Loss of pausing in spt5^m806 mutants regulates TGFβ and IFNγ signaling via distinct mechanisms. (A, left) An illustration for Pol II ChIP-PCR analysis to compare the ratio of Pol II occupancy at promoter vs gene body. 1 and 2 represent PCR amplicons within the promoter and gene body, respectively. ↓ means decrease; ↑ means increase. (Right) Quantification of ChIP ratio in sibling controls and spt5^m806 mutants (n = 3, mean ± SEM, *P < .05, **P < .01). (B) Left, western blot to compare phosphorylated Smad2/3 (p-Smad2/3) level in 36-hpf embryos between DMSO and flavopiridol (4 μM) treatment. Right, quantification of western results by normalized to β-actin (n ≥ 3, mean ± SEM, *P < .05). (C) MNase protection experiments for crfb17 (left) and crfb13 (right) in 36-hpf sibling control and spt5^m806 mutants. qPCR was performed to tile through the promoter region with overlapping amplicons. Relative ratio of the amount of digested DNA to genomic control was used to determine the extent of MNase protection. The shaded area marks the nucleosome-depleted region around the promoter. n = 3, mean ± SEM, *P < .05, **P < .01. (D) Pol II ChIP experiments to compare promoter-associated Pol II on crfb17 and crfb13 between 36-hpf sibling control and spt5^m806 mutant embryos. β-actin serves as an unchanged control gene (n = 3, mean ± SEM, * P < .05).