Skip to main content
. Author manuscript; available in PMC: 2015 Dec 21.
Published in final edited form as: Nat Struct Mol Biol. 2015 Jul 13;22(8):618–626. doi: 10.1038/nsmb.3055

Figure 6.

Figure 6

MCM2 can handle new and old histones, including all H3 variants. (a) IP of Flag-HA-MCM2 WT and mutant from soluble fractions or DNase I–solubilized chromatin. (b,c) PLA of endogenous chromatin-bound ASF1a with MCM2 (b) or CDC45 (c). Soluble proteins were extracted by CSK-T. Positive control, PLA of CAF-1 p150 and PCNA; negative control, PLA in cells depleted for ASF1a. Dot plots show PLA interaction foci per nucleus and are representative of two independent cell cultures and stainings. In c, n = 270 cells (ASF1A siRNA), n = 260 cells (control siRNA, −HU) and n = 284 cells (control siRNA, +HU). ***P < 0.001 by unpaired two-tailed t test with Welch’s correction. (d) PLA of endogenous chromatin-bound ASF1a and MCM2 in U-2-OS cells transiently transfected with Flag-HA-MCM2 WT or Y81A Y90A mutant. Dot plot shows PLA interaction foci per nucleus and is representative of two independent cell cultures and stainings. n = 188 (WT); n = 44 (Y81A Y90A). *P < 0.05 by unpaired two-tailed t test with Welch’s correction. (Representative images are shown in Supplementary Fig. 7c.) (e) Pulldowns of GST-MCM2 HBD with H3–H4 or CENPA–H4 tetramers in different salt conditions. (f) IP of Flag-HA-MCM2 WT and mutant from DNase I–solubilized chromatin of cells stably expressing GFP-CENPA52. (Input material for IP is shown in Supplementary Fig. 5f. GFP-CENPA coprecipitation with MCM2 in CSK-T fractions is shown in Supplementary Fig. 8d.) Uncropped images of gels are shown in Supplementary Data Set 1.