Doherty JR , Nilsson LM , Kuliyev E , Zhu H , Matthew R , Cleveland JL , Mead PE , Roussel MF .

P01 CA071907 NCI NIH HHS , P01 CA096832 NCI NIH HHS , P30 CA021765 NCI NIH HHS , R01 DK044158 NIDDK NIH HHS

	Figure 1. Predicted amino acid sequence alignments of Ink4d proteins. The predicted amino acid sequences of Ink4d proteins from Xenopus tropicalis, Fugu (Fugu rubrides), mouse (Mus musculus) and human (Homo sapiens) were compared to the two Ink4d proteins of Xenopus laevis. The Xl-Ink4d1 cDNA was isolated from a library made from Xenopus laevis adult spleen, whereas Xl-Ink4d2 was identified by blasting the NCBI database for similar expressed sequences in Xenopus laevis and represents a second allele of Ink4d. Xl-Ink4d1 protein shares 63% amino acid identity with both the human and mouse Ink4d protein. The predicted four ankyrin repeats (green lines) as well as many of the amino acids that make contacts with CDK6 are conserved between mammals and Xenopus (red box = hydrogen bond interaction, blue box = non-polar interaction).
	Figure 2. Expression of Xl-Ink4d during Xenopus laevis development. Embryos were staged according Xenopus laevis normal tables of development [27] and RNA was harvested at the indicated stages. (a) Reverse transcription PCR was performed on total RNA from staged embryos to amplify Xl-Ink4d1, cyclin-D1, Cdk4 and ODC transcripts. Trace [α32P]-dCTP was added to the reactions and the PCR products were separated by gel electrophoresis. (b) Relative quantitative real-time PCR analysis of Xl-Ink4d1 mRNA levels was performed using iQ SYBR Green Supermix (Bio-Rad), run and detected using an iCycler thermocycler (Bio-Rad). (c) In situ hybridization (ISH) for Xl-Ink4d1 expression during early embryonic development. Xl-Ink4d1 expression is detected at low levels in the dorsal anterior region of the developing tadpole. Stage 19 (i–iii); (i) dorsal view, (ii) anterior view, (iii) posterior view. Stage 22 (iv–vi); (iv) lateral view, (v) dorsal view, (vi) anterior view. Stage 32 (vii), trunk somites (S), eye (E) and head (H). (d) Immunoblotting of Xl-Ink4d from whole embryo lysates at the indicated stages using a polyclonal antibody directed against the C-terminus of the protein. Embryos microinjected with synthesized Xl-Ink4d1 mRNA were harvested and run as a positive control (+).
	Figure 3. Xl-Ink4d is a cyclin D-dependent Cdk4 inhibitor. (a) The indicated GST fusion proteins were expressed in bacteria and purified on glutathione sepharose. The adsorbed sepharose was incubated with in vitro transcribed and translated [35S]-labeled Xenopus Cdk4 (Xl-Cdk4), washed and separated on an SDS-polyacrylamide gel. 50% of the input Xl-Cdk4 that was used for pull-downs was run to estimate the percent of starting material that bound to the GST fusion protein (50% input). (b) Cdk4 kinase reactions were run with increasing amounts of purified GST-Xl-Ink4d1 and GST-Mm-Ink4d (mouse Ink4d) fusion proteins. Active mouse Cdk4/cyclinD1 kinase complexes were generated from SF9 cells infected with baculoviruses encoding mouse Cdk4 and cyclin-D1. Kinase assays were performed with [γ-32P]-ATP using purified GST-Rb as substrate. (c) The cell cycle profile of NIH-3T3 mouse fibroblasts overexpressing vector-alone (black bars), Xenopus Xl-Ink4d1 (dark grey bars) or mouse Mm-Ink4d (light grey bars). NIH-3T3 cells were infected with retroviruses that co-express the indicated Ink4d genes and GFP, and the DNA content of GFP-positive cells was measured by propidium iodide staining and FACS analysis. The percentage of cells in G1/G0, S and G2/M phase is presented. Expression of either Xl-Ink4d1 (* p=0.006) or Mm-Ink4d (** p=0.05) shows a significant decrease in S phase compared to vector alone.
	cdkn2d (cyclin-dependent kinase inhibitor 2D) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.

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