Figure 5.

Positively selected L162 encompassing loop contributes to its interaction with the P-TEFb complex

(A) Cell proliferation of HeLa cells specifically expressing the WT or mutant PAD12 (single, T159A or W161A or L162A; double, L162A/W161A or triple, L162A/W161A/T159A). Data represent mean ± SEM of at least eight biological experiments. ∗p value <0.05; ∗∗p value <0.01.

(B) Representative image of immunoprecipitation with GFP-specific antibody or non-immune rabbit IgG of GFP-positive HeLa cells nuclear extracts expressing wild-type (WT) or mutant (single, L162A; double, L162A/W161A or triple, L162A/W161A/T159A) PADI2 followed by western blot with the indicated antibodies. The relative quantification is shown as a bar plot of the two biological replicates.

(C). Quantitative RT-qPCR validation in HeLa cells selectively expressing the WT or mutant PAD12 (as in B). Changes in mRNA levels were normalized to GAPDH mRNA. Data represent the mean ± SEM of n ≥ 3 biological experiments for all plots in the figure. Two-tailed unpaired Student’s t-test was used to determine the statistical significance between the groups.

(D) ChIP-qPCR assay performed in HeLa cells selectively expressing the WT or mutant PAD12 (as in B) with CDK9 antibody. Non-immune IgG was used as a negative control. Y axis: fold change over the input samples. Data represent mean ± SEM of three biological experiments, ∗p value <0.05; ∗∗p value <0.01 (related to Figure S5).