rabbit polyclonal anti pot1  (Novus Biologicals)

Journal: Cell Discovery

Article Title: Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells

doi: 10.1038/celldisc.2017.34

Figure Lengend Snippet: Human POT1 variants have important roles in maintaining telomere overhangs. ( a ) 293T cells transiently co-expressing GST-tagged TPP1 and Flag-tagged human POT1 variants were immunoprecipitated (IP) with anti-Flag antibodies and blotted as indicated. In addition to wild-type (wt) POT1 variant proteins, rescue constructs encoding POT1 variants that contained sgRNA-resistant silent mutations (rescue) were also examined. POT1 KO cells stably expressing the sgRNA-resistant rescue constructs were then induced with doxycycline for 6 days before being harvested for the assays described below. ( b ) For telomere ChIP analysis, the rescue cells were immunoprecipitated (IP) with anti-HA antibodies to bring down associated telomeric DNA for dot-blotting and hybridization with a telomere repeat probe (TTAGGG) 3 (left panel). IgG was used as a negative control. Telomeric signals were quantified and normalized against Alu repeat signals and graphed on the right. Error bars indicate s.e. P -values were obtained using one-way analysis of variance (ANOVA). * P <0.05, ** P <0.01. ( c ) To assess RPA1 recruitment to telomeres, cells were immunostained with antibodies against RPA1 along with a telomere PNA probe. The data were quantified and graphed as shown. Three independent experiments were carried out for each cell line with at least 100 cells in each experiment. Error bars indicate s.e. ( n =3). P -values were obtained using the Student’s t -test. ** P <0.01, *** P <0.001. ( d ) To assess possible changes in TIFs, cells were immunostained using antibodies against 53BP1 along with a telomere PNA probe. The data were quantified and graphed as shown. Three independent experiments were carried out for each cell line with at least 100 cells in each experiment. Only cells with at least five TIFs were counted as positive. Error bars indicate s.e. ( n =3). P -values were obtained using the Student’s t -test. * P <0.05, *** P <0.001. ( e ) Genomic DNA was extracted from the cells for processing in the presence (+) or absence (−) of Exonuclease I (ExoI) before hybridization analysis of ss G overhangs in the native gel using the (CCCTAA) 3 probe. Total telomeric DNA and genomic DNA (Alu) signals were determined under denaturing conditions. ( f ) Overhang signals from e were quantified and normalized against Alu repeat signals and graphed as indicated. At least three independent experiments were performed for each cell line. Error bars indicate s.e. ( n =3). P -values were calculated using one-way analysis of variance (ANOVA).

Article Snippet: Antibodies used for immunoprecipitation and western blot analyses in this study are: horseradish peroxidase-conjugated anti-glutathione S-transferase (GST) polyclonal antibody (GE Healthcare Life Science, Pittsburgh, PA, USA), horseradish peroxidase-conjugated anti-FLAG M2 antibody and M2-conjugated agarose beads (Sigma, St Louis, MO, USA), rabbit anti-FLAG polyclonal antibody (Sigma), goat anti-actin polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-SMC1 antibody (Bethyl Laboratories, Montgometry, TX, USA), mouse anti-TRF2 monoclonal antibody (Calbiochem, San Diego, CA, USA), rabbit anti-RAP1 polyclonal antibody (Bethyl Laboratories), rabbit anti-POT1 polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti-TPP1 and anti-TIN2 polyclonal antibodies [ ], and goat anti-TRF1 antibody [ ], rabbit anti-p-Chk1(Ser317) and anti-Chk1 antibodies (Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-p-Chk2(Thr68) and anti-Chk2 antibodies (Cell Signaling), mouse anti-p-ATM (Ser1981) and rabbit anti-ATM antibodies (Cell Signaling), rabbit anti-p-ATR(Ser428) and anti-ATR antibodies (Cell Signaling) and rabbit anti-HA antibody (Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, Variant Assay, Construct, Stable Transfection, Hybridization, Negative Control

Journal: Cell Discovery

Article Title: Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells

doi: 10.1038/celldisc.2017.34

Figure Lengend Snippet: Deletion of individual subunits impacts metabolic pathways in the inducible KO cells. ( a ) Core metabolic pathways and key metabolites in mammalian cells are highlighted. G6P, glucose-6-phosphate. F6P, fructose-6-phosphate. FBP, fructose-1,6,-biphosphate. 3PG/2PG, 3-phosphoglycerate/2-phsphoglycerate. PEP, phosphoenolpyruvate. OAA, oxaloacetate. ( b , c ) Inducible KO cells were cultured in the presence of doxycycline for 6 days before being collected in replicates and processed for targeted metabolomic analysis by LC-MS. Cas9-inducible parental cells were grown in the presence of doxycycline and used as controls (Con). The results were normalized to internal standards and metabolites that consistently showed differences in two experiments with independent doxycycline inductions are presented here. Error bars correspond to s.d. of three independent experiments. P- values were calculated using one-way analysis of variance (ANOVA). * P <0.05; ** P <0.01; *** P <0.001. ( d ) The six telomere proteins may assemble and function on telomeres as a single unit (1), a TRF1-less five-protein subcomplex (2), or a four-protein subcomplex without TRF2/RAP1 (3). Non-telomere bound subcomplexes also exist (4). ( e ) A model of telomere protection that incorporates human POT1 isoforms highlights the unique features of the human system.

Article Snippet: Antibodies used for immunoprecipitation and western blot analyses in this study are: horseradish peroxidase-conjugated anti-glutathione S-transferase (GST) polyclonal antibody (GE Healthcare Life Science, Pittsburgh, PA, USA), horseradish peroxidase-conjugated anti-FLAG M2 antibody and M2-conjugated agarose beads (Sigma, St Louis, MO, USA), rabbit anti-FLAG polyclonal antibody (Sigma), goat anti-actin polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-SMC1 antibody (Bethyl Laboratories, Montgometry, TX, USA), mouse anti-TRF2 monoclonal antibody (Calbiochem, San Diego, CA, USA), rabbit anti-RAP1 polyclonal antibody (Bethyl Laboratories), rabbit anti-POT1 polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti-TPP1 and anti-TIN2 polyclonal antibodies [ ], and goat anti-TRF1 antibody [ ], rabbit anti-p-Chk1(Ser317) and anti-Chk1 antibodies (Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-p-Chk2(Thr68) and anti-Chk2 antibodies (Cell Signaling), mouse anti-p-ATM (Ser1981) and rabbit anti-ATM antibodies (Cell Signaling), rabbit anti-p-ATR(Ser428) and anti-ATR antibodies (Cell Signaling) and rabbit anti-HA antibody (Santa Cruz Biotechnology).

Techniques: Cell Culture, Liquid Chromatography with Mass Spectroscopy

Journal: Biomedical reports

Article Title: Telomere protein RAP1 levels are affected by cellular aging and oxidative stress.

doi: 10.3892/br.2016.707

Figure Lengend Snippet: Figure 2. Shelterin levels in young and old HDFn cells. (A) The levels of TRF2 (upper panel) and RAP1 (middle panel) were compared in young HDFn cells at PDL6 and old HDFn cells at PDL28. Various amounts of whole cell extracts were resolved by SDS‑PAGE, and the blotted proteins were detected with specific antibodies. Actin was used as a loading control (lower panel). The secondary antibodies used were for detection using the Odyssey CLx imager quantitation program (LI‑COR Biosciences). Asterisks indicate the appropriate bands for the proteins of interest. Measurements were done using the Odyssey CLx imager quantitation program. Values for TRF2 and RAP1 were normalized by those for actin. The values calculated for each lane of one protein were averaged for each PDL. TRF2 levels in the PDL28 samples were 36% of the PDL6 levels. RAP1 at PDL28 was 75% of the PDL6 levels. (B) The levels of POT1 (upper panel) and TIN2 (middle panel) were compared in young HDFn cells at PDL6 and old HDFn cells at PDL32. Various amounts of whole cell extracts were resolved by SDS‑PAGE, and the blotted proteins were detected with specific antibodies. Actin was used as a loading control (lower panel). Asterisks indicate the appropriate bands for the proteins of interest. Measurements for the appropriate bands on the film were done using NIH ImageJ. Values for POT1 and TIN2 were nor malized by those for actin. The values calculated for each lane of one protein were averaged for each PDL. Levels of both POT1 and TIN2 in the older cells were approximately the same as those in the younger cells. For both A and B, all portions of the figure were from the same blot with irrelevant information deleted for visual clarity.

Article Snippet: The primary antibodies used in the study were obtained from the following vendors: mouse monoclonal anti-human TRF2 (cat. no. 05-521) from Calbiochem (Billerica, MA, USA) used at 1:1,000 dilution; rabbit polyclonal anti-human RAP1 (cat. no. A300-306A) from Bethyl Laboratories (Montgomery, TX, USA) used at 1:2,000 dilution; rabbit polyclonal anti-human TIN2 (cat. no. ab82998) from Abcam Inc. (Cambridge, MA, USA) used at 1:1,000 dilution; rabbit polyclonal anti-human POT1 (cat. no. NB500-176) from Novus Biologicals (Littleton, CO, USA) used at 1:1,000 dilution; and goat polyclonal anti-human actin (cat. no. sc-1616) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) used at 1:2,000 dilution.

Techniques: Control, Quantitation Assay