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. 2022 Aug 22;23(16):9499. doi: 10.3390/ijms23169499

Figure 5.

Figure 5

Serine residues S98 and S100 are important for fission yeast PC4 function. (A) Phosphorylation of wild type, S98A and S98A/S100A PC4 polypeptides (500 ng) with 10 pmol of fission yeast Cka1, as described in Materials and Methods. Lower panel shows a Western blot of the different proteins (100 ng) used in the upper panel (lanes 1–3). (B) The signals of each lane from (A) were quantified and plotted using the Image J software. (C) Wild type PC4 and mutants (40 ng) were assayed in transcription assays using fission yeast whole cell extracts adding wild type PC4 or combinations of Ck1a (4.0 pmol) or BSA (4.0 pmol), as indicated on the top of the figure. The lanes 2, 5 and 12, labeled control, indicate basal transcription from the whole cell extract in the absence of exogenous proteins. PC4 100 °C indicates that PC4 was inactivated by heat. (D) The signals of each lane from (A) were quantified and plotted using the Image J program. (E) The effect of CKb1 on Cka1 activity on the transcriptional functions of wild type and mutant PC4 polypeptides was measured in the transcription assay using different proteins and combination of proteins, as indicated at the top of the panel. Control (lanes 1 and 5) indicates basal transcription in which no exogenous proteins were added to the assay. In this assay the amounts of PC4 polypeptides and Cka1 were the same as used in (C). (F) The signals of each lane were quantified and plotted using the Image J program, as described in Materials and Methods; ns indicates no significative, * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001. (G) EMSA assay showing that phosphorylation of PC4 by Ck1a inhibits its dsDNA binding activity. In this assay, 40 ng of wild type PC4 was incubated with dsDNA oligonucleotide and increasing amounts of Cka1 (0.5, 1 and 2 pmol; lanes 3–5, respectively) were added in the presence of 0.5 mM ATP. In addition, the PC4 mutant S98A/S100A (40 ng) was assayed in the presence of ATP and increasing amounts of Cka1 (1 and 2 pmol, lanes 7 and 8, respectively) were added. Lanes 2 and 6 represent controls in which Cka1 was not added. Lane 1 does not contain protein.