). Bar represents 20 μm. (E) Hoxa transcript levels were measured by real-time reverse transcription-PCR and normalized to housekeeping genes GAPDH and β2M and then to empty vector-infected MEFs by the ΔΔCt method. (F) Chromatin immunoprecipitation analysis of di-me H3K79 in the promoter regions of the Hoxa cluster genes. Hoxa amplification was measured by real-time PCR as a percent of input and then normalized to the vector control. For panels E-F, results are shown as mean ± SEM from separately generated MEF lines for each construct (n = 3). White bars represent empty vector, gray bars are CALM-AF10, and black bars are CALM(mutNES1+2)-AF10. Statistical analyses were performed using one-way ANOVA for each Hoxa gene followed by Dunnett’s multiple comparison test. Only CALM-AF10 was found to be significantly different from the vector control: **P < .01, *P < .05.