Figure 5.

Nonphosphorylatable S838A/T841A RB prevents CAPH2 chromatin unloading upon T-cell receptor cross-linking.A, jurkat cells were treated as indicated for 30 min, whole cell extracts and chromatin fractions were prepared, and expression levels of the indicated proteins were detected by western blotting. Histone levels serve as loading control and were detected by Coomassie Blue staining. ∗ indicates nonspecific band. B, relative levels of CAPH2 in chromatin fractions was normalized to SMC1 and compared between treated and untreated conditions (n = 3, ∗ p < 0.05, Student’s t-test). C, the open reading frame for WT and mutant RBLP constructs (aa 379–928) are shown depicting their relevant coding regions and HA tags. The mutant construct labeled RBLP AA carries double alanine substitutions at S838 and T841. D, lentiviruses were used to transduce RBLP and the AA mutant construct into Jurkat cells. Expression of exogenous RBLP was detected by western blotting for HA. Tubulin blots serve as loading controls. E, jurkat cells expressing either WT or RBLP, or untransduced controls (UT), were subjected to TCR cross-linking for 30 min. Chromatin fractions were prepared, and levels of the indicated proteins were detected by western blotting. F, levels of chromatin-associated CAPH2 were normalized to SMC1 and compared between PBS and TCR cross-linked conditions (n = 3, ∗ p < 0.05, Two-way ANOVA and Sidak’s multiple comparisons test).