Fig 1: CCNI2 interacts with CDK5.Western blots show that CCNI and CCNI2 were co-immunoprecipitated with CDK5. Expression vectors were transfected into HEK293 cells to express epitope-tagged human (A) or chicken (B) CDK5, CCNI, and CCNI2 proteins, and cell lysates were subjected to immunoprecipitation. Human CCNA1 was included as a negative control. IP indicates antibody used for immunoprecipitation, and WB indicates antibody used for detection.
Fig 2: Knockdown of CCNI2 decreases cell cycle progression and cell proliferation of HeLa cells.The specificity and efficiency of siRNAs against CCNI (A) and CCNI2 (B) were examined by quantitative PCR. HeLa cells were transfected with siRNAs at day 0 and day 1, and the expression level of CCNI or CCNI2 was determined by qPCR analysis at day 2. (C) Cell cycle profiles of CCNI-depleted and CCNI2-depleted HeLa cells. (D) BrdU incorporation of CCNI-depleted and CCNI2-depleted HeLa cells. (E) Cell proliferation of CCNI-depleted and CCNI2-depleted HeLa cells was examined by MTT assay. The bar graphs and the table show quantification of the results, with each value represents the mean ± SD of three independent experiments. Statistical significance is shown using the Student t test analysis; *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 3: Protein sequence and tissue expression pattern of CCNI2.(A) Amino acid sequence alignment of CCNI and CCNI2 from different species. Amino acid sequences of Homo sapiens CCNI, Mus musculus CCNI, Gallus gallus CCNI and Homo sapiens CCNI2, Gallus gallus CCNI2 were aligned using the ClustalW method. Dashed box indicates the cyclin box. Solid box indicates the PEST region. (B) Schematic representation of the domain structures of CCNI and CCNI2. Cyclin box and PEST region are indicated. (C) Tissue expression pattern of chicken CCNI2 was examined by western blot. Total proteins from E18.5 chicken tissues were extracted and separated by PAGE and detected with antibodies against CDK5, CCNI2, and GAPDH.
Fig 4: Expression analysis of CCNI2 during cell cycle.HeLa cells were arrested at late G1 phase with double-thymidine block, then released to enter S phase. Total RNA from different time point was extracted and used as template for reverse transcription. Quantitative PCR (A) and semiquantitative PCR (B) were performed using this cDNA as template. CCNE and CCNB were used as cell cycle markers of G1/S and G2/M phases, respectively. (C) Lysates from cells at different time points were subjected to SDS-PAGE, followed by western blot with different antibodies as indicated.
Fig 5: Colocalization of CCNI2 and CDK5 in transiently transfected cells.COS-7 cells were transfected with expression vectors that express human CCNI, CCNI2 and CDK5 with GFP or mCherry tag. (A) When expressed alone, mCherry-CDK5 localizes diffusely in the cell. (B) GFP-CCNI mainly localizes in the nuclei. (C) GFP-CCNI2 mainly localizes in the cytoplasm and the plasma membrane. (D) When expressed together with GFP-CCNI, mCherry-CDK5 moves into the nuclei and colocalizes with CCNI. (E) When expressed together with GFP-CCNI2, mCherry-CDK5 mainly localizes in the cytoplasm as well as on the plasma membrane. Nuclei were stained with DAPI. The fluorescent intensity was quantified using Image J. Scale bar: 20 µm.
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