293t cells  (OriGene)

Journal: Cell reports

Article Title: Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells

doi: 10.1016/j.celrep.2019.05.073

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For High Five (Thermo Fisher Scientific) insect cell expression and purification of human POT1 the pFBHTb-Smt3star-hPOT1 was used as described previously ( Kocak et al., 2014 ).

Techniques: Recombinant, Genome Wide, RNA Sequencing Assay, CRISPR, FLAG-tag, Primer Extension Assay, Software, Mutagenesis, Negative Control, Positive Control

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Primary structure of POT1 and TPP1 showing domains and their functional properties. POT1C disease mutations (L343F, P446Q, P475L, R477T, A532P, C591W and Q623H) discussed in this manuscript are also indicated. ( b ) SDS–page gel of the purified POT1C and TPP1(PBD) proteins used in structural and biochemical studies. ( c ) Stereo image of a portion (TPP1 bound to the OB-fold of POT1) of the simulated annealing omit map at 1 sigma contour level. ( d ) X-ray Crystal structure of the human POT1C—TPP1(PBD) complex; The OB-fold and HJR of POT1C are shown in blue and red colours respectively; TPP1(PBD) is shown in green colour. The Zn 2+ ion coordinated by 4 cysteins (C382, C385, C503 and C506—stick) is shown as a cyan sphere. ( e ) Overlay of the POT1C and the Oxytricha nova telomere end binding protein alpha subunit (OnTEBPα—beige colour, PDB ID: 1OTC (ref. )) OB folds. ( f ) Overlay of the POT1C HJR (red colour) with its closest structural homologue Archaeoglobus fulgidus HJR (AfHJR—cyan colour; PDB ID: 2WIW).

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Functional Assay, SDS Page, Purification, Binding Assay

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) SDS–PAGE gel of super-telomerase extract (STE) and E. coli purified full-length POT1 (flPOT1) and TPP1(87) proteins used in the direct assay of ( c ). The red arrow indicates where TERT is expected to run (∼130 kDa). The assay in c was carried out in the presence of 150 nM of purified POT1–TPP1 complex. ( b ) Western blot of HEK293T lysates used in the direct assay of panel ( d ). The gel shows the levels of transiently expressed POT1 and TPP1 are much higher than those of TERT, confirming that the direct assays were carried out at saturating levels of POT1 and TPP1. ( c ) Telomerase direct activity assay using 2 μg of STE; 150 nM purified WT or mutant (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) flPOT1; 150 nM WT TPP1(87); and 20 nM of primer A5 (TTAGGGTTAGCGTTAGGG). ( d ) Telomerase direct activity assay using 5 μg of STE co-transfected with WT or mutant (L343F, P466Q, P475L, R477T, A532P, C591W and Q623H) flPOT1 and WT full-length TPP1 (flTPP1). The STE was supplemented with 20 nM of A5 primer. The number of telomeric repeats added to the primer are indicated on the right of the gels ( c and d , e and f ) Quantification of telomerase processivity of panels ( c ) and ( d ) respectively. The values are the average of four independent experiments; error bars indicate s.d.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: SDS Page, Purification, Western Blot, Activity Assay, Mutagenesis, Transfection

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) FP assays of POT1C and the POT1C–TPP1(PBD) complex with a single-stranded DNA probe consisting of 3 telomeric repeats (18mer). ( b ) FP assays of the WT and mutant (those that partially disrupt the POT1–TPP1 complex P446Q, C591W and Q623H) flPOT1 with the 18mer DNA probe. ( c ) FP assays of the WT and mutant flPOT1−TPP1(87) complex with the 18mer. ( d ) Bar graph showing the differences in Kd (nM) between the WT and mutant flPOT1 and flPOT1–TPP1(87) complex. The values are the average of three independent measurements and a two-tailed Student's t -test was performed with respect to WT POT1–TPP1(PBD) complex: * P <0.05, ** P <0.01.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Western blot showing the levels of YFP tagged, WT and mutant (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) full length POT1. We used GFP (αGFP) or POT1 (αPOT1) to detect the levels of YFP-POT1 in each cell line. The band at 71 kDa and below the YFP-aPOT1 is a non specific protein. GAPDH was used as a loading control. ( b ) Maximum projection images of co-localization of YFP-POT1 (green) and Cherry-TRF2 (red) proteins are shown. Merged images include DAPI. Scale bar, 5 μm. The data clearly shows co-localization of WT and mutant flPOT1 proteins to telomeres.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Western Blot, Mutagenesis, Control

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Quantitative RT-PCR showing knock down of endogenous POT1 mRNA, normalized to GAPDH transcript levels, after lentiviral infection of HEK293T cells with shPOT1 and WT Flag-POT1; selection was carried out with puromycin and blasticidin;±s.d. ( n =3). ( b ) Western blot showing WT and mutant POT1 protein expression levels in HEK293T cells expressing shPOT1 and Flag-POT1. Cells infected with the empty pLU and pLKO.1 lentiviral vectors are used as controls. ( c , d , e ) Southern blot of HEK293T cells expressing shPOT1 and WT or mutant flPOT1. DNA from 5, 50 and 120 populations doublings (PD) is shown. DNA length standards are indicated along the left and right of the gels. The white dashed line indicates the average telomere length of HEK293T cells transfected with the vector alone and no shPOT1 after 5, 50 and 120 PD. The vector (- shPOT1), vector (+shPOT1) and WT POT1 (+shPOT1) controls are indicated with a green and yellow dot respectively. The POT1 mutants (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) are indicated with red dots. ( f ) Quantification of the mean telomere length (kb) from panels ( c , d and e ). The same colour scheme as in panels c , d and e is used. Black bars indicate the range of telomere restriction fragments (TRF).

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Quantitative RT-PCR, Knockdown, Infection, Selection, Western Blot, Mutagenesis, Expressing, Southern Blot, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Telomeric FISH metaphase spreads of cells expressing WT and mutant (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) flPOT1 proteins after 50 population doublings (Red, TelC-Tamra; blue, DAPI). Endogenous POT1 levels were reduced with shPOT1. Telomere fusions, fragile telomeres and missing telomeres are indicated by red, pink and green arrows respectively. Scale bar is 5 μm. ( b - d ), Quantification of telomere fusions, fragile, and missing telomeres from the metaphase spreads of ( a ). Bars indicate the per cent of metaphase events. Error bars are indicating s.d. An average of 500 chromosomes were counted in each experiment.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Expressing, Mutagenesis

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Primary structure of POT1 and TPP1 showing domains and their functional properties. POT1C disease mutations (L343F, P446Q, P475L, R477T, A532P, C591W and Q623H) discussed in this manuscript are also indicated. ( b ) SDS–page gel of the purified POT1C and TPP1(PBD) proteins used in structural and biochemical studies. ( c ) Stereo image of a portion (TPP1 bound to the OB-fold of POT1) of the simulated annealing omit map at 1 sigma contour level. ( d ) X-ray Crystal structure of the human POT1C—TPP1(PBD) complex; The OB-fold and HJR of POT1C are shown in blue and red colours respectively; TPP1(PBD) is shown in green colour. The Zn 2+ ion coordinated by 4 cysteins (C382, C385, C503 and C506—stick) is shown as a cyan sphere. ( e ) Overlay of the POT1C and the Oxytricha nova telomere end binding protein alpha subunit (OnTEBPα—beige colour, PDB ID: 1OTC (ref. )) OB folds. ( f ) Overlay of the POT1C HJR (red colour) with its closest structural homologue Archaeoglobus fulgidus HJR (AfHJR—cyan colour; PDB ID: 2WIW).

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Functional Assay, SDS Page, Purification, Binding Assay

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) SDS–PAGE gel of super-telomerase extract (STE) and E. coli purified full-length POT1 (flPOT1) and TPP1(87) proteins used in the direct assay of ( c ). The red arrow indicates where TERT is expected to run (∼130 kDa). The assay in c was carried out in the presence of 150 nM of purified POT1–TPP1 complex. ( b ) Western blot of HEK293T lysates used in the direct assay of panel ( d ). The gel shows the levels of transiently expressed POT1 and TPP1 are much higher than those of TERT, confirming that the direct assays were carried out at saturating levels of POT1 and TPP1. ( c ) Telomerase direct activity assay using 2 μg of STE; 150 nM purified WT or mutant (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) flPOT1; 150 nM WT TPP1(87); and 20 nM of primer A5 (TTAGGGTTAGCGTTAGGG). ( d ) Telomerase direct activity assay using 5 μg of STE co-transfected with WT or mutant (L343F, P466Q, P475L, R477T, A532P, C591W and Q623H) flPOT1 and WT full-length TPP1 (flTPP1). The STE was supplemented with 20 nM of A5 primer. The number of telomeric repeats added to the primer are indicated on the right of the gels ( c and d , e and f ) Quantification of telomerase processivity of panels ( c ) and ( d ) respectively. The values are the average of four independent experiments; error bars indicate s.d.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: SDS Page, Purification, Western Blot, Activity Assay, Mutagenesis, Transfection

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) FP assays of POT1C and the POT1C–TPP1(PBD) complex with a single-stranded DNA probe consisting of 3 telomeric repeats (18mer). ( b ) FP assays of the WT and mutant (those that partially disrupt the POT1–TPP1 complex P446Q, C591W and Q623H) flPOT1 with the 18mer DNA probe. ( c ) FP assays of the WT and mutant flPOT1−TPP1(87) complex with the 18mer. ( d ) Bar graph showing the differences in Kd (nM) between the WT and mutant flPOT1 and flPOT1–TPP1(87) complex. The values are the average of three independent measurements and a two-tailed Student's t -test was performed with respect to WT POT1–TPP1(PBD) complex: * P <0.05, ** P <0.01.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Western blot showing the levels of YFP tagged, WT and mutant (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) full length POT1. We used GFP (αGFP) or POT1 (αPOT1) to detect the levels of YFP-POT1 in each cell line. The band at 71 kDa and below the YFP-aPOT1 is a non specific protein. GAPDH was used as a loading control. ( b ) Maximum projection images of co-localization of YFP-POT1 (green) and Cherry-TRF2 (red) proteins are shown. Merged images include DAPI. Scale bar, 5 μm. The data clearly shows co-localization of WT and mutant flPOT1 proteins to telomeres.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Western Blot, Mutagenesis, Control

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Quantitative RT-PCR showing knock down of endogenous POT1 mRNA, normalized to GAPDH transcript levels, after lentiviral infection of HEK293T cells with shPOT1 and WT Flag-POT1; selection was carried out with puromycin and blasticidin;±s.d. ( n =3). ( b ) Western blot showing WT and mutant POT1 protein expression levels in HEK293T cells expressing shPOT1 and Flag-POT1. Cells infected with the empty pLU and pLKO.1 lentiviral vectors are used as controls. ( c , d , e ) Southern blot of HEK293T cells expressing shPOT1 and WT or mutant flPOT1. DNA from 5, 50 and 120 populations doublings (PD) is shown. DNA length standards are indicated along the left and right of the gels. The white dashed line indicates the average telomere length of HEK293T cells transfected with the vector alone and no shPOT1 after 5, 50 and 120 PD. The vector (- shPOT1), vector (+shPOT1) and WT POT1 (+shPOT1) controls are indicated with a green and yellow dot respectively. The POT1 mutants (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) are indicated with red dots. ( f ) Quantification of the mean telomere length (kb) from panels ( c , d and e ). The same colour scheme as in panels c , d and e is used. Black bars indicate the range of telomere restriction fragments (TRF).

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Quantitative RT-PCR, Knockdown, Infection, Selection, Western Blot, Mutagenesis, Expressing, Southern Blot, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Telomeric FISH metaphase spreads of cells expressing WT and mutant (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) flPOT1 proteins after 50 population doublings (Red, TelC-Tamra; blue, DAPI). Endogenous POT1 levels were reduced with shPOT1. Telomere fusions, fragile telomeres and missing telomeres are indicated by red, pink and green arrows respectively. Scale bar is 5 μm. ( b - d ), Quantification of telomere fusions, fragile, and missing telomeres from the metaphase spreads of ( a ). Bars indicate the per cent of metaphase events. Error bars are indicating s.d. An average of 500 chromosomes were counted in each experiment.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Expressing, Mutagenesis

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Primary structure of POT1 and TPP1 showing domains and their functional properties. POT1C disease mutations (L343F, P446Q, P475L, R477T, A532P, C591W and Q623H) discussed in this manuscript are also indicated. ( b ) SDS–page gel of the purified POT1C and TPP1(PBD) proteins used in structural and biochemical studies. ( c ) Stereo image of a portion (TPP1 bound to the OB-fold of POT1) of the simulated annealing omit map at 1 sigma contour level. ( d ) X-ray Crystal structure of the human POT1C—TPP1(PBD) complex; The OB-fold and HJR of POT1C are shown in blue and red colours respectively; TPP1(PBD) is shown in green colour. The Zn 2+ ion coordinated by 4 cysteins (C382, C385, C503 and C506—stick) is shown as a cyan sphere. ( e ) Overlay of the POT1C and the Oxytricha nova telomere end binding protein alpha subunit (OnTEBPα—beige colour, PDB ID: 1OTC (ref. )) OB folds. ( f ) Overlay of the POT1C HJR (red colour) with its closest structural homologue Archaeoglobus fulgidus HJR (AfHJR—cyan colour; PDB ID: 2WIW).

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Functional Assay, SDS Page, Purification, Binding Assay

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) SDS–PAGE gel of super-telomerase extract (STE) and E. coli purified full-length POT1 (flPOT1) and TPP1(87) proteins used in the direct assay of ( c ). The red arrow indicates where TERT is expected to run (∼130 kDa). The assay in c was carried out in the presence of 150 nM of purified POT1–TPP1 complex. ( b ) Western blot of HEK293T lysates used in the direct assay of panel ( d ). The gel shows the levels of transiently expressed POT1 and TPP1 are much higher than those of TERT, confirming that the direct assays were carried out at saturating levels of POT1 and TPP1. ( c ) Telomerase direct activity assay using 2 μg of STE; 150 nM purified WT or mutant (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) flPOT1; 150 nM WT TPP1(87); and 20 nM of primer A5 (TTAGGGTTAGCGTTAGGG). ( d ) Telomerase direct activity assay using 5 μg of STE co-transfected with WT or mutant (L343F, P466Q, P475L, R477T, A532P, C591W and Q623H) flPOT1 and WT full-length TPP1 (flTPP1). The STE was supplemented with 20 nM of A5 primer. The number of telomeric repeats added to the primer are indicated on the right of the gels ( c and d , e and f ) Quantification of telomerase processivity of panels ( c ) and ( d ) respectively. The values are the average of four independent experiments; error bars indicate s.d.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: SDS Page, Purification, Western Blot, Activity Assay, Mutagenesis, Transfection

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) FP assays of POT1C and the POT1C–TPP1(PBD) complex with a single-stranded DNA probe consisting of 3 telomeric repeats (18mer). ( b ) FP assays of the WT and mutant (those that partially disrupt the POT1–TPP1 complex P446Q, C591W and Q623H) flPOT1 with the 18mer DNA probe. ( c ) FP assays of the WT and mutant flPOT1−TPP1(87) complex with the 18mer. ( d ) Bar graph showing the differences in Kd (nM) between the WT and mutant flPOT1 and flPOT1–TPP1(87) complex. The values are the average of three independent measurements and a two-tailed Student's t -test was performed with respect to WT POT1–TPP1(PBD) complex: * P <0.05, ** P <0.01.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Western blot showing the levels of YFP tagged, WT and mutant (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) full length POT1. We used GFP (αGFP) or POT1 (αPOT1) to detect the levels of YFP-POT1 in each cell line. The band at 71 kDa and below the YFP-aPOT1 is a non specific protein. GAPDH was used as a loading control. ( b ) Maximum projection images of co-localization of YFP-POT1 (green) and Cherry-TRF2 (red) proteins are shown. Merged images include DAPI. Scale bar, 5 μm. The data clearly shows co-localization of WT and mutant flPOT1 proteins to telomeres.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Western Blot, Mutagenesis, Control

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Quantitative RT-PCR showing knock down of endogenous POT1 mRNA, normalized to GAPDH transcript levels, after lentiviral infection of HEK293T cells with shPOT1 and WT Flag-POT1; selection was carried out with puromycin and blasticidin;±s.d. ( n =3). ( b ) Western blot showing WT and mutant POT1 protein expression levels in HEK293T cells expressing shPOT1 and Flag-POT1. Cells infected with the empty pLU and pLKO.1 lentiviral vectors are used as controls. ( c , d , e ) Southern blot of HEK293T cells expressing shPOT1 and WT or mutant flPOT1. DNA from 5, 50 and 120 populations doublings (PD) is shown. DNA length standards are indicated along the left and right of the gels. The white dashed line indicates the average telomere length of HEK293T cells transfected with the vector alone and no shPOT1 after 5, 50 and 120 PD. The vector (- shPOT1), vector (+shPOT1) and WT POT1 (+shPOT1) controls are indicated with a green and yellow dot respectively. The POT1 mutants (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) are indicated with red dots. ( f ) Quantification of the mean telomere length (kb) from panels ( c , d and e ). The same colour scheme as in panels c , d and e is used. Black bars indicate the range of telomere restriction fragments (TRF).

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Quantitative RT-PCR, Knockdown, Infection, Selection, Western Blot, Mutagenesis, Expressing, Southern Blot, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Structural and functional analysis of the human POT1-TPP1 telomeric complex

doi: 10.1038/ncomms14928

Figure Lengend Snippet: ( a ) Telomeric FISH metaphase spreads of cells expressing WT and mutant (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) flPOT1 proteins after 50 population doublings (Red, TelC-Tamra; blue, DAPI). Endogenous POT1 levels were reduced with shPOT1. Telomere fusions, fragile telomeres and missing telomeres are indicated by red, pink and green arrows respectively. Scale bar is 5 μm. ( b - d ), Quantification of telomere fusions, fragile, and missing telomeres from the metaphase spreads of ( a ). Bars indicate the per cent of metaphase events. Error bars are indicating s.d. An average of 500 chromosomes were counted in each experiment.

Article Snippet: For the western blot analysis of HEK293T cells co-transfected with hTERT, hTER, POT1 and TPP1, standard immunoblot protocols were used with the following antibody dilutions: anti-human TERT (abx120550, Abbexa, 1:1,000, dilution), anti-human POT1 antibody (ab21283, Abcam, 1:1,000 dilution), anti-human ACD (SAB2100024, Sigma, 1:1,000 dilution), anti-Actin antibody conjugated to HRP (A3854, Sigma, 1:1,000 dilution), and secondary HRP-conjugated anti-sheep IgG (sc-2924, Santa Cruz, 1:1,000) and anti-rabbit IgG (A0545, Sigma, 1:1,000 dilution) were used to detect the human TERT and human POT1 and TPP1 antibody, respectively.

Techniques: Expressing, Mutagenesis