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Bethyl rabbit polyclonal anti tpp1 antibody

( A ) Schematic representation of the interactions between shelterin proteins and telomerase at chromosome ends. ( B ) Sequence alignment of the human <t>TPP1</t> POT1- and TIN2-binding domains with indicated mammalian orthologs. Residues of human TPP1 that were mutated in this screen are shown above the alignment. TPP1 mutants defective in binding POT1 and TIN2 are highlighted in red. Brackets indicate 2 residues simultaneously mutated (double mutant). Asterisks, colons, and periods beneath the sequence lineups represent identical residues, strongly conserved residues, and weakly conserved residues, respectively, as described by the MUSCLE algorithm. Cylinders underneath the sequence alignment indicate α helices. The structure of the POT1 C-terminus bound to the TPP1-PBD (Protein Data Bank [PDB]: 5UN7) is shown above the TPP1 domain diagram with POT1 shown in gray and TPP1 shown in yellow. Structure of the TIN2 TRFH -TPP1 TBM -TRF2 TBM complex (PBD: 5XYF) is shown below the TPP1 domain diagram with TIN2 TRFH represented in gray, TRF2 TBM represented in purple, and TPP1 TBM represented in pink. TPP1 amino acids whose mutation resulted in POT1- and TIN2-binding defects are shown in red in the structures.

Rabbit Polyclonal Anti Tpp1 Antibody, supplied by Bethyl, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis"

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

Journal: JCI Insight

doi: 10.1172/jci.insight.138059

Figure Legend Snippet: ( A ) Schematic representation of the interactions between shelterin proteins and telomerase at chromosome ends. ( B ) Sequence alignment of the human TPP1 POT1- and TIN2-binding domains with indicated mammalian orthologs. Residues of human TPP1 that were mutated in this screen are shown above the alignment. TPP1 mutants defective in binding POT1 and TIN2 are highlighted in red. Brackets indicate 2 residues simultaneously mutated (double mutant). Asterisks, colons, and periods beneath the sequence lineups represent identical residues, strongly conserved residues, and weakly conserved residues, respectively, as described by the MUSCLE algorithm. Cylinders underneath the sequence alignment indicate α helices. The structure of the POT1 C-terminus bound to the TPP1-PBD (Protein Data Bank [PDB]: 5UN7) is shown above the TPP1 domain diagram with POT1 shown in gray and TPP1 shown in yellow. Structure of the TIN2 TRFH -TPP1 TBM -TRF2 TBM complex (PBD: 5XYF) is shown below the TPP1 domain diagram with TIN2 TRFH represented in gray, TRF2 TBM represented in purple, and TPP1 TBM represented in pink. TPP1 amino acids whose mutation resulted in POT1- and TIN2-binding defects are shown in red in the structures.

Techniques Used: Sequencing, Binding Assay, Mutagenesis

( A ) Pulldown of transiently expressed FLAG-TPP1 PBD mutants on anti-FLAG–conjugated beads with Myc-POT1. ( B ) Pulldown of transiently expressed FLAG-TPP1 C-terminus mutants on anti-FLAG–conjugated beads with Myc-TIN2. These data are representative of at least 2 independent transfections and co-IPs ( n = 2).

Figure Legend Snippet: ( A ) Pulldown of transiently expressed FLAG-TPP1 PBD mutants on anti-FLAG–conjugated beads with Myc-POT1. ( B ) Pulldown of transiently expressed FLAG-TPP1 C-terminus mutants on anti-FLAG–conjugated beads with Myc-TIN2. These data are representative of at least 2 independent transfections and co-IPs ( n = 2).

Techniques Used: Transfection

( A ) Anti-Myc antibody–bound Myc-TIN2 was pulled down on protein A/G agarose beads with transiently expressed FLAG-TRF2 and indicated FLAG-TPP1 construct. ( B ) Sequence conservation of a portion of the TIN2 TRFH domain. TIN2 residues examined in this study are labeled above the sequence alignment, with red denoting amino acids whose mutation impaired the TPP1-TIN2 interaction and black denoting amino acids whose mutation did not affect TPP1-TIN2 binding. Asterisks, colons, and periods beneath the sequence lineups represent identical residues, strongly conserved residues, and weakly conserved residues, respectively, as described by the MUSCLE algorithm. ( C ) Pulldown of indicated FLAG-TIN2 construct on anti-FLAG–conjugated beads with WT Myc-TRF2 and Myc-TPP1. ( D ) Structure of the TIN2 TRFH (gray)-TPP1 TBM (pink)- TRF2 TBM (lilac) complex (PDB: 5XYF, ref. ). TIN2 residues whose mutation impacts binding to TPP1 and that likely contact the TPP1 TBM-ext are shown in yellow, TIN2 K160A is shown in green, and TPP1 TBM mutants are shown in red. Data in A and C are representative of at least 5 independent transfections and coimmunoprecipitations (co-IPs) ( n = 5).

Figure Legend Snippet: ( A ) Anti-Myc antibody–bound Myc-TIN2 was pulled down on protein A/G agarose beads with transiently expressed FLAG-TRF2 and indicated FLAG-TPP1 construct. ( B ) Sequence conservation of a portion of the TIN2 TRFH domain. TIN2 residues examined in this study are labeled above the sequence alignment, with red denoting amino acids whose mutation impaired the TPP1-TIN2 interaction and black denoting amino acids whose mutation did not affect TPP1-TIN2 binding. Asterisks, colons, and periods beneath the sequence lineups represent identical residues, strongly conserved residues, and weakly conserved residues, respectively, as described by the MUSCLE algorithm. ( C ) Pulldown of indicated FLAG-TIN2 construct on anti-FLAG–conjugated beads with WT Myc-TRF2 and Myc-TPP1. ( D ) Structure of the TIN2 TRFH (gray)-TPP1 TBM (pink)- TRF2 TBM (lilac) complex (PDB: 5XYF, ref. ). TIN2 residues whose mutation impacts binding to TPP1 and that likely contact the TPP1 TBM-ext are shown in yellow, TIN2 K160A is shown in green, and TPP1 TBM mutants are shown in red. Data in A and C are representative of at least 5 independent transfections and coimmunoprecipitations (co-IPs) ( n = 5).

Techniques Used: Construct, Sequencing, Labeling, Mutagenesis, Binding Assay, Transfection

( A ) Schematic of representative TPP1 PBD (L191A/L193A), TPP1 TBM (Y376A/Y378A), or TPP1 TBM-ext (R393A/L394A) mutations in mice, with their equivalent in humans. ( B ) Experimental scheme. BM was harvested from 5-fluorouracil–treated (5-FU–treated) Mx-Cre + Acd fl/– B6-CD45.2 mice and retrovirally cotransduced with NUP98-HOXA10HD-mCherry and a TPP1 rescue construct expressing an EGFP reporter (vs. EGFP only). Transduced BM progenitors were transplanted into lethally irradiated congenic B6-CD45.1 recipients. Six weeks later, endogenous Acd was inactivated via poly(I:C) administration to induce Mx-Cre expression. ( C ) Survival and ( D ) complete blood counts of transplanted mice at days 6, 13, and 34. n = 10 per group; remaining numbers of mice are noted in D . *** P < 0.001 by log-rank Mantel-Cox test ( C ). ** P < 0.01, *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means in D . Mean and 1 SD reported.

Figure Legend Snippet: ( A ) Schematic of representative TPP1 PBD (L191A/L193A), TPP1 TBM (Y376A/Y378A), or TPP1 TBM-ext (R393A/L394A) mutations in mice, with their equivalent in humans. ( B ) Experimental scheme. BM was harvested from 5-fluorouracil–treated (5-FU–treated) Mx-Cre + Acd fl/– B6-CD45.2 mice and retrovirally cotransduced with NUP98-HOXA10HD-mCherry and a TPP1 rescue construct expressing an EGFP reporter (vs. EGFP only). Transduced BM progenitors were transplanted into lethally irradiated congenic B6-CD45.1 recipients. Six weeks later, endogenous Acd was inactivated via poly(I:C) administration to induce Mx-Cre expression. ( C ) Survival and ( D ) complete blood counts of transplanted mice at days 6, 13, and 34. n = 10 per group; remaining numbers of mice are noted in D . *** P < 0.001 by log-rank Mantel-Cox test ( C ). ** P < 0.01, *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means in D . Mean and 1 SD reported.

Techniques Used: Construct, Expressing, Irradiation

( A ) Donor-derived NUP98-HOXA10HD-mCherry + CD11b + Gr-1 + myeloid cells were assessed by flow cytometric analysis of EGFP before poly(I:C)-induced loss of endogenous Acd (d0) or at day 6 after poly(I:C) induction. EGFP reports expression of WT TPP1 versus TPP1 mutants versus EGFP only control (MigR1). ( B ) Relative enrichment of EGFP + myeloid cells at day 6 and 13 is noted and summarized. *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means ( B ). n = 10 per group.

Figure Legend Snippet: ( A ) Donor-derived NUP98-HOXA10HD-mCherry + CD11b + Gr-1 + myeloid cells were assessed by flow cytometric analysis of EGFP before poly(I:C)-induced loss of endogenous Acd (d0) or at day 6 after poly(I:C) induction. EGFP reports expression of WT TPP1 versus TPP1 mutants versus EGFP only control (MigR1). ( B ) Relative enrichment of EGFP + myeloid cells at day 6 and 13 is noted and summarized. *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means ( B ). n = 10 per group.

Techniques Used: Derivative Assay, Expressing, Control

( A ) Immunoblot analysis of the indicated Acd fl/fl MEF cell lines showing reduction of TPP1 protein levels 72 hours after Cre-ER activation with 4-hydroxytamoxifen (4-OHT) and a uniform rescue of protein levels upon infection with indicated TPP1 WT or mutant lentiviruses. Asterisk indicates nonspecific bands detected by the antibody that serve as loading controls. n = 1. ( B ) TIF analysis was performed on the cell lines described in A using peptide nucleic acid–FISH for telomeres (red) and immunofluorescence for 53BP1 (green). DAPI was used to stain the nucleus (blue). Appearance of orange foci in the “Merge” panel indicates TIFs. Arrowheads point to 2 representative TIFs in the panel. ( C ) Quantitation of TIF data of which B is representative. Mean and SD for n = 3 sets of images (each set containing 15–20 cells) are plotted for the indicated cell lines. * P ≤ 0.05 using 2-tailed Student’s t test.

Figure Legend Snippet: ( A ) Immunoblot analysis of the indicated Acd fl/fl MEF cell lines showing reduction of TPP1 protein levels 72 hours after Cre-ER activation with 4-hydroxytamoxifen (4-OHT) and a uniform rescue of protein levels upon infection with indicated TPP1 WT or mutant lentiviruses. Asterisk indicates nonspecific bands detected by the antibody that serve as loading controls. n = 1. ( B ) TIF analysis was performed on the cell lines described in A using peptide nucleic acid–FISH for telomeres (red) and immunofluorescence for 53BP1 (green). DAPI was used to stain the nucleus (blue). Appearance of orange foci in the “Merge” panel indicates TIFs. Arrowheads point to 2 representative TIFs in the panel. ( C ) Quantitation of TIF data of which B is representative. Mean and SD for n = 3 sets of images (each set containing 15–20 cells) are plotted for the indicated cell lines. * P ≤ 0.05 using 2-tailed Student’s t test.

Techniques Used: Western Blot, Activation Assay, Infection, Mutagenesis, Immunofluorescence, Staining, Quantitation Assay

( A ) Schematic of human mutations and their equivalents in mice. ( B ) Experimental scheme, similar to . ( C ) Survival of mice reconstituted with BM containing TPP1 rescue constructs noted in B . ( D ) EGFP expression in donor NUP98-HOXA10HD-mCherry + CD11b + Gr-1 + myeloid cells before poly(I:C)-induced loss of endogenous TPP1 (d0) and subsequent time points. ( E and F ) EGFP expression in donor NUP98-HOXA10HD-mCherry + LSK progenitor cells from BM at day 239 after poly(I:C) induction. n = 10 per group. *** P < 0.001 by log-rank Mantel-Cox test ( C ). * P < 0.05, *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means ( D ). Mean and 1 SD reported.

Figure Legend Snippet: ( A ) Schematic of human mutations and their equivalents in mice. ( B ) Experimental scheme, similar to . ( C ) Survival of mice reconstituted with BM containing TPP1 rescue constructs noted in B . ( D ) EGFP expression in donor NUP98-HOXA10HD-mCherry + CD11b + Gr-1 + myeloid cells before poly(I:C)-induced loss of endogenous TPP1 (d0) and subsequent time points. ( E and F ) EGFP expression in donor NUP98-HOXA10HD-mCherry + LSK progenitor cells from BM at day 239 after poly(I:C) induction. n = 10 per group. *** P < 0.001 by log-rank Mantel-Cox test ( C ). * P < 0.05, *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means ( D ). Mean and 1 SD reported.

Techniques Used: Construct, Expressing

RNA Sequencing:

Article Title: Two Separation-of-Function Isoforms of Human TPP1 Dictate Telomerase Regulation in Somatic and Germ Cells
Article Snippet: .. The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval. (B) Schematic for the different stages of spermatogenesis. (C) Archived RNA-seq data reveal an increase in TPP1-L abundance in human round spermatids relative to spermatocytes. (D) Immunoblot analysis using an anti-rabbit TPP1 polyclonal antibody (Bethyl Labs) of the chromatin-associated fraction of cells from a biopsy of a human testis reveals a band corresponding to TPP1-L. FLAG-tagged TPP1-L and TPP1-S overexpression cell extracts were run in parallel as size markers. (E) RNAscope staining for TERT mRNA in formaldehyde fixed-paraffin embedded (FFPE) sections of human testes tissue. ..

Western Blot:

Over Expression:

RNAscope:

Staining:

Formalin-fixed Paraffin-Embedded:

Image Search Results

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Schematic representation of the interactions between shelterin proteins and telomerase at chromosome ends. ( B ) Sequence alignment of the human TPP1 POT1- and TIN2-binding domains with indicated mammalian orthologs. Residues of human TPP1 that were mutated in this screen are shown above the alignment. TPP1 mutants defective in binding POT1 and TIN2 are highlighted in red. Brackets indicate 2 residues simultaneously mutated (double mutant). Asterisks, colons, and periods beneath the sequence lineups represent identical residues, strongly conserved residues, and weakly conserved residues, respectively, as described by the MUSCLE algorithm. Cylinders underneath the sequence alignment indicate α helices. The structure of the POT1 C-terminus bound to the TPP1-PBD (Protein Data Bank [PDB]: 5UN7) is shown above the TPP1 domain diagram with POT1 shown in gray and TPP1 shown in yellow. Structure of the TIN2 TRFH -TPP1 TBM -TRF2 TBM complex (PBD: 5XYF) is shown below the TPP1 domain diagram with TIN2 TRFH represented in gray, TRF2 TBM represented in purple, and TPP1 TBM represented in pink. TPP1 amino acids whose mutation resulted in POT1- and TIN2-binding defects are shown in red in the structures.

Article Snippet: The following antibodies were used for detection with chemiluminescence by ECL plus reagents (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific): mouse monoclonal anti-FLAG M2-HRP conjugate (MilliporeSigma; A8592; 1:10,000), mouse monoclonal anti–c-Myc (9E10) HRP conjugate (Santa Cruz Biotechnology; sc-40 HRP; 1:10,000), rabbit polyclonal anti-TPP1 antibody (Bethyl Laboratories, A303-069A, 1:500), and anti-rabbit HRP-conjugated secondary antibody (Jackson ImmunoResearch; 111035045).

Techniques: Sequencing, Binding Assay, Mutagenesis

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Pulldown of transiently expressed FLAG-TPP1 PBD mutants on anti-FLAG–conjugated beads with Myc-POT1. ( B ) Pulldown of transiently expressed FLAG-TPP1 C-terminus mutants on anti-FLAG–conjugated beads with Myc-TIN2. These data are representative of at least 2 independent transfections and co-IPs ( n = 2).

Techniques: Transfection

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Anti-Myc antibody–bound Myc-TIN2 was pulled down on protein A/G agarose beads with transiently expressed FLAG-TRF2 and indicated FLAG-TPP1 construct. ( B ) Sequence conservation of a portion of the TIN2 TRFH domain. TIN2 residues examined in this study are labeled above the sequence alignment, with red denoting amino acids whose mutation impaired the TPP1-TIN2 interaction and black denoting amino acids whose mutation did not affect TPP1-TIN2 binding. Asterisks, colons, and periods beneath the sequence lineups represent identical residues, strongly conserved residues, and weakly conserved residues, respectively, as described by the MUSCLE algorithm. ( C ) Pulldown of indicated FLAG-TIN2 construct on anti-FLAG–conjugated beads with WT Myc-TRF2 and Myc-TPP1. ( D ) Structure of the TIN2 TRFH (gray)-TPP1 TBM (pink)- TRF2 TBM (lilac) complex (PDB: 5XYF, ref. ). TIN2 residues whose mutation impacts binding to TPP1 and that likely contact the TPP1 TBM-ext are shown in yellow, TIN2 K160A is shown in green, and TPP1 TBM mutants are shown in red. Data in A and C are representative of at least 5 independent transfections and coimmunoprecipitations (co-IPs) ( n = 5).

Techniques: Construct, Sequencing, Labeling, Mutagenesis, Binding Assay, Transfection

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Schematic of representative TPP1 PBD (L191A/L193A), TPP1 TBM (Y376A/Y378A), or TPP1 TBM-ext (R393A/L394A) mutations in mice, with their equivalent in humans. ( B ) Experimental scheme. BM was harvested from 5-fluorouracil–treated (5-FU–treated) Mx-Cre + Acd fl/– B6-CD45.2 mice and retrovirally cotransduced with NUP98-HOXA10HD-mCherry and a TPP1 rescue construct expressing an EGFP reporter (vs. EGFP only). Transduced BM progenitors were transplanted into lethally irradiated congenic B6-CD45.1 recipients. Six weeks later, endogenous Acd was inactivated via poly(I:C) administration to induce Mx-Cre expression. ( C ) Survival and ( D ) complete blood counts of transplanted mice at days 6, 13, and 34. n = 10 per group; remaining numbers of mice are noted in D . *** P < 0.001 by log-rank Mantel-Cox test ( C ). ** P < 0.01, *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means in D . Mean and 1 SD reported.

Techniques: Construct, Expressing, Irradiation

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Donor-derived NUP98-HOXA10HD-mCherry + CD11b + Gr-1 + myeloid cells were assessed by flow cytometric analysis of EGFP before poly(I:C)-induced loss of endogenous Acd (d0) or at day 6 after poly(I:C) induction. EGFP reports expression of WT TPP1 versus TPP1 mutants versus EGFP only control (MigR1). ( B ) Relative enrichment of EGFP + myeloid cells at day 6 and 13 is noted and summarized. *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means ( B ). n = 10 per group.

Techniques: Derivative Assay, Expressing, Control

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Immunoblot analysis of the indicated Acd fl/fl MEF cell lines showing reduction of TPP1 protein levels 72 hours after Cre-ER activation with 4-hydroxytamoxifen (4-OHT) and a uniform rescue of protein levels upon infection with indicated TPP1 WT or mutant lentiviruses. Asterisk indicates nonspecific bands detected by the antibody that serve as loading controls. n = 1. ( B ) TIF analysis was performed on the cell lines described in A using peptide nucleic acid–FISH for telomeres (red) and immunofluorescence for 53BP1 (green). DAPI was used to stain the nucleus (blue). Appearance of orange foci in the “Merge” panel indicates TIFs. Arrowheads point to 2 representative TIFs in the panel. ( C ) Quantitation of TIF data of which B is representative. Mean and SD for n = 3 sets of images (each set containing 15–20 cells) are plotted for the indicated cell lines. * P ≤ 0.05 using 2-tailed Student’s t test.

Techniques: Western Blot, Activation Assay, Infection, Mutagenesis, Immunofluorescence, Staining, Quantitation Assay

Journal: JCI Insight

Article Title: TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

doi: 10.1172/jci.insight.138059

Figure Lengend Snippet: ( A ) Schematic of human mutations and their equivalents in mice. ( B ) Experimental scheme, similar to . ( C ) Survival of mice reconstituted with BM containing TPP1 rescue constructs noted in B . ( D ) EGFP expression in donor NUP98-HOXA10HD-mCherry + CD11b + Gr-1 + myeloid cells before poly(I:C)-induced loss of endogenous TPP1 (d0) and subsequent time points. ( E and F ) EGFP expression in donor NUP98-HOXA10HD-mCherry + LSK progenitor cells from BM at day 239 after poly(I:C) induction. n = 10 per group. *** P < 0.001 by log-rank Mantel-Cox test ( C ). * P < 0.05, *** P < 0.001 by 2-way ANOVA with post hoc Tukey’s test to assess differences in means ( D ). Mean and 1 SD reported.

Techniques: Construct, Expressing

(A) Schematic showing a TPP1-centric view of the shelterin complex in humans.

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: (A) Schematic showing a TPP1-centric view of the shelterin complex in humans.

Article Snippet: The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval. (B) Schematic for the different stages of spermatogenesis. (C) Archived RNA-seq data reveal an increase in TPP1-L abundance in human round spermatids relative to spermatocytes. (D) Immunoblot analysis using an anti-rabbit TPP1 polyclonal antibody (Bethyl Labs) of the chromatin-associated fraction of cells from a biopsy of a human testis reveals a band corresponding to TPP1-L. FLAG-tagged TPP1-L and TPP1-S overexpression cell extracts were run in parallel as size markers. (E) RNAscope staining for TERT mRNA in formaldehyde fixed-paraffin embedded (FFPE) sections of human testes tissue.

Techniques:

(A) HeLa-EM2-11ht cells expressing TPP1-S, TPP1-L M87A, or TPP1ΔNOB were analyzed for telomerase recruitment to telomeres by immunofluorescence (IF) and fluorescence in situ hybridization (FISH). “FLAG (TPP1)” indicates IF signal of the indicated TPP1 construct at telomeres (green). Telomerase RNA (“TR”) was detected by FISH with a fluorescently tagged DNA probe (red). The “merge” panel depicts the extent of telomerase recruitment to telomeres (yellow).

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: (A) HeLa-EM2-11ht cells expressing TPP1-S, TPP1-L M87A, or TPP1ΔNOB were analyzed for telomerase recruitment to telomeres by immunofluorescence (IF) and fluorescence in situ hybridization (FISH). “FLAG (TPP1)” indicates IF signal of the indicated TPP1 construct at telomeres (green). Telomerase RNA (“TR”) was detected by FISH with a fluorescently tagged DNA probe (red). The “merge” panel depicts the extent of telomerase recruitment to telomeres (yellow).

Techniques: Expressing, Immunofluorescence, Fluorescence, In Situ Hybridization, Construct

(A) Cartoon representation of contrasting outcomes of the in vivo IF-coFISH telomere extension assay involving FLAG-TPP1 (green), TR (cyan), and newly synthesized mutant telomeric repeats “mutant telomere” (red). White foci in merge of TR and mutant telomere signals depict colocalization.

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: (A) Cartoon representation of contrasting outcomes of the in vivo IF-coFISH telomere extension assay involving FLAG-TPP1 (green), TR (cyan), and newly synthesized mutant telomeric repeats “mutant telomere” (red). White foci in merge of TR and mutant telomere signals depict colocalization.

Techniques: In Vivo, Synthesized, Mutagenesis

(A) Existing high-throughput sequencing information for the ACD locus was visualized using the UCSC genome browser. Vertical gray bar denotes the 5′ end of TPP1-S mRNA. See also Figure S4A and STAR Methods.

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: (A) Existing high-throughput sequencing information for the ACD locus was visualized using the UCSC genome browser. Vertical gray bar denotes the 5′ end of TPP1-S mRNA. See also Figure S4A and STAR Methods.

Techniques: Next-Generation Sequencing

(A) Deposited Burge lab RNA-seq data from the indicated tissues or organs were visualized on the UCSC genome browser. The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval.

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: (A) Deposited Burge lab RNA-seq data from the indicated tissues or organs were visualized on the UCSC genome browser. The conspicuous abundance of TPP1-L-specific reads in testes relative to other tissues is highlighted with a gray oval.

Techniques: RNA Sequencing

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

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

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: (A) Schematic showing a TPP1-centric view of the shelterin complex in humans.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-TPP1 antibody Bethyl Bethyl Cat# A303-069A; RRID:AB_10890369 Monoclonal anti-β-actin antibody Sigma-Aldrich (Sigma; Cat# A5441); RRID:AB_476744 Monoclonal ANTI-FLAG M2-Peroxidase (HRP) Sigma-Aldrich (Sigma-Aldrich Cat# A8592; RRID:AB_439702) Goat anti-rabbit IgG-HRP Secondary antibody Santa Cruz Biotechnology Santa Cruz Biotechnology Cat# sc-2004; RRID:AB_631746 Goat anti-mouse IgG-HRP Secondary antibody Santa Cruz Biotechnology Santa Cruz Biotechnology Cat# sc-2005; RRID:AB_631736 c-MYC (9E10) HRP antibody Santa Cruz Biotechnology Santa Cruz Biotechnology Cat#sc-40 HRP; RRID: N/A Monoclonal ANTI-FLAG M2 antibody Sigma-Aldrich Sigma-Aldrich Cat# F1804; RRID:AB_262044 53BP1 Antibody Novus Novus Cat# NB100-304; RRID:AB_10003037 Goat anti-mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fisher Scientific Thermo Fisher Scientific Cat# A-11001; RRID:AB_2534069 Goat Anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 Thermo Fisher Scientific Thermo Fisher Scientific Cat# A-21244; RRID:AB_2535812 Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 Thermo Fisher Scientific Thermo Fisher Scientific Cat# A-21235; RRID:AB_2535804 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 Thermo Fisher Scientific Thermo Fisher Scientific Cat# A-11011; RRID:AB_143157 Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 Thermo Fisher Scientific Thermo Fisher Scientific Cat# A-11004; RRID:AB_2534072 Bacterial and Virus Strains BL21 (DE3) Millipore Sigma Cat# 70235 High Five insect cells Thermo Fisher Scientific Cat# {"type":"entrez-nucleotide","attrs":{"text":"B85502","term_id":"2926634","term_text":"B85502"}} B85502 Chemicals, Peptides, and Recombinant Proteins TPP1 1-334 This paper N/A TPP1 90-334 ( Wang et al., 2007 ) N/A POT1 ( Kocak et al., 2014 ) N/A Deposited Data GENCODE ( Harrow et al., 2012 ) N/A Genome-wide ChIP studies in 91 human cell lines ( Gerstein et al., 2012 ; Wang et al., 2012 ; Wang et al., 2013 ) N/A GRO-cap and GRO-seq data ( Core et al., 2014 ; Core et al., 2008 ) N/A RNA-seq from a large number of human cell lines ( ENCODE Project Consortium, 2012 ) (data deposited by Wold Lab at Caltech) N/A RIKEN CAGE ( Carninci et al., 1996 ) N/A Histone ChIP from a large number of human cell lines Bernstein Lab at the Broad Institute as part of ENCODE N/A Tissue/organ RNA-seq Burge Lab ( Wang et al., 2008 ).

Techniques:

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

Techniques: Expressing, Immunofluorescence, Fluorescence, In Situ Hybridization, Construct

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

Techniques: In Vivo, Synthesized, Mutagenesis

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

Techniques: Next-Generation Sequencing

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

Techniques: RNA Sequencing

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

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

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: (A) Schematic showing a TPP1-centric view of the shelterin complex in humans.

Techniques:

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

Techniques: Expressing, Immunofluorescence, Fluorescence, In Situ Hybridization, Construct

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

Techniques: In Vivo, Synthesized, Mutagenesis

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

Techniques: Next-Generation Sequencing

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

Techniques: RNA Sequencing Assay

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

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

(A) HTC75 cells treated with triciribine or mock treated with DMSO for 3 hours were analyzed by western blotting using the indicated antibodies. Actin was used as loading control. Arrows indicate the two POT1 isoforms. (B) Cells from (A) were examined by telomere chromatin immunoprecipitation (ChIP) using antibodies against TPP1, POT1 and RAP1, followed by dot-blotting with probes against telomere sequences or Alu repeats. Rabbit IgG served as control. (C) Quantification of data from (B) (two independent experiments). ChIP signal intensities were normalized against input DNA. Error bars represent s.d. P-values were determined by the Student t-test.

Journal: Aging cell

Article Title: Akt Regulates TPP1 Homodimerization and Telomere Protection

doi: 10.1111/acel.12137

Figure Lengend Snippet: (A) HTC75 cells treated with triciribine or mock treated with DMSO for 3 hours were analyzed by western blotting using the indicated antibodies. Actin was used as loading control. Arrows indicate the two POT1 isoforms. (B) Cells from (A) were examined by telomere chromatin immunoprecipitation (ChIP) using antibodies against TPP1, POT1 and RAP1, followed by dot-blotting with probes against telomere sequences or Alu repeats. Rabbit IgG served as control. (C) Quantification of data from (B) (two independent experiments). ChIP signal intensities were normalized against input DNA. Error bars represent s.d. P-values were determined by the Student t-test.

Article Snippet: The antibodies used for ChIP are: rabbit polyclonal POT1 (N-terminal) ( O'Connor et al., 2006 ), rabbit polyclonal TPP1 (467) ( Liu et al., 2004 ), polyclonal anti-RAP1 (Bethyl Laboratories) and rabbit IgG (Santa Cruz, SC2027).

Techniques: Western Blot, Control, Chromatin Immunoprecipitation

(A) HTC75 cells co-expressing YFPn-tagged TPP1 with YFPc-tagged TPP1 or TIN2 were examined by BiFC assays. The percentage of cells displaying fluorescence complementation was quantitated by flow cytometry. Error bars indicate standard error (n=3). (B) 293T cells transiently co-expressing Myc-tagged TPP1 with Flag-tagged TPP1 or TIN2 were immunoprecipitated with anti-Flag antibodies. The immunoprecipitates were western blotted as indicated. (C) 293T cells transiently co-expressing Myc-tagged TPP1 with Flag-tagged TPP1 or TPP1 OB-fold deletion mutant (TPP1ΔOB) were immunoprecipitated with anti-Flag antibodies. The immunoprecipitates were western blotted as indicated. (D) Bacterially purified GST-tagged TPP1 OB fold only mutant (TPP1 OB) was incubated with MBP-tagged TPP1 OB for GST pull-down assays. The precipitates were resolved by SDS-PAGE and visualized by Coomassie staining or western blotting. GST-tagged Raf-1 Ras-binding domain (RBD) and MBP-tagged FAM118B were used as controls. (E) HTC75 cells were transfected with siRNA oligos against Akt1 (siAKT1-1) in combination with Flag-tagged wildtype TPP1 or TPP1 OB fold deletion mutant (TPP1ΔOB), and then analyzed by Western blotting using the indicated antibodies. Actin was used as loading control. (F) Cells from (E) were examined by immunostaining using anti-53BP1 (red) and TRF2 (green) antibodies. Arrows indicate overlapping foci. (G) Quantification of data from (F). Only cells with >4 co-localized foci were scored. Error bars indicate s.e.m. (n=3). P-values were determined by the Student t-test.

Journal: Aging cell

Article Title: Akt Regulates TPP1 Homodimerization and Telomere Protection

doi: 10.1111/acel.12137

Figure Lengend Snippet: (A) HTC75 cells co-expressing YFPn-tagged TPP1 with YFPc-tagged TPP1 or TIN2 were examined by BiFC assays. The percentage of cells displaying fluorescence complementation was quantitated by flow cytometry. Error bars indicate standard error (n=3). (B) 293T cells transiently co-expressing Myc-tagged TPP1 with Flag-tagged TPP1 or TIN2 were immunoprecipitated with anti-Flag antibodies. The immunoprecipitates were western blotted as indicated. (C) 293T cells transiently co-expressing Myc-tagged TPP1 with Flag-tagged TPP1 or TPP1 OB-fold deletion mutant (TPP1ΔOB) were immunoprecipitated with anti-Flag antibodies. The immunoprecipitates were western blotted as indicated. (D) Bacterially purified GST-tagged TPP1 OB fold only mutant (TPP1 OB) was incubated with MBP-tagged TPP1 OB for GST pull-down assays. The precipitates were resolved by SDS-PAGE and visualized by Coomassie staining or western blotting. GST-tagged Raf-1 Ras-binding domain (RBD) and MBP-tagged FAM118B were used as controls. (E) HTC75 cells were transfected with siRNA oligos against Akt1 (siAKT1-1) in combination with Flag-tagged wildtype TPP1 or TPP1 OB fold deletion mutant (TPP1ΔOB), and then analyzed by Western blotting using the indicated antibodies. Actin was used as loading control. (F) Cells from (E) were examined by immunostaining using anti-53BP1 (red) and TRF2 (green) antibodies. Arrows indicate overlapping foci. (G) Quantification of data from (F). Only cells with >4 co-localized foci were scored. Error bars indicate s.e.m. (n=3). P-values were determined by the Student t-test.

Techniques: Expressing, Fluorescence, Flow Cytometry, Immunoprecipitation, Western Blot, Mutagenesis, Purification, Incubation, SDS Page, Staining, Binding Assay, Transfection, Control, Immunostaining

(A) HTC75 cells co-expressing YFPn-tagged TPP1 with YFPc-tagged TPP1 or TIN2 were either serum starved (0.01% FBS) or treated with triciribine, and then examined by BiFC assays. Error bars indicate standard error (n=3). (B) 293T cells transiently co-expressing TPP1-Flag with Myc-TPP1 were treated with DMSO or triciribine. Cells co-expressing SFB-Akt1 and Myc-TPP1 were also examined. Cell lysates were immunoprecipitated with anti-Flag antibodies and blotted as indicated. (C) HTC75 cells co-expressing YFPn-TPP1 with YFPc-TPP1 were transiently transfected with siRNA oligos against Akt1 and analyzed in BiFC assays. Error bars indicate standard errors (n=3). (D) Cells from (C) were examined by western blotting for Akt1 knockdown efficiency. Actin was used as loading control. (E) HTC75 cells co-expressing YFPn-TPP1 and YFPc-TPP1 were transiently co-transfected with control oligos or siRNA oligos against Akt1 in combination with siRNA-resistant SFB-tagged wildtype (WT) Akt1 or kinase-dead Akt1 (Akt1 K179M). The cells were then examined by western blotting using the indicated antibodies. Actin was used as a loading control. (F) Cells from (E) were analyzed in BiFC assays. P-values were determined by the Student t-test.

Journal: Aging cell

Article Title: Akt Regulates TPP1 Homodimerization and Telomere Protection

doi: 10.1111/acel.12137

Figure Lengend Snippet: (A) HTC75 cells co-expressing YFPn-tagged TPP1 with YFPc-tagged TPP1 or TIN2 were either serum starved (0.01% FBS) or treated with triciribine, and then examined by BiFC assays. Error bars indicate standard error (n=3). (B) 293T cells transiently co-expressing TPP1-Flag with Myc-TPP1 were treated with DMSO or triciribine. Cells co-expressing SFB-Akt1 and Myc-TPP1 were also examined. Cell lysates were immunoprecipitated with anti-Flag antibodies and blotted as indicated. (C) HTC75 cells co-expressing YFPn-TPP1 with YFPc-TPP1 were transiently transfected with siRNA oligos against Akt1 and analyzed in BiFC assays. Error bars indicate standard errors (n=3). (D) Cells from (C) were examined by western blotting for Akt1 knockdown efficiency. Actin was used as loading control. (E) HTC75 cells co-expressing YFPn-TPP1 and YFPc-TPP1 were transiently co-transfected with control oligos or siRNA oligos against Akt1 in combination with siRNA-resistant SFB-tagged wildtype (WT) Akt1 or kinase-dead Akt1 (Akt1 K179M). The cells were then examined by western blotting using the indicated antibodies. Actin was used as a loading control. (F) Cells from (E) were analyzed in BiFC assays. P-values were determined by the Student t-test.

Techniques: Expressing, Immunoprecipitation, Transfection, Western Blot, Knockdown, Control

Review

rabbit polyclonal anti tpp1 antibody (Bethyl)

Structured Review

Images

1) Product Images from "TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis"

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