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. 2022 Jul 13;608(7924):819–825. doi: 10.1038/s41586-022-04930-8

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 1 — a, Silver-stained SDS-PAGE of one of the HEK cell CST–Polα-primase preparations used in this study (HEK CST-PP), recombinant human CST overexpressed in insect cells (Insect CST), and recombinant human Polα-primase overexpressed in insect cells (Insect PP). M, marker proteins. The HEK cell and insect cell STN1 proteins have slightly different mobilities due to a Myc tag on the former. b, Western blot with antibodies to STN1 and to epitope tags on the HEK cell CTC1 and TEN1 subunits (thus no signal for CTC1 or TEN1 in the Insect CST). The HEK cell-expressed CTC1 consistently runs as a doublet. Amount of protein loaded (pmoles) is based on direct determination of protein concentration for Insect CST but is calculated from the STN1 western blots for HEK CST-PP. HEK FLAG/HA CST-PP indicates double immunopurification via FLAG and HA tags. c and d, Quantification of STN1 from the western blot gives (c) the phosphorimager counts of STN1 per µL for the HEK cell preparation and (d) the counts of STN1 per pmol for purified insect cell CST; the calculation below the graphs gives the concentration of STN1 in the HEK cell CST–Polα-primase preparation. e, Western blot with antibodies to the two POLA subunits. The endogenous POLA1 from HEK cells consistently runs as a doublet, likely due to processing between lys 123 and lys 124 producing a stable 165 kDa species³⁹; in contrast, the POLA1 in the Polα-primase overexpressed in insect cells is mostly a single species with a minor smaller species. Amount of protein loaded (pmoles) is based on direct determination of protein concentration for Insect PP but is calculated from the POLA western blots for HEK CST-PP. f, g, Quantification of POLA1 and POLA2 from the western blot gives (f) the counts of POLA1 and POLA2 per µL of the HEK cell preparation and (g) the counts per pmol for the purified insect cell Polα-primase; the calculation below the graphs gives the concentration of POLA in the HEK cell CST–Polα-primase preparation. Dividing the concentration of POLA from f and g by the concentration of CST from c and d gives the stoichiometry of 0.21 POLA per CST in the HEK cell preparation. These results were consistent between two biological replicates of the entire set of experiments. For gel source data, see Supplementary Fig. 1

Source Data