mouse monoclonal antibody against trf2  (Novus Biologicals)

Journal: Nucleic Acids Research

Article Title: Aurora Kinase B, a novel regulator of TERF1 binding and telomeric integrity

doi: 10.1093/nar/gkx904

Figure Lengend Snippet: AURKB localization at telomere is linked to stem cell pluripotency. ( A ) AURKB localizes to the telomeres of mitotic mouse ES129.1 cells (arrowheads in (i)), but is lost in ES129.1 cells subjected to retinoic acid treatment differentiation (ii). Note that AURKB localization at pericentric heterochromatin is not lost in differentiated cells (arrows in Ai-ii). ( B ) AURKB localizes to the pericentric heterochromatin (arrows) but not to the telomeres of somatic, non-ESCs including mouse NIH3T3 (i) and human HT1080 (ii), telomerase-negative SKLU1 ALT cancer (iii) and telomerase overexpressing HT1080 (iv) cells. In mouse cells, TERF1 was used as a telomere marker. In human cells, TERF2 antibody was used as the telomere marker as the TERF1 antibody did not work in human cell types. Scalebars represent 5μm.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal antisera against mouse TERF1 ( ); mouse monoclonal antisera against AURKB (BD Transduction Laboratories, #611082); mouse monoclonal antisera against GFP (Roche, #11814460001), rabbit polyclonal antisera against phosphorylated H3.3 serine 31 (Active Motif, #39637), mouse monoclonal antisera against TERF2 (Santa Cruz, #sc-47693) and rat monoclonal antisera against hemagglutinin (HA) tag (Roche, #11867423001).

Techniques: Marker

Journal: Nucleic Acids Research

Article Title: Aurora Kinase B, a novel regulator of TERF1 binding and telomeric integrity

doi: 10.1093/nar/gkx904

Figure Lengend Snippet: Loss of AURKB activity in ESCs results in the formation of MTS. ( A ) Examples of MTS (obtained with APH treatment) shown. ( B ) Representative metaphase images of untreated control mouse ES129.1 cells (i) and those treated with either 0.2 µM APH (ii) or 1 µM AURKB inhibitor ZM447439 (iii) for 24 h. TEL-FISH analyses indicated that 24 h of 1 µM ZM447439 treatment resulted in an increase in MTS formation from an average of 2.3 of MTS/metaphase in untreated control cells to 8.5 MTS/metaphase in ZM447439 treated cells ( P < 0.0001; N = 1000 chromosomes from three biological replicates), compared to an average of 7.3 MTS/metaphase in cells treated with 0.2 µM APH ( P < 0.0001; N = 1000 chromosomes from three biological replicates) (iv and v). ( C ) Western blot analyses of AURKB and actin in ES129.1 cells subjected to scramble control siRNA and siRNA depletion of TERF1, TERF2 and AURKB, respectively (i). Representative images of metaphase ES129.1 cells subjected to scramble control siRNA (ii; negative control), 72 h of AURKB (iii) and TERF1 siRNA depletion (iv), respectively. About 72 h of AURKB depletion resulted in aberrant MTS formation, increasing from an average of 2.3 MTS/metaphase in cells subjected to scramble control siRNA depletion to 5.1 MTS/metaphase in AURKB-depleted cells ( P = 0.0006, N = 1200 chromosomes from three biological replicates) (v and vi). As a comparison, 72 h of TERF1 siRNA depletion caused an average of 19.95 MTS/metaphase ( P < 0.0001; Cv and vi). Magnified images of the boxed chromosomes in B and C are shown in the inset, with examples of MTS indicated by the arrowheads. Each point in scatterplots (Biv and Cv) represents of the number of MTS in a single metaphase spread, with error bars showing Q1, Q2 and Q3 values. P -values are indicated in column graphs (Biv and Cv). Scalebars represent 5 μm.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal antisera against mouse TERF1 ( ); mouse monoclonal antisera against AURKB (BD Transduction Laboratories, #611082); mouse monoclonal antisera against GFP (Roche, #11814460001), rabbit polyclonal antisera against phosphorylated H3.3 serine 31 (Active Motif, #39637), mouse monoclonal antisera against TERF2 (Santa Cruz, #sc-47693) and rat monoclonal antisera against hemagglutinin (HA) tag (Roche, #11867423001).

Techniques: Activity Assay, Control, Western Blot, Negative Control, Comparison