Supplementary Figure 4: RPAP2 phosphatase activity in vitro. Related to Figure 6.

(a) In vitro phosphatase assays using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) substrate showing the effects of RPRD1A and RPRD1B on RPAP2 (1-334) activity. The reaction includes 1 μM RPAP2 (1-334), 10 μM DiFMUP and indicated concentration of RPRD1A or RPRD1B. The fluorescence level induced by RPAP2 (1-334) was set as 100%. Error bars: s.d. from four technical replicates. (b) In vitro DiFMUP phosphatase assays showing the effects on RPAP2 phosphatase activity by the phosphatase inhibitor vanadate. RPAP2 (1-334): 2 μM. DiFMUP: 10 μM. Vanadate: 1 mM. The fluorescence level induced by RPAP2 (1-334) was set as 100%. *: p<0.01 as compared with the no inhibitor controls (two-tailed Student’s t-test). Error bars: s.d. from five technical replicates. (c) In vitro DiFMUP phosphatase assays showing the effects on RPAP2 phosphatase activity by indicated metals. RPAP2 (1-334): 2 μM. DiFMUP: 10 μM. Indicated metals: 10 mM. The fluorescence level induced by RPAP2 (1-334) in the absence of added metals was set as 100%. *: p<0.01 as compared with the no metal controls (one-way ANOVA Tukey's Multiple Comparison test). Error bars: s.d. from six technical replicates. (d) In vitro phosphatase ELISAs showing the effects of vanadate and the indicated metals on RPRD1A-stimulated RPAP2 phosphatase activity on the indicated biotinylated CTD peptide. The reactions contain three µM RPAP2, 10 μM RPRD1A and biotinylated S2-5-2P CTD peptide, with or without 1 mM vanadate or 10 mM of the indicated metals, incubated at 37 °C for 3 hr, followed by measurement of the phosphorylation levels using the S5P monoclonal antibody (3E8). No protein controls were set as 100%. *: p<0.01 as compared with the no inhibitor controls (two-tailed Student’s t-test). Error bars; s.d. from four technical replicates. (e) In vitro phosphatase ELISAs showing the effects on the antibody recognition at S5P by adjacent S7P. The reactions contain the indicated biotinylated CTD peptides incubated with 3 µM RPAP2 and 10 µM RPRD1A at 37 °C for 3 hours. Phosphorylation levels were measured using the indicated antibodies. *: p<0.01 (two-tailed Student’s t-test). Error bars: s.d. from three technical replicates. (f) In vitro phosphatase ELISAs assays showing the effects of RPRD1A or RPRD1B stimulating RPAP2 phosphatase activity on S5P located between two S7P-containing CTD repeats. The reactions contain the indicated biotinylated CTD peptides, indicated proteins incubated at 37 °C for 3 hours, followed by measurement of the phosphorylation levels using the indicated antibodies. No protein controls were set as 100%. *: p<0.01 (two-tailed Student’s t-test) as compared to no protein control. Error bars: s.d. from four technical replicates.