Fig 1: TREX2 caused polymerase slippage-induced mutations in MMR-mutant ESCs and in human-cancer-derived cells(A–F) Polymerase slippage was measured with a puro reporter with the following out-of-frame inserts: (A)10, (T)11, (T)10, (GT)5, (GT)4, and (AC)5.(G) Polymerase slippage was measured in human-derived cancer cells. A GFP(A)10 reporter was transfected into HCT116 cells, derived from human colorectal carcinoma (ATCC CCL-247), shown on the left. A Luciferase(A)10 reporter was transfected into SK-OV-3 cells, derived from ovarian adenocarcinoma (ATCC HTB-77), and into CCRF-CEM (ATCC CCL-199) cells, derived from acute lymphoblastic leukemia, shown in the middle and right, respectively.(H) RAD18 deletion reduced polymerase slippage of puro(A)10. Statistics for (A)–(D) are the average of three replicates using the unpaired Student’s t test and are listed in the text; mean ± SD; n = 3.
Fig 2: Identification of plasma membrane calcium ATPase isoform PMCA4b as a renalase binding protein.A, HK-2 cells incubated with either labeled RP-Scr220 or RP-220, biotin-labeled proteins purified using streptavidin column, separated by SDS-PAGE and visualized by western blot using streptavidin-HRP; * = regions evaluated by mass spectrometry in samples labeled with either RP-Scr220 or RP-220; # = RP-220 band containing the plasma membrane calcium ATPase isoform PMCA4b. B, Endogenous expression of PMCA4b in HK-2 cells, western immunoblot using isoform specific monoclonal; CCL-119: human leukemic cell line; thyroid tumor = human thyroid tumor cell line (ATCC, CRL-1803) 10 μg protein loaded in each lane. C, co-immunolocalization of PMCA4b and renalase in HK-2 cells, images acquired using a Zeiss laser scanning confocal microscope, scale bar = 9 μm; arrow = plasma membrane. D, Co-Immunoprecipitation of PMCA4b and renalase from HK-2 cell lysates; renalase-Ab-beads = renalase antibody coated beads; PMCA4b-Ab-beads = PMCA4b antibody coated beads.
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