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D. Whole cellular extracts. Chromatin fractions. -. +. +. -. Myc-HBO1. -. +. +. -. control. 1.2 1.1 0.98 0.97 1.1 - 3.5. MCM3 MCM5 ORC1 Cdt1 Cdc6 Myc-HBO1 HBO1. Supplementary Figure 3. A. B. G1/S. G2/M. MW(bp). Chromatin fractions. Whole cellular extracts.

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Presentation Transcript
slide1

D

Whole cellular

extracts

Chromatin

fractions

-

+

+

-

Myc-HBO1

-

+

+

-

control

1.2

1.1

0.98

0.97

1.1

-

3.5

MCM3

MCM5

ORC1

Cdt1

Cdc6

Myc-HBO1

HBO1

Supplementary Figure 3

A

B

G1/S

G2/M

MW(bp)

Chromatin

fractions

Whole cellular

extracts

HBO1

100

2n

4n

Asy.

G1/S

G2/M

Asy.

G1/S

G2/M

G1/S staged

(hydroxyurea)

ORC1

HBO1

100

Cdt1

Cdt1

200

ORC1

MCM2

100

Cdc6

2n

4n

G2/M staged

(nocodazole)

300

ORC2

Actin

C

Chromatin

fractions

Whole cellular

extracts

Actinomycin D

+

+

-

-

Cdt1

HBO1

ORC1

ORC2

Cdc6

MCM3

Actin

Levels of mRNA and protein bound to bulk chromatin at different stages of the cell cycle, in response to actinomycin D treatment, and upon HBO1 overexpression. (A) Expression level of HBO1, MCM2, ORC1, and Cdt1 mRNAs in CCL-156 cells staged in early S phase (hydroxyurea block) or at the G2/M transition (nocodozole block). Efficient staging was confirmed by FACS analysis. (B) Western blot analysis of HBO1, Cdt1, Cdc6, ORC1, and ORC2 expression in CCL-156 cells staged in early S phase and at the G2/M transition. (C) Expression level and chromatin association of HBO1 and replication factors in the absence or presence of actinomycin D (actD). Note that, for unknown reasons, soluble HBO1 is degraded following ActD treatment, whereas the level of chromatin-bound HBO1 is unaffected. (D) Expression level and chromatin association of HBO1 and replication factors under conditions of HBO1 overexpression. Values for each protein indicate fold-difference upon HBO1 overexpression