Journal: Nucleic Acids Research

Article Title: Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ

doi: 10.1093/nar/gkab980

Figure Lengend Snippet: Phospho-Ku70 causes rapid dissociation from sites of DNA damage; inhibition of neddylation compromises both pKu70 release and DNA repair following laser micro-irradiation. ( A ) Ku70 release from DNA damage sites after the inhibition of neddylation by MLN4924 (3 μM, 1 h), followed by laser micro-irradiation as in Figure . Relative fluorescence intensity fold change (established as in Figure ). Unpaired t-test, NS = non-significant. ( B ) Western blot of γ-H2AX after irradiation alone (a) or after combined cell treatment (b) with MLN4924 (3 μM, 1 h), followed by irradiation (4Gy). Anti-Ku70 (clone N3H10) was used as the loading control. ( C , a) Western blot analyses of the neddylation patterns of untreated, irradiated (4 Gy), or MLN4924 (3 μM, 1 h) pre-treated and irradiated (4Gy) cells expressing eGFP-ser- or -ala-Ku70-fusion proteins. Cells were lysed 2 h post-irradiation. After SDS-PAGE, the membranes were probed with anti-NEDD8 or anti-Ku70 antibodies. The band below the 100 KDa-MW marker with decreased intensity upon MLN4924 treatment indicates decreased neddylation of cullin. (b) The ubiquitylation pattern analysed in protein extracts from cells expressing eGFP-Ku70-fusion proteins. Cells expressing eGFP-ser-Ku70 or eGFP-ala-Ku70 were irradiated with or without MLN4924 or TZ9 pre-treatment and lysed 2 h post-irradiation. Protein extracts were immunoprecitpitated by using a GFP-Trap_MA kit (Chromotek). After SDS-PAGE, the membranes were probed with an anti-ubiquitin, anti-Ku70, or anti-GFP antibody.

Article Snippet: eGFP-ser-Ku70 and eGFP-ala-Ku70 fusion proteins were immunoprecipitated using the ChromoTek GFP-Trap kit according to the supplier's protocol, except for the cell lysis step.

Techniques: Inhibition, Irradiation, Fluorescence, Western Blot, Expressing, SDS Page, Marker

Journal: Nucleic Acids Research

Article Title: Phospho-Ku70 induced by DNA damage interacts with RNA Pol II and promotes the formation of phospho-53BP1 foci to ensure optimal cNHEJ

doi: 10.1093/nar/gkab980

Figure Lengend Snippet: Ser-Ku70 stimulates HR activity while ala-Ku70 promotes DNA DSB distal end junctions. ( A ) HR activity and the cell cycle were assessed by flow cytometry of U2OS cell line containing the integrated reporter construct pDR-GFP (a and b). This cell line was transfected using ser-, ala-, or glu-Ku70 vectors as described in the methods section. (c) pKu70 expression was verified by western blot and is shown as cropped blots. The reporter is composed of two inactive eGFP genes. The upstream GFP gene is truncated; the gene downstream of the promoter has an integrated I-SceI cleavage site and is therefore inactive. Upon I-SceI expression, as verified by western blotting (c) (α-tubulin was used as the loading control), the cleaved GFP gene recombines with the truncated GFP gene on the sister chromatid, resulting in the expression of GFP, which was measured by flow cytometry 72h after I-SceI transfection (exemplified in a). The results (d) represent values that were calculated as follows: (I-SceI – transfection events) – (control – transfection events). n = 6. Unpaired t -test, * P < 0.01. ( B ) NHEJ activity was measured (a) using the human fibroblast cell line GC92 (SV40-transformed) expressing ser-, ala-, or glu-Ku70 [controlled by western cropped (b) blots] that contains the intrachromosomally integrated pCOH-CD4 (cohesive ends) reporter construct. Upon cleavage by I-SceI, a fragment containing the H2Kd and CD8 genes was excised. Re-joining of the flanking ends by NHEJ brought the pCMV promoter closer to the CD4 gene, which promoted its expression. (a) CD4 expression on the cell surface was quantified by flow cytometry 72 h after I-SceI transfection. (c) The results represent values that were calculated as follows: (I-SceI – transfection events) – (control – transfection events). n = 4. Unpaired t -test, * P < 0.01. Differences between values of cells expressing ser-Ku70 or glu-Ku70 are statisticaly non significant.

Article Snippet: eGFP-ser-Ku70 and eGFP-ala-Ku70 fusion proteins were immunoprecipitated using the ChromoTek GFP-Trap kit according to the supplier's protocol, except for the cell lysis step.

Techniques: Activity Assay, Flow Cytometry, Construct, Transfection, Expressing, Western Blot, Transformation Assay

Journal: Science Advances

Article Title: PES1 is a critical component of telomerase assembly and regulates cellular senescence

doi: 10.1126/sciadv.aav1090

Figure Lengend Snippet: ( A ) Cell extracts from MCF7 cells stably expressing Flag (control) or Flag-tagged hTERT (Flag-hTERT) were immunopurified with anti-Flag affinity gel, and the purified complex was subject to SDS–polyacrylamide gel electrophoresis (PAGE) and silver-stained. The differential protein bands were retrieved and analyzed by MS. n , number of peptides identified by MS analysis; MM, molecular mass. ( B ) HEK293T cells transiently transfected with Myc-tagged hTERT (Myc-hTERT) and Flag-PES1 or Flag-DKC1 were treated with or without RNase A (0.1 mg/ml), and immunoprecipitated (IP) with anti-Flag, followed by immunoblotting (IB) with the indicated antibodies. ( C ) MCF7 cell lysates were immunoprecipitated with anti-hTERT from Abbexa and immunoblotted with anti-hTERT from Abcam and anti-PES1. IgG, immunoglobulin G. ( D ) Glutathione-Sepharose beads bound with GST-hTERT were incubated with purified His-tagged wild-type (WT) or mutated PES1. After washing the beads, the bound proteins were subjected to SDS-PAGE and immunoblotted with anti-GST antibody. Also shown on top of the graph is a schematic diagram of the PES1 protein, illustrating the location of the BRCT domain. Asterisks indicate the positions of the expected full-length fusion proteins. ( E ) Glutathione-Sepharose beads bound with WT or truncated hTERT were incubated with purified His-tagged PES1. After washing the beads, the bound proteins were examined by immunoblot with anti-His. Schematic diagram of the hTERT deletion constructs used is shown at the top. Asterisks indicate the positions of the expected full-length fusion proteins. TEN, telomerase N-terminal; TRBD, TR-binding domain; RT, reverse transcriptase; CTE, C-terminal extension.

Article Snippet: Immunohistochemical staining was performed as described previously ( ) using rabbit anti-PES1 (A300-902A, Bethyl Laboratories) as primary antibody.

Techniques: Stable Transfection, Expressing, Purification, Polyacrylamide Gel Electrophoresis, Staining, Transfection, Immunoprecipitation, Western Blot, Incubation, SDS Page, Construct, Binding Assay

Journal: Science Advances

Article Title: PES1 is a critical component of telomerase assembly and regulates cellular senescence

doi: 10.1126/sciadv.aav1090

Figure Lengend Snippet: ( A ) MCF7 cells transfected with the indicated plasmids were immunoprecipitated with anti-Flag. The immune complexes were eluted with Flag peptide and re-IP using anti-Myc or normal IgG. The resulting precipitates were used for detection of Flag-hTERT and Myc-PES1 expression by immunoblot. hTR levels were determined by RT-PCR. ( B ) MCF7 cell extracts were fractionated on Superose 6 size exclusion columns. Fast protein liquid chromatography (FPLC) chromatographic elution profiles are indicated. The elution positions of calibration proteins with known molecular masses (kDa) are shown by arrows, and an equal volume from each chromatographic fraction was used for determination of protein expression by immunoblot with the indicated antibodies. To increase the specificity of hTERT detection, cells were immunoprecipitated with anti-hTERT from Abbexa, followed by immunoblot with anti-hTERT from Abcam (hTERT after pre-purification). hTR levels were determined by reverse transcription polymerase chain reaction (RT-PCR) and telomerase activity by telomerase repeat amplification protocol (TRAP). CHAPS buffer was used as a negative control for TRAP assay. IC, internal control; mAu, milli-Absorbance unit. Red frame indicates a potential complex with hTERT/hTR/DKC1/PES1. ( C ) MCF7 and U2OS cells were transfected with Flag-tagged PES1 or hTERT or empty vector, and immunoprecipitated with anti-Flag agarose. The resulting precipitates were used for assessment of Flag-PES1 or Flag-hTERT expression by immunoblot. hTR levels were determined by RT-PCR. β-Actin was used as a negative control for hTR determination. ( D ) MCF7-Flag-PES1 +shPES1 cells or control (MCF7-Flag +shCon ) cells were transfected with control small interfering RNA (siRNA) or hTERT siRNA as indicated and were analyzed as in (C). ( E ) Biotinylated hTR was incubated with purified GST-hTERT, GST-PES1, or GST-hTERT plus GST-PES1. The resulting complexes were subject to RNA pull-down assay. The amount of biotin-hTR was detected by the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific).

Article Snippet: Immunohistochemical staining was performed as described previously ( ) using rabbit anti-PES1 (A300-902A, Bethyl Laboratories) as primary antibody.

Techniques: Transfection, Immunoprecipitation, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Fast Protein Liquid Chromatography, Purification, Activity Assay, Amplification, Negative Control, TRAP Assay, Plasmid Preparation, Small Interfering RNA, Incubation, Pull Down Assay

Journal: Science Advances

Article Title: PES1 is a critical component of telomerase assembly and regulates cellular senescence

doi: 10.1126/sciadv.aav1090

Figure Lengend Snippet: ( A ) PES1 WT or KO MCF7 cells (left) and MEFs (right) were transiently transfected with the indicated plasmids and immunoprecipitated with anti-Flag. The precipitates were used for assessment of Flag-h/mTERT, PES1, and β-actin expression by immunoblot. TR levels were determined by RT-PCR. Flag-h/mTERT, Flag-tagged human or mouse TERT. Flag-hTERT was used for MCF7 cells, and Flag-mTERT was used for MEFs. ( B ) siRNA-mediated PES1 KD MCF7 or HepG2 cells were transfected with Flag-hTERT and siRNA-resistant Myc-tagged PES1, PES1 (ΔBRCT), or PES1 (W397R) as indicated and were analyzed as in (A). ( C ) siRNA-mediated PES1 KD MEFs were transfected with Flag-mTERT and siRNA-resistant Myc-tagged PES1, PES1 (ΔBRCT), or PES1 (W397R) as indicated and were analyzed as in (A). Data shown are mean ± SD of three independent experiments (A to C). ** P < 0.01. ( D ) Biotinylated hTR was incubated with purified GST-hTERT and WT or mutant His-PES1. The resulting complexes were subject to RNA pull-down assay. Unlabeled hTR was used as a negative control. Asterisks indicate the positions of the expected full-length fusion proteins.

Article Snippet: Immunohistochemical staining was performed as described previously ( ) using rabbit anti-PES1 (A300-902A, Bethyl Laboratories) as primary antibody.

Techniques: Transfection, Immunoprecipitation, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation, Purification, Mutagenesis, Pull Down Assay, Negative Control

Journal: Science Advances

Article Title: PES1 is a critical component of telomerase assembly and regulates cellular senescence

doi: 10.1126/sciadv.aav1090

Figure Lengend Snippet: ( A ) Direct telomerase assay in PES1 KO MCF7 cells or MEFs transfected with Flag-hTERT, hTR, and PES1 or its mutants. Transfected cells were immunoprecipitated with anti-Flag, followed by telomerase activity detection. Parental cells without Flag-hTERT transfection were used as a negative control. Values shown are mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01. ( B ) PES1 WT and KO mouse hepatocytes were isolated from liver-specific PES1 KO mice generated by mating conditional PES1 KO mice with Alb-Cre transgenic mice and were analyzed by TRAP assay. ( C ) IP-TRAP analysis of PES1 KO MCF7 cells transfected with Flag-tagged WT or mutant PES1. When the binding of Flag-tagged proteins to anti-Flag agarose beads was complete, the reaction mixtures were centrifuged and the supernatants were collected for TRAP assay. The precipitates were washed, lysed, and used for TRAP assay. Flag-tagged PES1 or endogenous PES1 levels were analyzed by immunoblot with anti-PES1. ( D ) IP-TRAP analysis of siRNA-mediated PES1 KD HepG2 cells transfected with Flag-PES1 or empty vector. ( E ) TRAP assay was performed by mixing in vitro transcribed hTR and in vitro translated hTERT with purified WT and mutated His-PES1. ( F ) Representative MCF7 monoclones stably expressing PES1 shRNA or control shRNA and WT or mutant PES1 were harvested at indicated PDLs. Reexpression of WT or mutant PES1 was performed after 25 PDLs by splitting the same clone. Telomere lengths were measured using terminal restriction fragment (TRF) analysis. Representative immunoblot reveals PES1 expression. PES1-R, shRNA-resistant PES1. PES1 W397R-R, shRNA-resistant PES1 W397R. ( G ) PES1 WT and KO mouse hepatocytes isolated from liver-specific PES1 KO mice at 3 months were subjected to TRF analysis. Left and right panels indicate two nest mice. fl, flox.

Article Snippet: Immunohistochemical staining was performed as described previously ( ) using rabbit anti-PES1 (A300-902A, Bethyl Laboratories) as primary antibody.

Techniques: Telomerase Assay, Transfection, Immunoprecipitation, Activity Assay, Negative Control, Isolation, Generated, Transgenic Assay, TRAP Assay, Mutagenesis, Binding Assay, Western Blot, Plasmid Preparation, In Vitro, Purification, Stable Transfection, Expressing, shRNA

Journal: Science Advances

Article Title: PES1 is a critical component of telomerase assembly and regulates cellular senescence

doi: 10.1126/sciadv.aav1090

Figure Lengend Snippet: ( A ) Cells from were captured by microscopy (top) and subjected to SA-β-gal staining (bottom). The percentage of SA-β-gal–positive cells on the right was calculated from five randomly chosen fields. At least 150 cells were analyzed per experiment. Scale bars, 50 μm. Data shown are mean ± SD of three independent experiments. ** P < 0.01. ( B ) Representative frozen liver sections from liver-specific PES1 KO mice at 3 months were subjected to SA-β-gal staining followed by eosin staining. The PES1 +/+ and PES1 +/f mice were littermates of the PES1 f/f mice. Scale bars, 100 μm. * P < 0.05. ( C ) Growth curves of representative MCF7 monoclones stably expressing PES1 shRNA (shPES1) or control shRNA (shCon) and shRNA-resistant WT or mutated PES1 from 34 PDLs. ( D ) Cells from were labeled with BrdU and stained with anti-BrdU. Scale bars, 100 μm. Data shown are mean ± SD of three independent experiments. ** P < 0.01.

Article Snippet: Immunohistochemical staining was performed as described previously ( ) using rabbit anti-PES1 (A300-902A, Bethyl Laboratories) as primary antibody.

Techniques: Microscopy, Staining, Stable Transfection, Expressing, shRNA, Labeling

Journal: Science Advances

Article Title: PES1 is a critical component of telomerase assembly and regulates cellular senescence

doi: 10.1126/sciadv.aav1090

Figure Lengend Snippet: ( A ) Representative immunohistochemical staining of PES1, telomerase activity, and telomere length in 43 human breast cancer samples. Scale bars, 100 μm. Correlation of PES1 levels with relative telomerase activity and telomere length was generated by Mann-Whitney test. ( B ) Representative immunohistochemical staining of PES1 and SA-β-gal in frozen breast cancer tissue sections from (A). Scale bars, 100 μm. Correlation between PES1 expression and SA-β-gal staining was generated by Fisher’s exact test. ( C ) A proposed model underlying the role of PES1 in regulation of telomerase and senescence. Telomerase minimally comprises TERT and TR. When PES1 expression levels are elevated in some cases (e.g., cancer), it facilitates assembly of TERT and TR by forming a complex with TERT and TR, resulting in high telomerase activity, long telomere, and inhibition of senescence. PES1 depletion has opposite effects.

Article Snippet: Immunohistochemical staining was performed as described previously ( ) using rabbit anti-PES1 (A300-902A, Bethyl Laboratories) as primary antibody.

Techniques: Immunohistochemical staining, Staining, Activity Assay, Generated, MANN-WHITNEY, Expressing, Inhibition

Journal: bioRxiv

Article Title: The Pseudogene RPS27AP5 Reveals Novel Ubiquitin and Ribosomal Protein Variants Involved in Specialised Ribosomal Functions

doi: 10.1101/2024.02.05.578897

Figure Lengend Snippet: A. Polysome profiling assay with cells expressing either Myc-S27a or Myc-S27aP5 proteins (N=2). Cell lysates expressing RPS27a or RPS27aP5 and treated with CHX (cycloheximide) were layered over a continuous sucrose gradient, and proteins were separated by ultracentrifugation. Separated ribosomal units were visualised using a gradient fractionator. B. Immunoblot analysis of precipitated fractions corresponding to the 40S, 60S, 80S ribosomal and polysomal proteins collected from polysome profiling assay (N=3). Fractions from polysome profiling were collected, and proteins following precipitation were separated by SDS-PAGE. Ribosomal proteins were revealed with the Myc and RPS3 antibodies. C. Venn diagram representation of RNA-Seq results of total RNAs captured by TRAP assay using either Myc-RPS27a or Myc-RPS27aP5 as bait (N=3). Cells expressing RPS27a or RPS27aP5 and treated with CHX (cycloheximide) were subjected to Myc immunoprecipitation. Ribosome-bound RNAs were recovered and analysed using RNA-seq. Identified RNAs bound to either RPS27a or RPS27aP5 were compared using a Venn diagram. D. Comparison of RNA-Seq results of protein-coding RNAs identified from the TRAP assay using either Myc-RPS27a or Myc-RPS27aP5 as bait (N=3). Log2 Fold Change threshold=1 and Significance cut-off is at 1.3 (0.05% FDR). E. Immunoblot analysis of HeLa and hTERT-RPE-1 cell extracts. Lysates from un-transfected cells, or cells expressing either RPS27a or RPS27aP5, were separated by SDS-PAGE and detected using the Myc, H3K27me3 and p15/16 antibodies. F. qPCR quantification of H3K27me3 levels at two different sites within the promoter region of INK4A in wild type HeLa cells or cells expressing S27a or S27aP5 proteins. Results were normalised to a spike-in DNA control and are shown relative to the un-transfected control sample used. Error bars correspond to standard deviation (N=3).

Article Snippet: Primary antibody binding was then performed at 4°C for 2 h. For negative control rabbit IgG (DA1E) (#66362, Cell Signaling, USA), for positive control H3K4me3 (C42D8) (#9751, Cell Signaling, USA) and for the experimental reaction H3K27me3 (C36B11) (#9733, Cell Signaling, USA) antibodies were used.

Techniques: Expressing, Western Blot, SDS Page, RNA Sequencing, TRAP Assay, Immunoprecipitation, Comparison, Transfection, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Involvement of SRSF11 in cell cycle-specific recruitment of telomerase to telomeres at nuclear speckles

doi: 10.1093/nar/gkv844

Figure Lengend Snippet: Identification of SRSF11 as a TERC-interacting protein. ( A ) Lysates from HeLa S3 cells were incubated with biotinylated TERC bound to streptavidin agarose beads and assayed for protein binding by Coomassie staining of SDS-PAGE gel. Binding proteins were identified by nano LC-MS/MS. Molecular size markers are shown in kilodaltons. ( B and C ) HeLa S3 cells expressing Flag-TERT ( B ) or Flag-SRSF11 ( C ) were subjected to immunoprecipitation (IP) with anti-Flag antibody, followed by immunoblotting to detect various telomerase components and quantitative reverse transcriptase-PCR (RT-PCR) to detect TERC. The extracts were treated with 0.1 mg/ml RNase A during immunoprecipitation. ( D ) Lysates from HeLa S3 cells were immunoprecipitated with anti-TERT and anti-SRSF11 antibodies, followed by immunoblotting with anti-SRSF11 and anti-TERT antibodies and quantitative RT-PCR to detect TERC. IgG was used as a negative control. The extracts were treated with 0.1 mg/ml RNase A during immunoprecipitation. ( E ) The various GST fusion proteins were affinity-purified and incubated with the lysates from cells expressing Flag-TERT, followed by immunoblotting with anti-Flag antibody. In vitro transcribed TERC was added to the lysates before GST pulldown. The purified GST fusion proteins were visualized by Coomassie staining and indicated with arrowheads. Molecular mass markers are shown in kilodaltons. ( F ) Immunoprecipitates of endogenous SRSF11 and TCAB1 from HeLa S3 cells were analyzed for telomerase activity by the TRAP assay. IgG was used as a negative control. ITAS, internal telomerase assay standard.

Article Snippet: Cells were incubated with rabbit anti-SRSF11 (800 ng/ml, Abcam), rabbit anti-TERT (500 ng/ml, Rockland), rabbit anti-TCAB1 (200 ng/ml, Abcam), rabbit anti-dyskerin (800 ng/ml, Santa Cruz Biotechnology), rabbit anti-Son (400 ng/ml, Abnova), mouse anti-SC35 (1 μg/ml, Abcam), mouse anti-coilin (2 μg/ml, Abcam), rabbit anti-coilin (200 ng/ml Santa Cruz Biotechnology), mouse anti-nucleolin (200 ng/ml, Santa Cruz Biotechnology), goat anti-TRF1 (2 μg/ml, Santa Cruz Biotechnology), rabbit anti-TRF2 (500 ng/ml, Cell signaling), rabbit anti-RAP1 (500 ng/ml, Bethyl laboratory), rabbit anti-POT1 (2 μg/ml, Abcam), mouse anti-TPP1 (1 μg/ml, Abcam) and mouse anti-TIN2 (1 μg/ml, Abcam) for 16 h at 4°C.

Techniques: Incubation, Protein Binding, Staining, SDS Page, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Expressing, Immunoprecipitation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Negative Control, Affinity Purification, In Vitro, Purification, Activity Assay, TRAP Assay, Telomerase Assay

Journal: Nucleic Acids Research

Article Title: Involvement of SRSF11 in cell cycle-specific recruitment of telomerase to telomeres at nuclear speckles

doi: 10.1093/nar/gkv844

Figure Lengend Snippet: Identification of the domains in SRSF11 and TERT that are required for their interaction. ( A ) Schematic representation of the human SR protein family. RRM, RNA recognition motif; RS, arginine/serine-rich domain. ( B ) Lysates from HeLa S3 cells expressing Flag-TERT were immunoprecipitated with anti-Flag antibody, followed by immunoblotting to detect various SR proteins as indicated. ( C ) Schematic representation of the region of SRSF11 involved in TERC binding. ( D ) Lysates from HeLa S3 cells expressing Flag-SRSF11, Flag-RRM and Flag-RS were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-TERT antibody and quantitative RT-PCR to detect TERC. The asterisk marks the position of nonspecific immunoglobulin chains. ( E ) Schematic representation of the region of TERT involved in SRSF11 binding. ( F ) Lysates from HeLa S3 cells expressing the various Flag-TERT domains were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with anti-SRSF11 antibody and quantitative RT-PCR to detect TERC. The asterisks mark the positions of nonspecific immunoglobulin chains.

Article Snippet: Cells were incubated with rabbit anti-SRSF11 (800 ng/ml, Abcam), rabbit anti-TERT (500 ng/ml, Rockland), rabbit anti-TCAB1 (200 ng/ml, Abcam), rabbit anti-dyskerin (800 ng/ml, Santa Cruz Biotechnology), rabbit anti-Son (400 ng/ml, Abnova), mouse anti-SC35 (1 μg/ml, Abcam), mouse anti-coilin (2 μg/ml, Abcam), rabbit anti-coilin (200 ng/ml Santa Cruz Biotechnology), mouse anti-nucleolin (200 ng/ml, Santa Cruz Biotechnology), goat anti-TRF1 (2 μg/ml, Santa Cruz Biotechnology), rabbit anti-TRF2 (500 ng/ml, Cell signaling), rabbit anti-RAP1 (500 ng/ml, Bethyl laboratory), rabbit anti-POT1 (2 μg/ml, Abcam), mouse anti-TPP1 (1 μg/ml, Abcam) and mouse anti-TIN2 (1 μg/ml, Abcam) for 16 h at 4°C.

Techniques: Expressing, Immunoprecipitation, Western Blot, Binding Assay, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Involvement of SRSF11 in cell cycle-specific recruitment of telomerase to telomeres at nuclear speckles

doi: 10.1093/nar/gkv844

Figure Lengend Snippet: Telomerase localizes to nuclear speckles specifically during S phase. ( A ) HeLa S3 cells were synchronized in mid/late S phase and analyzed by indirect immunofluorescence for co-localization of SRSF11 with SC35 (nuclear speckle marker), coilin (Cajal body marker) or nucleolin (nucleolus marker). Co-localization channels were calculated using Meta Imaging Series R MetaMorph software and appear in white in all figures. DNA was stained with DAPI. Coloc, co-localization. ( B ) The average percentage of cells showing co-localization of SRSF11 with SC35, coilin or nucleolin and standard deviation were determined by analyzing >100 nuclei for each cell cycle phase. Statistical analyses were performed using a two-tailed Student's t test. AS, asynchronous cells. ( C ) HeLa S3 cells were synchronized in mid/late S phase and analyzed by indirect immunofluorescence for co-localization of SRSF11 or SC35 with telomeric sites marked by TTAGGG-specific FISH probe. ( D ) The average percentage of telomere foci co-localizing with SRSF11 or SC35 per cell and standard deviation were determined by analyzing >100 nuclei for each cell cycle phase. ( E ) HeLa S3 cells were synchronized in mid/late S phase and analyzed by indirect immunofluorescence for co-localization of TERT, dyskerin, TCAB1 or TERC with SC35. ( F ) The average percentage of cells showing co-localization of TERT, dyskerin, TCAB1 or TERC with SC35 and standard deviation were determined by analyzing >100 nuclei for each cell cycle phase.

Article Snippet: Cells were incubated with rabbit anti-SRSF11 (800 ng/ml, Abcam), rabbit anti-TERT (500 ng/ml, Rockland), rabbit anti-TCAB1 (200 ng/ml, Abcam), rabbit anti-dyskerin (800 ng/ml, Santa Cruz Biotechnology), rabbit anti-Son (400 ng/ml, Abnova), mouse anti-SC35 (1 μg/ml, Abcam), mouse anti-coilin (2 μg/ml, Abcam), rabbit anti-coilin (200 ng/ml Santa Cruz Biotechnology), mouse anti-nucleolin (200 ng/ml, Santa Cruz Biotechnology), goat anti-TRF1 (2 μg/ml, Santa Cruz Biotechnology), rabbit anti-TRF2 (500 ng/ml, Cell signaling), rabbit anti-RAP1 (500 ng/ml, Bethyl laboratory), rabbit anti-POT1 (2 μg/ml, Abcam), mouse anti-TPP1 (1 μg/ml, Abcam) and mouse anti-TIN2 (1 μg/ml, Abcam) for 16 h at 4°C.

Techniques: Immunofluorescence, Marker, Imaging, Software, Staining, Standard Deviation, Two Tailed Test

Journal: PLoS ONE

Article Title: Human Golgi phosphoprotein 3 is an effector of RAB1A and RAB1B

doi: 10.1371/journal.pone.0237514

Figure Lengend Snippet: (A-B) Pulldowns of the indicated variants of GST-RAB1A (A) or of GST-RAB1B (B) incubated with soluble protein extracts from human neuroglioma H4 cells. Samples of the pulldowns (upper two panels) and of the protein extracts used in the pulldowns (lower two panels; input ) were processed by SDS-PAGE followed by immunoblotting. Before the immunoblotting of the pulled down samples, nitrocellulose membranes were subjected to Ponceau S staining (upper panels). The position of GST (used as pulldown control) and of the GST-tagged RAB1A or RAB1B variants is indicated on the right. Immunoblottings were carried out using antibodies to detect the proteins indicated on the right of the bottom three panels. The position of molecular mass markers is indicated on the left. (C-D) Densitometry quantification of the amount of GOLPH3 pulled down as shown in the second panel of A and B. The immunoblot signal of anti-β-actin was used as loading control. Bar represents the mean ± standard deviation (n = 3 independent experiments). * P < 0.05; ** P < 0.01; ns , not statistically significant.

Article Snippet: H4 human neuroglioma cells and HeLa cells were obtained from the American Type Culture Collection, and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin (ThermoFisher), and 5 μg/ml plasmocin (InvivoGen), in a humidified incubator with 5% CO 2 at 37°C.

Techniques: Incubation, SDS Page, Western Blot, Staining, Control, Standard Deviation

Journal: PLoS ONE

Article Title: Human Golgi phosphoprotein 3 is an effector of RAB1A and RAB1B

doi: 10.1371/journal.pone.0237514

Figure Lengend Snippet: (A-B) Soluble protein extracts from human H4 neuroglioma cells transiently expressing the indicated variants of GFP-RAB1A (A) or of GFP-RAB1B (B) were subjected to the GFP-Trap assay. Samples of the GFP traps (upper two panels) and of the protein extracts used in the pulldowns (lower two panels; input ) were processed by SDS-PAGE followed by immunoblotting. Immunoblottings were carried out using antibodies to detect the proteins indicated on the right. Antibody to GFP was used to detect GFP (used as GFP-Trap control) and the GFP-tagged RAB1A and RAB1B variants. The position of molecular mass markers is indicated on the left. (C-D) Densitometry quantification of the amount of GOLPH3 pulled down as shown in the third panel of A and B. The immunoblot signal of anti-β-actin was used as loading control. Bar represents the mean ± standard deviation (n = 3 independent experiments). * P < 0.05; ** P < 0.01; ns , not statistically significant.

Article Snippet: H4 human neuroglioma cells and HeLa cells were obtained from the American Type Culture Collection, and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin (ThermoFisher), and 5 μg/ml plasmocin (InvivoGen), in a humidified incubator with 5% CO 2 at 37°C.

Techniques: Expressing, TRAP Assay, SDS Page, Western Blot, Control, Standard Deviation

Journal: Nature Communications

Article Title: CDK1 dependent phosphorylation of hTERT contributes to cancer progression

doi: 10.1038/s41467-020-15289-7

Figure Lengend Snippet: a Detection of endogenous hTERT. The cells were treated with DMSO (denoted as “non M”) or nocodazole to manipulate cells in mitosis (denoted as “M”), and immunoprecipitated with anti-hTERT mouse mAb (10E9-2) or anti-hTERT sheep pAbs (abx120550). The hTERT proteins were detected by anti-hTERT mouse mAb (2E4-2) (two left panels) or anti-hTERT rabbit mAb (ab32020) (two right panels). b hTERT proteins were isolated by immunoprecipitation with 10E9-2 and detected by 2E4-2 (upper panel). Cells arrested in mitosis with nocodazole were confirmed by anti-phospho-histone H3 (Ser10) antibodies (lower panel). c Endogenous hTERT immunoprecipitated from 293T cells were treated with λ phosphatase to remove the phosphate groups and detected by 2E4-2 (upper panel). d Summary of MS analysis to identify phosphorylation sites in hTERT. The numbers of phosphopeptides and nonphosphopeptides are based on pep_expect (Expectation value for PSM) < 0.05 used as a cutoff value. Bold letters indicate amino-acid residues identified as phosphorylated residues. See the details in Supplementary Data . e Multiple sequence alignment of telomerases using COBALT . Red letters denote conserved residues and blue letters denote non-conserved residues with no gaps. Yellow is the phosphorylated threonine residue detected in this study. f The immune complexes were treated with λ phosphatase. Phosphorylation of hTERT at T249 was detected by anti-249T-P rabbit pAbs. g Endogenous hTERT proteins from cells transfected with two different siRNAs specific for hTERT . Phosphorylation of hTERT at T249 was detected by anti-249T-P pAbs (upper panel). Whole-hTERT proteins were detected by 2E4-2 (lower panel). h The recombinant hTERT fragment proteins (191–306 a.a) were phosphorylated by CDK1-cyclinB or IKK2_2-664 in vitro and phosphorylated proteins were detected in Phos-Tag SDS-PAGE (upper panel). Phosphorylation of hTERT at threonine 249 by CDK1-cyclinB was confirmed by anti-249T-P pAbs (lower panel). The protein bands enclosed with square were analyzed by MS to confirm the phosphorylation sites. i Phosphorylation of threonine 249 by CDK1-cyclinB was confirmed by anti-249T-P pAbs and TpMab-1 mouse mAb. Experiments were repeated three times (for a , g , i ), five times (for b , c , f ) and twice (for h ) with similar results. Source data are provided in the Source Data file.

Article Snippet: Anti-hTERT mAb (clone 10E9-2: MBL Co., Ltd., M216-3) and anti-hTERT sheep pAbs (Abbexa Ltd., abx120550) were used for immunoprecipitation (IP).

Techniques: Immunoprecipitation, Isolation, Phospho-proteomics, Sequencing, Residue, Transfection, Recombinant, In Vitro, SDS Page

Journal: Nature Communications

Article Title: CDK1 dependent phosphorylation of hTERT contributes to cancer progression

doi: 10.1038/s41467-020-15289-7

Figure Lengend Snippet: a – e Representative images of precancerous lesions stained with anti-249T-P pAbs. N-small, intercalated duct to intralobular duct; N-large, interlobular duct to main pancreatic duct; PanIN, pancreatic intraepithelial neoplasia; Cancer, pancreatic invasive ductal adenocarcinoma. Original magnification x400, scale bar: 50 µm. f Phosphorylation of hTERT T249 was detected in the nucleus of pancreatic adenocarcinomas. Original magnification x400, scale bar: 50 µm. g , h Both normal mitotic (pointed by arrows, g ) and atypical multipolar mitotic images (pointed by arrow, h ) exhibited phosphorylation of hTERT T249 (inserts). Original magnification x400. i Expression of CDK1 was detected in the nucleus of pancreatic adenocarcinomas. Original magnification x400, scale bar: 50 µm. j Relationship between pancreatic ductal lesions and hTERT phosphorylation at T249. Box-and-whisker plot of hTERT phosphorylation for each of the pancreatic ductal lesions surgically resected from patients with pancreatic cancer ( n = 47). The box portion of the box plot is defined by two lines at the 25th percentile and 75th percentile. A line is drawn inside the box at the median. The two whisker boundaries are the 5th percentile and 95th percentile. p < 0.05 by Speaman’s rank correlation test. k – p IHC staining with anti-249T-P pAbs in non-cancerous lesions ( k , l ) and HCC lesions ( m , n ) obtained from two individual patients. IHC staining with anti-CDK1 pAbs in HCC lesions ( o , p ) obtained from two identical patients. Original magnification x200, scale bar: 100 µm. q , r Kaplan–Meier survival analysis of primary patients with HCC grouped for 249T-P-positive (red) or negative (blue). Phosphorylation status of hTERT T249 ( q ) and CDK1 expression ( r ) showed statistically significant correlations with overall survival (OS, p = 0.048 and 0.036, respectively, log rank test, two-sided) in patients.

Article Snippet: Anti-hTERT mAb (clone 10E9-2: MBL Co., Ltd., M216-3) and anti-hTERT sheep pAbs (Abbexa Ltd., abx120550) were used for immunoprecipitation (IP).

Techniques: Staining, Phospho-proteomics, Expressing, Whisker Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: CDK1 dependent phosphorylation of hTERT contributes to cancer progression

doi: 10.1038/s41467-020-15289-7

Figure Lengend Snippet: a Direct telomerase assay (left panel) and IP-RdRP assay (right panel) using HeLa cells. Immune complexes were treated with λ phosphatase to remove the phosphate groups from hTERT proteins. The relative activities of telomerase and RdRP are noted below the panel, respectively. b In vitro CDK1 kinase assay of hTERT immune complexes treated with λ phosphatase, followed by IP-IB assay (left panel) and IP-RdRP assay (right panel). The relative RdRP activities are noted below the panel. c Detection of endogenous hTERT proteins using HeLa cells transfected with four different siRNAs specific for CDK1 followed by nocodazole treatment. Phosphorylation of hTERT proteins were detected by anti-249T-P pAbs (upper panel). Suppression of CDK1 proteins was confirmed by anti-CDK1 mAb (lower panel). d Direct telomerase assay (left panel) and IP-RdRP assay (right panel) using HeLa cells transfected with four different siRNAs specific for CDK1 . e Detection of phosphorylated hTERT proteins using HeLa cells treated with 5 µM of CDK1 inhibitor, RO-3306. The cells were then treated with nocodazole. Phosphorylation of hTERT proteins was detected by anti-249T-P pAbs (upper panel) and hTERT proteins isolated by immunoprecipitation were detected by anti-hTERT mAb (clone 2E4-2) (lower panel). f Direct telomerase assay (left panel) and IP-RdRP assay (right panel) using HeLa cells treated with 5 µM of CDK1 inhibitor, RO-3306. The relative RdRP activities are noted below the panel. Experiments were repeated three times (for a (right panel), c , e , f (right panel)) and twice (for a (left panel), b , d , f (left panel)) with similar results. Source data are provided in the Source Data file.

Article Snippet: Anti-hTERT mAb (clone 10E9-2: MBL Co., Ltd., M216-3) and anti-hTERT sheep pAbs (Abbexa Ltd., abx120550) were used for immunoprecipitation (IP).

Techniques: Telomerase Assay, In Vitro, Kinase Assay, Transfection, Phospho-proteomics, Isolation, Immunoprecipitation

Journal: Nature Communications

Article Title: CDK1 dependent phosphorylation of hTERT contributes to cancer progression

doi: 10.1038/s41467-020-15289-7

Figure Lengend Snippet: a TRAP assay using BJ cells stably introduced with control retrovirus, hTERT mutants or wild-type hTERT (left panel). hTERT mutants were created by substitution of the threonine 249 with alanine (T249A) or glutamic acid (T249E). Telomere length in the BJ cells was determined by Southern blot analysis for telomere restriction fragments (right panel). White bars indicate the position of mean radioactivity and the TRF average length were noted below the panel. Telomere structure in BJ cells was analyzed at 8 PD after infection. b Direct telomerase assay using hTERT proteins immunoprecipitated from 293T cells transfected with control vector, wild-type hTERT, T249A, or T249E mutants. The relative telomerase activities are noted below the panel. c IP-RdRP assay using the cells prepared with identical manipulations as used in the b . The relative RdRP activities are noted below the panel. d Immunoblotting to confirm ectopically overexpressed hTERT mutant proteins in PEO1, PEO14 and MT cells infected with hTERT-T249A, hTERT-T249E, or control retroviruses. hTERT proteins were detected by anti-hTERT mAb (clone 2E4-2) (upper panel). β-actin was used as an internal control (lower panel). e Cell proliferation assay using PEO1, PEO14 and MT cells infected with hTERT-T249A (red line), hTERT-T249E (blue line) or control retrovirus (black line). This assay was done in triplicate and data are shown as the mean ± SD. Asterisk: statistically significant values ( p < 0.05 by Student’s t -test, two-sided). f MT cells introduced a control retroviral vector (MT-Control) or hTERT-T249A (MT+T249A) were implanted into eight NOD/SCID mice for each and tumors generated from the cells are presented (left panel). The volume of tumors from mice harboring MT-Control (black line) or MT+T249A (red line) is shown as the mean ± SEM ( n = 8, p = 0.0028 by unpaired t -test, two-sided) (right panel). Experiments were repeated three times (for c , d ) and twice (for a , b ) with similar results. Source data are provided in the Source Data file.

Article Snippet: Anti-hTERT mAb (clone 10E9-2: MBL Co., Ltd., M216-3) and anti-hTERT sheep pAbs (Abbexa Ltd., abx120550) were used for immunoprecipitation (IP).

Techniques: TRAP Assay, Stable Transfection, Control, Southern Blot, Radioactivity, Infection, Telomerase Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Proliferation Assay, Retroviral, Generated

Journal: Nature Communications

Article Title: CDK1 dependent phosphorylation of hTERT contributes to cancer progression

doi: 10.1038/s41467-020-15289-7

Figure Lengend Snippet: a Genome editing strategy to introduce alanine or glutamic acid substitution in the endogenous hTERT locus. Green letters denote the PAM (protospacer adjacent motif) sequence for genome editing by CRISPR-Cas9. b PCR fragments surrounding target regions (1,184 bp for T249A and 456 bp for T249E) were amplified from genomic DNA of genome-edited 293T clones. PCR fragments from pBABE-puro-hTERT (plasmid control), 293T control cells (Control-CRISPR) and mutant cells (T249A-CRISPR, T249E-CRISPR) were digested with the indicated restriction enzymes and electrophoresed in 5% polyacrylamide gels. The bands enclosed with square and indicated by arrows show that the cleavage patterns of PCR fragment were changed in CRISPR-mutant clones. c Sanger sequencing traces of a control and two mutant clones generated from gel-purified PCR fragments of the genomic DNAs of the respective clones. The sequences boxed in red and blue are the base triplet coding for alanine and glutamic acid in mutant clones, respectively. d IP-RdRP assay using hTERT proteins immunoprecipitated from 293T CRISPR clones (upper panel). The relative RdRP activities are noted below the panel. hTERT proteins isolated by immunoprecipitation from the cells were detected by anti-hTERT mAb (clone 2E4-2) (lower panel). e Tumors generated from 1 × 10 5 cells of 293T Control-CRISPR or 293T-T249A-CRISPR in eight NOD/SCID mice for each are presented (upper panel). The volume of tumors from mice with Control-CRISPR (black line) or T249A-CRISPR (red line) is shown as the mean ± SEM ( n = 8, p = 0.0469 by unpaired t -test, two-sided) (lower panel). f Direct telomerase assay using hTERT proteins immunoprecipitated from Control-CRISPR or T249A-CRISPR. The relative telomerase activities are noted below the panel (upper panel). Immune complexes were isolated from Control-CRISPR and T249A-CRISPR with anti-hTERT mAb (clone 10E9-2) and associated hTERC RNA were subjected to RT-PCR (lower panel). Experiments were repeated five times (for d , f ) with similar results. Source data are provided in the Source Data file.

Article Snippet: Anti-hTERT mAb (clone 10E9-2: MBL Co., Ltd., M216-3) and anti-hTERT sheep pAbs (Abbexa Ltd., abx120550) were used for immunoprecipitation (IP).

Techniques: Introduce, Sequencing, CRISPR, Amplification, Clone Assay, Plasmid Preparation, Control, Mutagenesis, Generated, Purification, Immunoprecipitation, Isolation, Telomerase Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Development (Cambridge, England)

Article Title: Distinct and mutually inhibitory binding by two divergent ?-catenins coordinates TCF levels and activity in C. elegans

doi: 10.1242/dev.069054

Figure Lengend Snippet: Binding to WRM-1 requires the last 39 amino acids of POP-1. (A) POP-1 C terminus is required for WRM-1 binding. Co-expression of Flag-tagged POP-1 variants with either Myc-tagged WRM-1 or Myc-tagged SYS-1 in HeLa cells. Immunoprecipitation was performed using anti-Myc antibody and western blots were probed with 94I. Top panels, input; bottom panels, IP. Arrows indicate POP-1; asterisks indicate POP-1Δ39. (B) POP-1 binds to WRM-1 in embryos. Immunoprecipitation was performed using GFP-Trap and wild-type embryo extracts (lane 1), embryo extracts expressing GFP::WRM-1 (lane 2) or using a control antibody (RFP-Trap, which does not crossreact with GFP) and embryo extracts expressing GFP::WRM-1 (lane 3). Western blots were probed with anti-POP-1 (94I) or anti-GFP antibodies. POP-1 was detected only in lane 2 of immunoprecipitated products. (C) Phosphorylation of POP-1 by LIT-1 reduces the POP-1/WRM-1 interaction. FLAG-tagged WRM-1 co-transfected with HA-tagged POP-1 (wild-type or POP-1 ExpA) and Myc-tagged LIT-1 (wild-type or two kinase-dead variants). Input lysates and anti-FLAG pulldown products were probed with anti-HA or anti-FLAG antibodies as indicated. The SDS gel was run for a short period of time such that a migration difference due to phosphorylation was not observed.

Article Snippet: Embryo extract preparation, immunoprecipitation using the camelid anti-GFP antibody (GFP-Trap, Chromotek) and western blot analyses were performed as described previously ( Lo et al., 2004 ).

Techniques: Binding Assay, Expressing, Immunoprecipitation, Western Blot, Control, Phospho-proteomics, Transfection, SDS-Gel, Migration

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A EHMT1 interacting proteins identified by mass spectrometric analysis with details indicating coverage and peptide score. B Sequential IP in HEK293 cells demonstrating EHMT1, EHMT2, and LMNB1 are a part of the same complex. C LMNB1 interacts with EHMT1 via SET domain. Recombinant GST or GST-LMNB1 was incubated with Ni-NTA bound His-EHMT1 SET protein. Post washing eluents were loaded for immunoblotting using GST or His antibody. Recombinant pure proteins GST-LMNB1 (lane 1), GST (lane 2), EHMT1-SET (lane 3) were used as controls. D Venn diagram showing unique and overlapping reads obtained from EHMT1 and LMNB1 ChIP-Sequencing. E Composite profile of EHMT1 and LMNB1 read density around the transcription start site (TSS). F Genomic distribution of EHMT1 and LMNB1 peaks. The majority of binding sites obtained were enriched in an intronic region or distal regions from a gene. G, H Representative figure showing normalized ChIP-seq read density (above 1.5-fold over expected) of EHMT1 and LMNB1 in 1MB bin for chromosome 1 & 9.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Recombinant, Incubation, Western Blot, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A EHMT1 interacts with EHMT2, LMNB1, and HP1. Whole cell lysates from fetal HDFs were subjected to IP reaction using EHMT1 or LMNB1 antibody. Subsequently, western blot was performed using the IPed material to detect EHMT1, EHMT2, LMNB1, and HP1. ASH2L did not show any interaction with EHMT1 and LMNB1 thus acted as a negative control. B EHMT1 interacts with LMNB1 and EHMT2 via SET domain. HEK293 cells were transfected with pEGFP-Ankyrin (pEGFPC1-ANK) or pEGFPC1-SET domains of EHMT1 to determine domain specific association with LMNB1. Cell extracts were subjected to IP using GFP-antibody and the bound complexes were then analyzed by immunoblotting using LMNB1, EHMT2, and GFP antibodies. HEK-293 whole cell extract represents 1% input. pEGFPC1 empty vector transfected HEK293 or untransfected HEK293 cells were used as control reactions. Arrows indicate specific band. C Coomassie stained SDS-PAGE gel showing 6X His EHMT1-SET purified protein used for methyltransferase assays. D, E, F Gene Ontology (GO) analysis of EHMT1 and LMNB1 bound genes. Representative figure showing enriched GO terms for EHMT1 ( D ), LMNB1 ( E ) and EHMT1 and LMNB1 ( F ) co-bound genes. The length of the bar (y-axis) denotes total genes falling within GO term. G Circos plot showing genome wide peak density of EHMT1 (green) and LMNB1 (red).

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Negative Control, Transfection, Plasmid Preparation, Staining, SDS Page, Purification, Genome Wide

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Increasing concentrations of LMNB1-GST showed a greater degree of methylation by EHMT1-SET. Methyltransferase assay was performed using a fixed concentration of recombinant 6X His EHMT1-SET as an enzyme source, and SAM as a methyl group donor. Recombinant GST-LMNB1 (4.5 ng and 9 ng) and Histone H3 peptide (10 ng) were used as substrates in the assay. Histone H3 peptide was used as a positive control. The mean relative fluorescence unit (RFU) values represented in the graph were obtained after subtracting the values for controls EHMT1-SET only, SAM only, LMNB1-GST (4.5 ng) with those of the LMNB1-GST (4.5 ng and 9 ng) and Histone H3 (10 ng). B Coomassie stained SDS-PAGE gel showing 6X His LMNB1-CT purified protein used for methyltransferase assays. C mWasabi expression in fetal HDFs transduced with Wt.LMNB1 and K417A-LMNB1 mutant construct. (Scale bar: 20μm). Arrows indicate the cells zoomed in the far-right image presented. D MFI for mWasabi expression in cells transfected with Wt.LMNB1 and K417A-LMNB1 mutant constructs. Each biological replicate is represented as spread of technical replicates around the mean value. E MFI for mWasabi expression in cells transfected with Wt.LMNB1 and K417A-LMNB1 mutant constructs. MFI has been represented as center vs periphery of the nuclei. Individual biological replicates are plotted with the mean values. F Quantitation for percentage distorted nuclei in cells transfected with K417A-LMNB1 mutant construct compared to Wt.LMNB1 construct. Individual biological replicates are plotted with the median. G Immunostaining for LMNA/C in fetal HDFs transduced with Wt.LMNB1 and K417A-LMNB1 mutant construct. (Scale bar: 20μm). Arrows indicate the cells zoomed in the far-right image presented.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Methylation, Concentration Assay, Recombinant, Positive Control, Fluorescence, Staining, SDS Page, Purification, Expressing, Transduction, Mutagenesis, Construct, Transfection, Quantitation Assay, Immunostaining

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A, B Western blot probed with the anti-Methyl-K antibody. Lanes to the left of the ladder are control reactions containing 2 μg LMNB1-CT + 50 μM SAM (lane 1) or 2 μg of EHMT1-SET/EHMT2-SET + 2 μg LMNB1-CT (lane 2) and 2 μg LMNB1-CT only (lane 3). Lanes to the right of the ladder are methyltransferase reactions containing 1, 2 and 4 μg of LMNB1-CT and EHMT1-SET/EHMT2-SET along with 50 μM SAM. Lower panel: Coomassie stained gel representing all the reactions mentioned above. C, D Nuclear lysates from HEK-293 were immunoprecipitated with anti-Methyl-K or anti-LMNB1 antibody followed by western blotting with anti-Pan H3, anti-LMNB1 and anti-Methyl-K antibodies. Asterisk indicates the band of interest. E Reduced methylation of LMNB1 by EHMT1 and EHMT2 upon single amino acid substitution from lysine (RKR) to alanine (RAR). Mutant LMNB1 peptides were synthesized and subjected to methyltransferase reaction containing EHMT1-SET and EHMT2-SET with SAM as a methyl donor. H3 peptide was used as a positive control for the reaction. F Immunostaining for LMNB1 in fetal HDFs transfected with Wt.LMNB1 and K417A-LMNB1 constructs. (Scale bar: 20μm). Arrows indicate the cells zoomed in the far-right image presented.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Staining, Immunoprecipitation, Methylation, Mutagenesis, Synthesized, Positive Control, Immunostaining, Transfection, Construct

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Western blot analysis for EHMT1 and EHMT2 in fetal HDFs upon knock down of EHMT1 and EHMT2. GAPDH was used as a loading control. B, C Relative protein levels of EHMT1 (B) and EHMT2 (C) upon knockdown. Each biological replicate is represented as spread of technical replicates around the mean value. D Percentage of distorted nuclei increases in human fibroblasts transduced with shEHMT1 and shEHMT2 virus compared to UT or shCnt. (n=3), For UT, (n=43); shCnt, (n=23); shEHMT1, (n=18); shEHMT2, (n=20) nuclei counted. shCnt vs shEHMT1, **p=0.0048; shCnt vs shEHMT2, #p=0.0153. (Kruskal-Wallis test, post-hoc test: Dunn’s multiple comparison test)

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Transduction

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Western blot analysis for LMNB1 and LMNA/C in fetal HDFs transduced with shEHMT1 and shEHMT2 virus. Untransduced (UT) and control shRNA (shCnt) were used as controls. B, C Fetal HDFs were transduced with shCnt, shEHMT1 and shEHMT2 virus. Post transduction cells were immunostained for EHMT1 or EHMT2 and co-stained for NL using LMNB1 antibody. Distortion of nuclear architecture was seen upon loss of EHMT1 and EHMT2 proteins. (Scale bar: 20μm). Arrows indicate the cells zoomed in the far-right image presented. D Relative expression of LMNA and LMNB1 in fetal HDFs upon EHMT1 and EHMT2 knockdown compared to UT/shCnt cells. (n=3) For LMNB1: UT vs shEHMT1, ***p<0.0001 and UT vs shEHMT2, ***p<0.0001. (One Way ANOVA, post-hoc test: Bonferroni’s multiple comparison test).

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Transduction, shRNA, Staining, Expressing

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Western blot analysis for H3K9me2 and H3K9me3 in UT, shCnt, shEHMT1 and shEHMT2 transduced HDFs. GAPDH was used as loading control. B Quantification of H3K9me2 upon EHMT1 and EHMT2 knockdown. Each biological replicate is represented as spread of technical replicates around the mean value. C Immunostaining for H3K9me2 co-stained for nuclear lamina using LMNB1 antibody in fetal HDFs transduced with shCnt, shEHMT1 and shEHMT2 virus. (Scale bar: 20μm). Insets are zoomed images of cells in the merge. D H3K9me2 immunostaining in fetal HDFs transduced with shEHMT1.1 (lentivirus) and shEHMT2.1 (retrovirus). (Scale bar: 20μm). E MFI profile (Centre to periphery) with standard deviation for all the measured nuclei of shCnt, shEHMT1 and shEHMT2. F Immunostaining for H3K9me2 in fetal HDFs treated with or without BIX 01294 (1μM) for 48 h. (Scale bar: 20μm). G MFI plot for H3K9me2 staining in fetal HDFs treated with or without BIX 01294 (1μM) for 48 h. Each biological replicate is represented as spread of technical replicates around the mean value. H Western blot for LMNB1 and H3K9me2 on cells treated with BIX 01294 or vehicle control. BIX 01294 treatment resulted in a reduction of H3K9me2 levels without altering LMNB1 expression. I In vitro methylation assay was performed using recombinant LMNB1-CT and EHMT1-SET or EHMT2-SET domain in the presence or absence of BIX 01294. H3 peptide was used as a positive control. J TEM image for fetal HDFs treated with BIX 01294. BIX 01294 treatment did not affect the peripheral heterochromatin distribution. (Scale bar: 1μm) K MFI for H3K9me2 staining in cells transfected with Wt.LMNB1 and K417A-LMNB1 constructs. Each biological replicate is represented as spread of technical replicates around the mean value. L Western blot showing expression of endogenous LMNB1 (top panel lower band) and Wasabi tagged LMNB1 (top panel upper band). The lysates from cells transfected with Wt.LMNB1 and K417A-LMNBI were probed with the LMNB1 antibody. The lysate was also probed with H3K9me2 antibody and H3 (loading control). M MFI for H3K9me2 staining in cells transfected with Wt.LMNB1 and K417A-LMNB1 constructs. MFI has been represented as center vs periphery of the nuclei. Each biological replicate is represented as spread of technical replicates around the mean value.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Immunostaining, Staining, Transduction, Standard Deviation, Expressing, In Vitro, Methylation, Recombinant, Positive Control, Transfection, Construct

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A MFI for H3K9me2 staining in fetal HDFs transduced with shEHMT1 and shEHMT2. MFI has been represented as center vs. periphery of the nuclei and compared with respect to shCnt cells. Each biological replicate is represented as spread of technical replicates around the mean value. B, C, D Fetal HDFs were transduced with shCnt, shEHMT1 and shEHMT2 virus. TEM was performed to visualize the heterochromatin in EHMT1 and EHMT2 knockdown cells. Peripheral heterochromatin was intact at the NP beneath the NL in control cells while this distribution was drastically altered upon knockdown of EHMT1 and EHMT2. (Scale bar: 1μm). Arrows indicate the heterochromatin regions. E Immunostaining for H3K9me2 in fetal HDFs expressing Wt.LMNB1 or K417A-LMNB1 mutant plasmids. (Scale bar: 20μm). Arrows indicate the cells zoomed in the far-right image presented. F, G Fetal HDFs transfected with Wt.LMNB1 and K417A-LMNB1 plasmids were processed for electron microscopy. Cells transfected with Wt.LMNB1 plasmid showed intact peripheral heterochromatin while K417A-LMNB1 transfected cells showed loss of heterochromatin with nuclear envelope breaks. (Scale bar: 1μm). Arrows indicate the zoomed area presented in the inset below. H qRT-PCR to validate expression of EZH2 in UT, shEHMT1 and shEHMT2 transduced HDFs. (n=3), UT vs. shEHMT1 (**p=0.0067), UT vs. shEHMT2 (**p=0.0021). (One sample t-test, two-tailed). I Immunostaining for H3K27me3 mark and HP1 protein in fetal HDFs transduced with shCnt, shEHMT1, and shEHMT2. Knockdown of EHMT1 and EHMT2 leads to a reduction in the H3K27me3 mark, which corroborates with decreased Ezh2 expression while HP1 expression was reduced only in shEHMT1 cells. (Scale bar: 20μm)

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Staining, Transduction, Immunostaining, Expressing, Mutagenesis, Transfection, Electron Microscopy, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A, B Heat map for differential expression of (A) all genes and (B) chromatin modifiers obtained from RNA-seq analysis of shEHMT1 or shEHMT2 compared to shCnt transduced HDFs. Representative genes that were altered similarly or distinctly in EHMT1 vs EHMT2 are indicated from few clusters.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Heat map demonstrating differential expression of age related genes in EHMT1 and EHMT2 depleted fibroblasts. Representative genes that were altered similarly or distinctly in EHMT1 vs EHMT2 are indicated from few clusters. B. Validation for differential expression of candidate genes obtained from RNA-Seq analysis by semi-quantitative PCR. C. Validation for differential expression of candidate genes obtained from RNA-Seq analysis by qRT-PCR (n=3), UT vs FGFR1, p=0.05; UT vs CDKN2B, p= 0.0058; UT vs FOXM1, p=0.0018 One sample t-test, two tailed. D. Immunostaining for H3K9me3, H3K27me3 and HP1 in fetal, 18Y old and 65Y old HDFs followed by confocal imaging. (Scale bar: 20μm) E. Western blot analysis for H3K9me3 and H3K27me3 in fetal, 18Y and 65Y old HDFs.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Two Tailed Test, Immunostaining, Imaging, Western Blot

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A Western blot analysis for EHMT1, EHMT2 and GAPDH in HDF cell lysates derived from various age groups. B , Quantification of EHMT1 protein in HDFs of indicated age groups. One Sample t test (n=3). For fetal vs 65 (*p<0.05), Fetal vs 18Y and Fetal vs 31Y are not significant. C , Quantification of EHMT2 protein expression in HDFs of indicated age groups. One Sample t test (n=6). For Fetal vs 18Y (***p=0.0004), and Fetal vs 65Y (***p=0.0002). D Immunostaining for EHMT1, EHMT2 and Pan H3 in fibroblasts from indicated age groups (Scale bar: 20μm). E, F Western blot and immunostaining (Scale bar: 20μm) analysis for H3K9me2 in indicated age groups. G Representative TEM images for nuclei of 18Y, 31Y, 40Y and 65Y HDFs. Arrows indicate peripheral heterochromatin. Inset is the zoomed version of the same images. (Scale bar: 1μm). H Western blot analysis for LMNA/C and LMNB1 and in various age groups HDFs. GAPDH was used as an internal control. I Quantification of LMNB1 protein expression in HDFs of indicated age groups. One sample t test (n=6). For Fetal vs 18Y (**p=0.0069) and Fetal vs 65Y (**p=0.0016). J,K,L Reduced levels of methylated LMNB1 during physiological aging. Nuclear extracts from indicated age groups were subjected to IP using LMNB1 antibody. IPed material was divided into halves, Blot 1 (left,J) was probed with anti LMNB1 antibody, blot 2 was probed with anti-Methyl-K (middle, K) antibody. Antimethly-K blot was reprobed with LMNB1 antibody to demonstrate the methyl band indicated is indeed LMNB1 (Right, L). 80 μg of fetal HDFs derived nuclear lysate was used as input control.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Western Blot, Derivative Assay, Expressing, Immunostaining, Methylation

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A-C. MFI (Centre to periphery) with standard deviation for H3K9me2 staining in all the measured nuclei of fetal, 18Y and 65Y old HDFs. D. MFI for H3K9me2 staining in fetal, 18Y and 65Y old HDFs. MFI has been represented as center vs periphery. Individual biological replicates are plotted with mean values. E Relative expression of EHMT1 and EHMT2 at the mRNA level in fetal, 18Y and 65Y old HDFs. (n=3), For EHMT1: Fetal vs 65Y, *p=0.0219; For EHMT2: Fetal vs 65Y, #p=0.0219. (Kruskal-Wallis test, post-hoc test: Dunn’s multiple comparison test). F Western blot for EHMT1 and EHMT2 in 18Y and 65Y old HDFs treated with or without proteasomal degradation inhibitor MG132 (10μM for 6 h). G Quantification of EHMT1 and EHMT2 protein levels in 18Y old HDFs treated with or without MG132 treatment. (n=3), for 18Y EHMT1: UT vs MG132, p=0.5989, ns; For 18Y EHMT2: UT vs. MG132 (*p=0.0154). (One sample t-test, two-tailed). H-I. Cell lysates prepared from human fibroblasts of indicated age groups were subjected for IP using EHMT1/EHMT2 antibody. IPed material was analyzed by immunoblotting using LMNB1 and LMNA/C antibodies. 30 μg of fetal cell lysate was used as input control. Dotted lines indicate that different exposures were used for Input and IP of the same western blot.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Standard Deviation, Staining, Expressing, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A, B Immunostaining using V5 or Flag antibodies along with LMNB1 in 65Y HDFs transfected with V5-EHMT1 and Flag-EHMT2 overexpression constructs. Immunostaining using H3K9me2 antibody along with LMNB1 in 65Y HDFs transfected with V5-EHMT1 and Flag-EHMT2 overexpression constructs. (Scale bar: 20μm) Arrows indicate the cells zoomed in the far-right image presented. C, D, E 65Y Old HDFs overexpressing EHMT1 and EHMT2 were processed for electron microscopy. Overexpression of EHMT1 and EHMT2 causes restoration of peripheral heterochromatin in old cells compared with control cells (Scale bar: 1μm). Arrows indicate the area zoomed and presented in the inset format.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Immunostaining, Transfection, Over Expression, Construct, Electron Microscopy

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A-B. Quantitation of LMNB1 expression in aged HDFs upon overexpression of EHMT1 and EHMT2. Overexpression of EHMT1 significantly increased the LMNB1 expression while EHMT2 transfected cells did not show any change compared to untransfected cells. Biological replicates of V5-EHMT1 and Flag-EHMT2 expressing cells were plotted with the mean values. C, D,E. Old HDFs expressing Wt. LMNB1 + V5-EHMT1, K417A-LMNB1 + V5-EHMT1 and K417A-LMNB1 + Flag-EHMT2 were stained with H3K9me2 antibody. Mutation at lysine 417 position of LMNB1 affects the overall distribution H3K9me2 and morphology (Scale bar: 20μm). Arrows indicate the cells zoomed in the far right image presented.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Quantitation Assay, Expressing, Over Expression, Transfection, Staining, Mutagenesis

Journal: bioRxiv

Article Title: KMT1/Suv39 methyltransferase family regulates peripheral heterochromatin tethering via histone and non-histone protein methylations

doi: 10.1101/240952

Figure Lengend Snippet: A, B Knockdown of EHMT1 and EHMT2 reduces cell proliferation. An equal number of HDFs were seeded and transduced with shCnt, shEHMT1 and shEHMT2 virus. Forty-eight hours post transduction, the number of cells in the culture was counted over a period of four days as indicated. (n=3), for shEHMT1, Day3: shCnt vs. shEHMT1 (**p=0.0015); Day4: shCnt vs. shEHMT1 (***p<0.0001); for shEHMT2, Day1: shCnt vs. shEHMT2 (***p=0.0002), Day2: shCnt vs. shEHMT2 (***p<0.0001), Day3: shCnt vs. shEHMT2 (***p<0.0001), Day4: shCnt vs. shEHMT2 (***p<0.0001). (Two-way ANOVA, post-hoc: Tukey’s multiple comparison test) C Quantification of cell cycle analysis. Increase in sub-G1 population an indication of cell apoptosis upon EHMT2 knockdown. For shCnt, (n=3), shEHMT1, (n=2) and shEHMT2, (n=2) SubG1: shCnt vs. shEHMT2 (**p=0.0011) (Two-way ANOVA, post-hoc: Tukey’s multiple comparison test). D, E Equal number of HDFs were seeded and transduced with shCnt, shEHMT1 and shEHMT2 virus. β-Galactosidase (senescence) assay was performed to monitor the cellular senescence in cultures. Fold change in an average number of senescent cells per field was quantitated. F, G TRAP assay to detect telomerase activity in indicated age groups as well as in shCnt, shEHMT1 and shEHMT2 transduced cells. Fetal, 18Y and shCnt fibroblast lysates were heat inactivated (HI) as a negative control for the assay. Telomerase activity was reduced upon knockdown of EHMT1 and EHMT2 as well as during physiological aging from fetal to 65Y old cells.

Article Snippet: The following antibodies were used in the current study: EHMT1 (A301-642A, Bethyl Laboratories, Rabbit polyclonal), EHMT1 (NBP1-77400, Novus Biologicals, Rabbit polyclonal), EHMT2 (NBP2-13948, Novus Biologicals, Rabbit polyclonal), EHMT2 (07-551; Millipore, Rabbit polyclonal), H3K9me2 (ab1220, Abcam, Mouse monoclonal), LMNB1 (ab16048, Abcam, Rabbit polyclonal), LMNB2 (ab8983, Abcam, Mouse Monoclonal), LMNA/C (sc-20681, Santacruz, Rabbit polyclonal), HP1-β (ab101425, Abcam, Mouse monoclonal), H3K9me3 (ab8898, Abcam, Rabbit polyclonal), H3K27me3 (07-449, Millipore, Rabbit polyclonal), H3 (ab1791, Abcam, Rabbit polyclonal), GAPDH (G9545, Sigma, Rabbit polyclonal), Anti-Methyl lysine antibody (ICP0501, Immunechem, Rabbit polyclonal), Anti-6X His-tag antibody (ab9108, Abcam, Rabbit polyclonal), Anti-GST (ab9085, Abcam, Rabbit polyclonal), Anti-GFP (ab290, Abcam, Rabbit polyclonal), Ash2L (ab176334, Abcam, Rabbit monoclonal), p16 antiboy (ab54210, Abcam) Normal Rabbit IgG (12-370, Millipore), Normal Mouse IgG (12-371, Millipore).

Techniques: Transduction, Cell Cycle Assay, TRAP Assay, Activity Assay, Negative Control

Journal: Communications biology

Article Title: A functional interaction between liprin-α1 and B56γ regulatory subunit of protein phosphatase 2A supports tumor cell motility.

doi: 10.1038/s42003-022-03989-3

Figure Lengend Snippet: Fig. 2 Liprin-α1 directs cytoplasmic B56γ at PMAPs. MDA-MB-231 cells expressing B56γ-GFP treated with saponin and fixed with PFA and immunostained. a–d Accumulation of B56γ-GFP in ERC1-positive PMAPs, in the presence of control siRNA (siCtr) or anti-liprin-α1 siRNA (siLip). a Representative confocal images. b Quantification of the liprin-α1-derived fluorescence in ERC1-positive PMAPs, expressed as Liprin-α1/ERC1 ratio, revealed efficient silencing of Liprin-α1 in cells cotransfected with B56γ-GFP and siLip. c Quantification of B56γ-GFP in ERC1-positive PMAPs, represented as a ratio of the intensity of the two proteins, as revealed by immunofluorescence. d Quantification of B56γ-GFP signal in PMAPs in respect to its expression level, as determined by the fluorescence in the nucleus. e–h Accumulation of B56γ-GFP in Liprin-α1-positive PMAPs, in the presence of control siRNA (siCtr) or anti-ERC1 siRNA (siERC1). e Representative confocal images. f Quantification of B56γ-GFP in liprin-α1-positive PMAPs, represented as a ratio of the intensity of the two proteins, as revealed by immunofluorescence. g Quantification of the B56γ-GFP signal in PMAPs in respect to its expression level, as determined by the fluorescence in the nucleus. h Quantification of the ERC1-derived fluorescence in liprin-α1-positive PMAPs, expressed as ERC1/ liprin-α1 ratio, revealed efficient silencing of ERC1 in cells cotransfected with B56γ-GFP and siERC1. eLip, endogenous liprin; eERC1, endogenous ERC1; the same contrast was applied to confocal images in (a and e).

Article Snippet: For immunoprecipitation cell lysates were incubated with Protein-A–Sepharose beads (Cytiva), Pierce Protein G Agarose (Thermo Scientific) conjugated to antibodies, GFP-Trap (Chromotek), or anti-FLAG-M2 Affinity Gel (SigmaAldrich) before processing for SDS-PAGE and immunoblotting.

Techniques: Expressing, Control, Derivative Assay

Journal: Journal of biochemistry

Article Title: Human telomerase exists in two distinct active complexes in vivo.

doi: 10.1093/jb/mvm071

Figure Lengend Snippet: Fig. 4. Fractionation of HeLa cell lysate. (A) HeLa cell lysate was fractionated on HiLoad 16/60 Superdex 200 pg gel filtration columns in running buffer containing 200 mM NaCl and 0.1% Triton-X100. The telomerase activity peaks were at approximate molecular masses of 680 kDa (peak I) and 400 kDa (peak II). The fractions in peaks I and II were separated by 8% SDS–PAGE and subjected to Western blot analysis using anti- TERT (2C4) or anti-Hsp90 antibodies as indicated. Lane 1, fraction No. A2; lane 2, fraction No. A3; lane 3, fraction No. A4; lane 4, fraction No. A5; lane 5, fraction No. A6; lane 6, fraction No. B1; lane 7, fraction No. B2; lane 8, fraction No. B3; lane 9, fraction No. B4; lane 10, fraction No. B5; lane 11, fraction No. B6. (B) The quantification of hTERC in HeLa cells was measured by real time RT-PCR as shown in Fig. 2C. Relative telomerase activity of the fractions in peaks I and II (fraction No. A2–B6) was measured by TRAP ELISA.

Article Snippet: Antibodies and Western Blot Analysis—For western blot analysis, the total cell lysate and their fractions from gel filtration column were separated by SDS–PAGE and transferred to nitrocellulose membrane and probed with anti-FLAG M2, anti-Hsp 90a/b (Santa Cruz Biotechnology) or anti-TERT 2C4 (Novus Biologicals, Inc.) primary antibodies, followed by incubation with horseradish peroxidase conjugated goat anti-mouse IgG secondary antibody (Amersham Biosciences) for anti-FLAG M2 and anti-Hsp 90a/b antibodies, or horseradish peroxidase conjugated goat anti-mouse IgM secondary antibody (Pierce) for anti-TERT 2C4 antibody.

Techniques: Fractionation, Filtration, Activity Assay, SDS Page, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of biochemistry

Article Title: Human telomerase exists in two distinct active complexes in vivo.

doi: 10.1093/jb/mvm071

Figure Lengend Snippet: Fig. 5. Fractionation of HeLa cell lysate stably expressing FLAG-hTERT. (A) FLAG-hTERT was stably expressed in HeLa cells, and the lysate was fractionated on HiLoad 16/60 Superdex 200 pg gel filtration columns in running buffer containing 200 mM NaCl and 0.1% Triton-X100. The telomerase activity peaks were at approximate molecular masses of 680 kDa (peak I) and 400 kDa (peak II). The fractions in peaks I and II were separated by 8% SDS–PAGE and subjected to western blot analysis using anti-TERT (2C4), anti-FLAG M2 or anti-Hsp90 antibodies as indicated. Lane 1, fraction No. A2; lane 2, fraction No. A3; lane 3, fraction No. A4; lane 4, fraction No. A5; lane 5, fraction No. A6; lane 6, fraction No. B1; lane 7, fraction No. B2; lane 8, fraction No. B3; lane 9, fraction No. B4; lane 10, fraction No. B5; lane 11, fraction No. B6. (B) The quantification of hTERC in HeLa cell stably expressing FLAG-hTERT was measured by real time RT-PCR. Relative telomerase activity of the fractions in peaks I and II (fraction No. A2–B6) was measured by TRAP ELISA. (C) Total lysate, peak I and peak II fractions from HeLa cells stably expressing FLAG-hTERT were immunoprecipitated with anti-FLAG M2 resin. The bound proteins were separated by 8% SDS–PAGE and subjected to western blot analysis using anti-FLAG M2 or anti-Hsp90 antibodies as indicated. Western blot analysis of total lysate is equal to 5% of the input shown in lane 1 (¼ input). Lane 2, total lysate, IP; anti-FLAG antibody, lane 3, peak I fractions, IP; anti-FLAG antibody; and lane 4, peak II fractions, IP; anti-FLAG antibody.

Article Snippet: Antibodies and Western Blot Analysis—For western blot analysis, the total cell lysate and their fractions from gel filtration column were separated by SDS–PAGE and transferred to nitrocellulose membrane and probed with anti-FLAG M2, anti-Hsp 90a/b (Santa Cruz Biotechnology) or anti-TERT 2C4 (Novus Biologicals, Inc.) primary antibodies, followed by incubation with horseradish peroxidase conjugated goat anti-mouse IgG secondary antibody (Amersham Biosciences) for anti-FLAG M2 and anti-Hsp 90a/b antibodies, or horseradish peroxidase conjugated goat anti-mouse IgM secondary antibody (Pierce) for anti-TERT 2C4 antibody.

Techniques: Fractionation, Stable Transfection, Expressing, Filtration, Activity Assay, SDS Page, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: Journal of biochemistry

Article Title: Human telomerase exists in two distinct active complexes in vivo.

doi: 10.1093/jb/mvm071

Figure Lengend Snippet: Fig. 6. Characterization of the complexes in HeLa cells stably expressing FLAG-hTERT. (A) HeLa cells stably expressing FLAG-hTERT were treated for 4 h with the GA concentration indicated; where indicated, cells were pre-treated with MG132 at 10 mM for 2 h prior to treatment with GA. Each lysate were separated by 8% SDS–PAGE and then subjected to western blot analysis using anti-TERT (2C4), anti-FLAG M2 or anti-Hsp90 antibodies as indicated. The activity of each lysate was determined using the TRAP assay and TRAP ELISA. Lane 1, GA at 0 mg/ml; lane 2, GA at 50 mg/ml; lane 3, GA at 75 mg/ml; lane 4, GA at 100 mg/ml; lane 5, GA at 100 mg/ml plus MG132 at 10 mM. (B), (C) HeLa

Article Snippet: Antibodies and Western Blot Analysis—For western blot analysis, the total cell lysate and their fractions from gel filtration column were separated by SDS–PAGE and transferred to nitrocellulose membrane and probed with anti-FLAG M2, anti-Hsp 90a/b (Santa Cruz Biotechnology) or anti-TERT 2C4 (Novus Biologicals, Inc.) primary antibodies, followed by incubation with horseradish peroxidase conjugated goat anti-mouse IgG secondary antibody (Amersham Biosciences) for anti-FLAG M2 and anti-Hsp 90a/b antibodies, or horseradish peroxidase conjugated goat anti-mouse IgM secondary antibody (Pierce) for anti-TERT 2C4 antibody.

Techniques: Stable Transfection, Expressing, Concentration Assay, SDS Page, Western Blot, Activity Assay, TRAP Assay, Enzyme-linked Immunosorbent Assay