rabbit polyclonal antibodies against cdt1  (Proteintech)

Journal: Scientific Reports

Article Title: The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

doi: 10.1038/s41598-022-25201-6

Figure Lengend Snippet: The 23 genes associated with the recurrence of hepatocellular carcinoma (HCC) were extracted using AmpliSeq transcriptome analysis in Cohort 2. ( a ) Results of clustering analysis (display of 134 genes satisfying the p value cutoff of 0.01). Data were analyzed using GeneSpring GX software (version 2.0, Agilent Technologies, Inc., Santa Clara, CA, United States). Clustering analysis showed that samples of atypical hepatocytes and nonatypical hepatocytes belonged to different groups. ( b ) Results of the volcano plot and the 23 extracted genes were analyzed using GeneSpring GX v2.0 software (Agilent Technologies). Both open circles represent selected gene groups. ( c ) Twenty-three genes that showed P values less than 0.01 and fold changes greater than 4.0 were extracted. Data were analyzed by GeneSpring GX v.2.0 software (Agilent Technologies). A bioinformatics analysis strategy for RNA-sequencing analysis was evaluated with a correction for multiplicity of statistical test according to the manufacture’s instruction. AGFG1, ArfGAP with FG repeats 1; APOPT1, cytochrome c oxidase assembly factor 8 (COA8); BCAR4, breast cancer anti-estrogen resistance 4; BEND4, BEN domain containing 4; CDRT15P2, CMT1A duplicated region transcript 15 pseudogene 2; CDT1 , chromatin licensing and DNA replication factor 1; CDY2B, chromodomain Y-linked 2B; DPPA5, developmental pluripotency associated 5; FBLN1, fibulin 1; non-ATY; hepatocytes other than regenerative atypical hepatocytes.; SLC30A6, solute carrier family 30 member 6; GPC5, glypican 5; LOC100133612: LOC1134 (Gene ID: 100133612), long intergenic nonprotein coding RNA 1134; NLGN4X, neuroligin 4 X-linked; MBD3L1, methyl-CpG binding domain protein 3 like 1; OR2AT4, olfactory receptor family 2 subfamily AT member 4; OR56A5, olfactory receptor family 56 subfamily A member 5; OR5D13, olfactory receptor family 5 subfamily D member 13; OR7D4, olfactory receptor family 7 subfamily D member 4; PIGZ, phosphatidylinositol glycan anchor biosynthesis class Z; RPL23P8, ribosomal protein L23 pseudogene 8; SNHG3, small nucleolar RNA host gene 3; SYNPO, synaptopodin; ZNF98, zinc finger protein 98.

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies against CDT1 (Proteintech) at 4 °C for 16 h. Membranes were then incubated with anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare Life Sciences, Chalfont, UK) at room temperature for 1 h. Then, β-actin expression was incubated with mouse monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich) at 4 °C for 16 h. Membranes were then incubated with anti-mouse/human IgG-HRP (GE) at 4 °C for 16 h. Proteins were visualized using an ECL plus kit (GE Healthcare Life Sciences).

Techniques: Software, RNA Sequencing, Binding Assay, Glycoproteomics

Journal: Scientific Reports

Article Title: The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

doi: 10.1038/s41598-022-25201-6

Figure Lengend Snippet: Correlation between the extent of atypical hepatocytes and ΔCt value of the 23 genes in frozen noncancerous liver tissues in Cohort 3. (n = 62).

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies against CDT1 (Proteintech) at 4 °C for 16 h. Membranes were then incubated with anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare Life Sciences, Chalfont, UK) at room temperature for 1 h. Then, β-actin expression was incubated with mouse monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich) at 4 °C for 16 h. Membranes were then incubated with anti-mouse/human IgG-HRP (GE) at 4 °C for 16 h. Proteins were visualized using an ECL plus kit (GE Healthcare Life Sciences).

Techniques:

Journal: Scientific Reports

Article Title: The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

doi: 10.1038/s41598-022-25201-6

Figure Lengend Snippet: Genes affecting the presence or absence of the postoperative recurrence and time to postoperative recurrence in patients with hepatocellular carcinoma in Cohort 3 by Cox multivariate proportional hazards regression model (n = 62).

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies against CDT1 (Proteintech) at 4 °C for 16 h. Membranes were then incubated with anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare Life Sciences, Chalfont, UK) at room temperature for 1 h. Then, β-actin expression was incubated with mouse monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich) at 4 °C for 16 h. Membranes were then incubated with anti-mouse/human IgG-HRP (GE) at 4 °C for 16 h. Proteins were visualized using an ECL plus kit (GE Healthcare Life Sciences).

Techniques:

Journal: Scientific Reports

Article Title: The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

doi: 10.1038/s41598-022-25201-6

Figure Lengend Snippet: ( a ) Cumulative incidence of relapse free survival (RFS) in relation to the mRNA expression (ΔCt value) of CDRT15P2 , BCAR4 , ZNF98 , SNHG , PIG and CDT1 was evaluated according to three different levels of expression per gene in Cohort 3. The ΔCt value of CDRT15P2 (p = 0.8107), BCAR4 (p = 0.4459), ZNF98 (p = 0.0679), SNHG3 (p = 0.0843), PIGZ (p = 0.1722), and CDT1 (high versus low: p < 0.0001). Data were analyzed using the Kaplan–Meier method, and differences among the groups were analyzed using the log-rank test. Expression was analyzed by real-time reverse transcription-quantitative PCR (RT-qPCR). Quantification was performed using the Delta (Δ)Ct method. ΔCt = Ct for each gene of interest : Ct β-ACTIN . ( b ) Comparison of ΔCt value of CDT1 among early and late postoperative recurrence, categorized as postoperative recurrence within 2 years, equally or after 2 years, and no recurrence after 2 years in Cohort 3. There was a significant difference in ΔCt value of CTD1 between patients with recurrence within 2 years after liver resection and equally or after 2 years (p = 0.0244), patients with postoperative recurrence-free patients after 2 years and equally or after 2 years (p = 0.0315) and patients with recurrence within 2 years (p < 0.0001). Data were analyzed using the Kruskal–Wallis and Steel–Dwass tests. ( c ) The cumulative incidence of RFS was compared according to three different groups of ΔCt value of CDT1 in patients with early postoperative recurrence in Cohort 3. RFS was not significantly different among the three groups (p = 0.3181). ( d ) The cumulative incidence of RFS was compared according to three different groups of ΔCt value of CDT1 in patients with late postoperative recurrence in Cohort 3. RFS was significantly higher in the high ΔCt group than in the low ΔCt value group (high versus low: p < 0.0001).

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies against CDT1 (Proteintech) at 4 °C for 16 h. Membranes were then incubated with anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare Life Sciences, Chalfont, UK) at room temperature for 1 h. Then, β-actin expression was incubated with mouse monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich) at 4 °C for 16 h. Membranes were then incubated with anti-mouse/human IgG-HRP (GE) at 4 °C for 16 h. Proteins were visualized using an ECL plus kit (GE Healthcare Life Sciences).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison

Journal: Scientific Reports

Article Title: The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

doi: 10.1038/s41598-022-25201-6

Figure Lengend Snippet: Immunohistochemistry revealed that CDT1-positive cells corresponded to atypical hepatocytes. ( a ) Image show lower magnification of CDT1 staining. (Counterstained by hematoxylin, × 100). ( b ) The area circled by the square indicates CDT1-positive cell aggregation. (Counterstained by hematoxylin, × 400). ( c ) Image of HE staining shows almost the same section as the CDT1-staining image of ( a ) (HE, × 100). ( d ) The area circled by the square indicates atypical hepatocytes aggregation (HE, × 400).

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies against CDT1 (Proteintech) at 4 °C for 16 h. Membranes were then incubated with anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare Life Sciences, Chalfont, UK) at room temperature for 1 h. Then, β-actin expression was incubated with mouse monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich) at 4 °C for 16 h. Membranes were then incubated with anti-mouse/human IgG-HRP (GE) at 4 °C for 16 h. Proteins were visualized using an ECL plus kit (GE Healthcare Life Sciences).

Techniques: Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

doi: 10.1038/s41598-022-25201-6

Figure Lengend Snippet: Comparison of CDT1 staining cells and hematoxylin and eosin (HE) staining cells in almost the same area. ( a ) Images showing CDT1 staining (counterstained by hematoxylin, × 100). The square along the line indicates a cell population that is negative or weak for CDT1 staining. ( b ) Image of HE staining shows almost the same area as the CDT1-staining image of (a) (HE, × 100) of continuous sections. The dashed line along the line on the HE staining image indicates a positive cell population for CDT1 staining. ( c ) A higher magnification image of the square of ( a ) and ( b ) is presented (HE, × 400). Nonatypical hepatocytes population were observed. ( d ) CDT1 staining was consistently found in cells morphologically considered atypical hepatocytes (HE, × 400).

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies against CDT1 (Proteintech) at 4 °C for 16 h. Membranes were then incubated with anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare Life Sciences, Chalfont, UK) at room temperature for 1 h. Then, β-actin expression was incubated with mouse monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich) at 4 °C for 16 h. Membranes were then incubated with anti-mouse/human IgG-HRP (GE) at 4 °C for 16 h. Proteins were visualized using an ECL plus kit (GE Healthcare Life Sciences).

Techniques: Comparison, Staining

Journal: Scientific Reports

Article Title: The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

doi: 10.1038/s41598-022-25201-6

Figure Lengend Snippet: Comparison of Immunohistochemistry images of CDT1-positive and Ki-67-positive hepatocytes in lobules in continuous noncancerous formalin-fixed paraffin-embedded (FFPE) noncancerous liver sections in Cohort 4. ( a ) Localization of CDT1-positive cells in lobules (counterstained with hematoxylin, × 100). ( b ) Localization of Ki-67-positive cells in lobules (counterstained with hematoxylin, × 100). ( c ) Representative immunofluorescence images of CDT1, Ki-67 and Prolong, which contained 4',6-diamidino-2-phenylindole (DAPI), in noncancerous FFPE liver sections. Blue, nuclei counterstained with Hoechst 33342, green, CDT1, red, Ki-67 and merged image (right). Arrows indicate the colocalization of CDT1 with Ki-67 in the nuclei of hepatocytes. ( d ) Representative immunofluorescence images of CDT1, Ki-67 and DAPI in cancerous FFPE liver sections. Blue, nuclei counterstained with DAPI, green, CDT1, red, Ki-67 and merged image. Arrows indicate the colocalization of CDT1 with Ki-67 in the nuclei of cancer cells.

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies against CDT1 (Proteintech) at 4 °C for 16 h. Membranes were then incubated with anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare Life Sciences, Chalfont, UK) at room temperature for 1 h. Then, β-actin expression was incubated with mouse monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich) at 4 °C for 16 h. Membranes were then incubated with anti-mouse/human IgG-HRP (GE) at 4 °C for 16 h. Proteins were visualized using an ECL plus kit (GE Healthcare Life Sciences).

Techniques: Comparison, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Immunofluorescence

Journal: Scientific Reports

Article Title: The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

doi: 10.1038/s41598-022-25201-6

Figure Lengend Snippet: Comparison of ΔCt value of CDT1 at different hepatocytes populations in formalin-fixed paraffin-embedded (FFPE) tissues in Cohort 5. ( a ) The panel shows ΔCt value of CDT1 of the nonatypical hepatocyte population and that of the controls. Data were analyzed by Kruskal–Wallis tests. ( b ) The panel shows the distribution of ΔCt value of CDT1 in the nonatypical hepatocyte population, the atypical hepatocyte population and in cancer cells. Closed circles represent patients did not develop recurrence, and the others represent patients who became recurrence. ( c ) The relationship among ΔCt value of CDT1 and duration of observation in the nonatypical hepatocyte population, the atypical hepatocyte population, and cancer cells and time to recurrence. Closed circles represent nonrecurrent patients, and open circles represent recurrent patients. Data were analyzed by Spearman’s rank correlation test. ( d ) Comparison of the ΔCt value of CDT1 in the nonatypical hepatocyte population (closed circle) or similar atypical hepatocytes (squared circle) at multiple populations from the same section. Data were analyzed by the Kruskal–Wallis test and Spearman's rank correlation test.

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies against CDT1 (Proteintech) at 4 °C for 16 h. Membranes were then incubated with anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare Life Sciences, Chalfont, UK) at room temperature for 1 h. Then, β-actin expression was incubated with mouse monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich) at 4 °C for 16 h. Membranes were then incubated with anti-mouse/human IgG-HRP (GE) at 4 °C for 16 h. Proteins were visualized using an ECL plus kit (GE Healthcare Life Sciences).

Techniques: Comparison, Formalin-fixed Paraffin-Embedded

Journal: Scientific Reports

Article Title: The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

doi: 10.1038/s41598-022-25201-6

Figure Lengend Snippet: Representative images of Western blotting analysis of CDT1 siRNA (SiCDT1)- and control siRNA (Si–C)-transfected Huh7 cells. ( a ) Representative images of CDT1 expression in 24-h and 48-h incubated SiC- and SiCDT1-transfected Huh7 cells. Jurkat cells are a positive control. ( b ) Representative images of β-actin expression at 24 h and 48 h in the SiCDT1- and Si–C-transfected Huh7 cells. ( c ) Comparison of ΔCt value of CDT1 at 24 h, 48 h and 72 h between the Huh7 cells transfected with SiCDT1 and Si–C. (*; p = 0.0001, **; p < 0.0001, ***; p = 0.0008) Data were analyzed by the Kruskal–Wallis test. ( d ) Comparison of the results of the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 24 h, 48 h and 72 h using the SiCDT1 and Si–C in Huh7 cells. (*; p = 0.0011). Data were analyzed by Kruskal–Wallis tests.

Article Snippet: The membrane was incubated with rabbit polyclonal antibodies against CDT1 (Proteintech) at 4 °C for 16 h. Membranes were then incubated with anti-rabbit IgG HRP-linked secondary antibody (GE Healthcare Life Sciences, Chalfont, UK) at room temperature for 1 h. Then, β-actin expression was incubated with mouse monoclonal antibodies against β-actin (AC-15, Sigma-Aldrich) at 4 °C for 16 h. Membranes were then incubated with anti-mouse/human IgG-HRP (GE) at 4 °C for 16 h. Proteins were visualized using an ECL plus kit (GE Healthcare Life Sciences).

Techniques: Western Blot, Control, Transfection, Expressing, Incubation, Positive Control, Comparison, MTT Assay

Journal: FEBS Open Bio

Article Title: Analysis of a temperature-sensitive mutation in Uba1: Effects of the click reaction on subsequent immunolabeling of proteins involved in DNA replication

doi: 10.1016/j.fob.2015.02.004

Figure Lengend Snippet: Indirect immunolabeling of Cdt1 is inhibited by the click reaction. (A) Cells were grown on glass coverslips at 34 °C or 39 °C for 10 h, incubated with 10 μM EdU for 20 min, and then washed and fixed with 4% PFA. Cdt1 was indirectly immunolabeled with Alexa 594. After the click reaction without Alexa 488, cells were counterstained with Hoechst 33342. The intensity of fluorescence in the non-processed image, which is taken at the same microscope setting, is monitored along the white line and is shown below each image of Cdt1. Cdt1 was found in many discrete nuclear sites. (B) After the click reaction without Alexa 488, Cdt1 was stained with Alexa 594. Labeling of Cdt1 was significantly decreased. (C) Cdt1 was stained with Alexa 594 without the click reaction. Bar, 10 μm.

Article Snippet: Immunogen of a rabbit polyclonal antibody directed against Cdt1, which was used in the present study, is the synthetic peptide for amino acid position 448–490 of human Cdt1.

Techniques: Immunolabeling, Incubation, Fluorescence, Microscopy, Staining, Labeling

Journal: FEBS Open Bio

Article Title: Analysis of a temperature-sensitive mutation in Uba1: Effects of the click reaction on subsequent immunolabeling of proteins involved in DNA replication

doi: 10.1016/j.fob.2015.02.004

Figure Lengend Snippet: Cdt1 remained close to active replication sites in tsTM3 cells incubated at 39 °C. Cells were grown on glass coverslips at 34 °C or 39 °C for more than 10 h. DIG-dUTP was introduced into cells by a hypotonic shift to label nascent DNA, and cells were incubated for 30 min, washed, and fixed with 4% PFA. (A) Cdt1 was indirectly immunolabeled with Alexa 488. Next, nascent DNA labeled with DIG-dUTP was detected with rhodamine, and cells were also counterstained with Hoechst 33342. The merged views (right) are composed of Cdt1 (green channel) and DIG-dUTP (red channel). Cdt1 was found in many discrete nuclear sites, and most of these also contained DIG-dUTP, resulting in the yellow color in the merged images, indicating co-localization between Cdt1 and nascent DNA. (B) Nascent DNA labeled with DIG-dUTP was detected with rhodamine. Next, Cdt1 was indirectly immunolabeled with Alexa 488. Cdt1 was again found in many discrete nuclear sites, and most of these also contained DIG-dUTP, resulting in the yellow color in the merged images. Bar, 10 μm. (C) Quantitative analyses of the numbers of cells expressing Cdt1 and DIG-dUTP-incorporated cells. Cells expressing Cdt1 and cells labeled with DIG-dUTP, such as those shown in panels (A) and (B), were counted and are expressed as a ratio with standard deviation in the left and middle graphs, respectively ( n > 150). The proportion of cells expressing Cdt1 to cells labeled with DIG-dUTP is shown in the right graph.

Article Snippet: Immunogen of a rabbit polyclonal antibody directed against Cdt1, which was used in the present study, is the synthetic peptide for amino acid position 448–490 of human Cdt1.

Techniques: Incubation, Immunolabeling, Labeling, Expressing, Standard Deviation