Fig. 5.

Reactivity of anti-pY₁-CTD with CTD-peptides and endogenous RNAPII. a Representative images of ELISA results. Each column is coated with a different CTD peptide as indicated on the bottom of the columns. Each row is reacted with a different antibody as indicated to the left of the rows. 4G10 is a mouse monoclonal antibody that reacts with phosphor-tyrosine; 8WG16 is a mouse monoclonal antibody that reacts with un-phosphorylated CTD; α-pS₅ is rabbit polyclonal antibody that reacts with pS₅ in CTD; α-pS₂ is a rabbit polyclonal antibody that reacts with pS₂ in CTD; α-pY₁ is a rabbit polyclonal antibody raised against a pY₁pS₅-CTD peptide. b Quantification of ELISA results. Numbers shown are mean ± SD (n = 3). c Phosphotyrosine (pTyr) competed with the binding of α-pY₁ to pY₁-containing CTD peptides. d α-pY₁-CTD reacts with endogenous RNAPII. HEK293T cells transfected with vector or AblPPn were immunoprecipitated with 8WG16 or IgG and then probed with N20 or anti-pY₁. Transfection with AblPPn increased the pY₁-reactivity in whole cell lysate (input) and in 8WG16-precipitated RNAPII (upper panels). RNAPII CTD contains 52 CTD-repeats and not all 52 repeats are stoichiometrically phosphorylated in vivo, 8WG16 can react with RNAPII that contains some unphosphorylated CTD repeats and some pY₁-CTD repeats. In reciprocal immunoprecipitation (IP), whole cell lysates were reacted with anti-pY₁ and then immunoblotted with anti-pS₅-CTD or 8WG16. Note that anti-pY₁ immunoprecipitated RNAPII that reacted with anti-pS₅-CTD or 8WG16. IIo, RNAPII containing hyper-phosphorylated CTD; IIA, RNAPII with hypo-phosphorylated CTD. e The previously reported 3D12 antibody [17] does not react with pY₁-CTD. Increasing amounts of total lysates from HEK293T cells transfected with vector or AblPPn were immunoblotted with the indicated antibodies. Note that AblPPn did not alter the levels of pS₂ or pS₅ reactive RNAPII but increased the levels of pY₁ reactive RNAPII. The pY₁-reactivity was competed with phosphotyrosine (pTyr). Note that 3D13 reacts with the IIA form of RNAPII. Reactivity with 3D12 was not affected by the expression of AblPPn. Phosphotyrosine does not inhibit the 3D12 reactivity with RNAPIIA. f Anti-pY₁-CTD does not react with YF-CTD mutant. HEK293T cells were transfected with combinations of AblPPn, GST, GST-CTD or a RNAPII with a truncated CTD in which all of the Y₁ is mutated to F (phenylalanine). Whole cell lysates were immunoblotted with the indicated antibodies. Note that pS₅ reacted with the endogenous RNAPII, GST-CTD and the YF-RNAPII. The authenticity of the YF-RNAPII was established by its reactivity with B10 antibody [34]. The YF-RNAPII did not react with anti-pY₁-CTD