Samples of protein were collected following treatment using 6 × Laemmli buffer. Protein was subjected to SDS–PAGE on a 6.5% gel before transfer to nitrocellulose (GE Healthcare). Membranes were stained with affinity-purified, IR-labelled secondary antibodies against rat (680 nm; Alexa, Invitrogen) and mouse (800 nm; Rockford, Biomol), and stained with hrp-conjugated secondary antibodies against rat (Sigma) or mouse (Promega), and revealed by enhanced chemiluminescence.
The monoclonal antibodies specific for Rpb1 (Pol3.3 and 1C7; the latter recognizes non-modified CTD) and the different CTD phosphorylations (3E10, 3E8, 4E12) were diluted in a 5% milk solution (0.1% Tween in PBS) at a 1:10 ratio. The rat monoclonal antibody 6D7 against CTD-Thr4-P was applied in a 2% ECL Advance blocking agent solution (GE Healthcare) (0.1% Triton X-100 in PBS) at a 1:10 dilution as described previously37 (link)57 (link).
The monoclonal antibodies specific for Rpb1 (Pol3.3 and 1C7; the latter recognizes non-modified CTD) and the different CTD phosphorylations (3E10, 3E8, 4E12) were diluted in a 5% milk solution (0.1% Tween in PBS) at a 1:10 ratio. The rat monoclonal antibody 6D7 against CTD-Thr4-P was applied in a 2% ECL Advance blocking agent solution (GE Healthcare) (0.1% Triton X-100 in PBS) at a 1:10 dilution as described previously37 (link)57 (link).