tmg cap antibodies  (Santa Cruz Biotechnology)

Journal: Cell reports

Article Title: Loss of Human TGS1 Hypermethylase Promotes Increased Telomerase RNA and Telomere Elongation

doi: 10.1016/j.celrep.2020.01.004

Figure Lengend Snippet: (A) Schematic representation of the experiments. hTR was precipitated from nuclear extracts with an anti-TERT antibody, and purified TERT-hTR complexes were subjected to IP using an anti-TMG cap antibody. (B) hTR associated with TERT eluates from TGS1 M1 and two TGS1 -proficient CTR cells was detected by NB. IgG IP is a CTR for nonspecific binding. EtBr-stained rRNA is a loading CTR. Note that hTR is more abundant in TGS1 mutant cells than in CTR cells. (C) RNA IP with an anti-TMG antibody from TERT-hTR complexes. qRT-PCR on eluates indicates that TERT-associated hTR is not hypermethylated. The monomethylated beta-actin RNA is a negative CTR. Error bars represent standard deviations derived from two independent RNA IP experiements. (D) Top: telomeric repeat amplification protocol (TRAP) performed in 33 dilutions on extracts from cells of the indicated genotypes. IC, internal CTR. TGS1 M1 and M2 cells exhibit higher telomerase activities than both TGS1 -proficient CTR cells and TGS1 M1 rescued cells ( M1R ). Bottom: quantification of TRAP activity. Error bars, SEM. a.u., arbitrary units. (E) Mutations in TGS1 (C1, C2) were generated by CRISPR/Cas9 in the UMUC3 bladder tumor cell line. WB with anti-TGS1 or anti-FLAG antibodies shows reduced levels of endogenous TGS1 in mutant cells and the expression of the TGS1-FLAG rescue construct. See also . (F) qRT-PCR showing that TGS1 CRISPR clones stably expressing FLAG-GFP exhibit increased hTR abundance compared to the same clones stably expressing the rescue construct FLAG-TGS1. Data are from three biological replicates, are normalized to GAPDH and relative to the parental cell line (*p < 0.05; **p < 0.01; one-way ANOVA). (G) TGS1 loss induces telomere lengthening in UMUC3 cells. Telomere restriction fragment (TRF) analysis was performed on genomic DNA extracted from TGS1 mutant (C1, C2) and CTR cell lines kept in culture for the indicated time. PD, population doublings. The growth kinetics were similar for all lines (average doubling time: 1.8 days).

Article Snippet: TMG cap specific (R1131) polyclonal antibody , Synaptic Systems , Cat# 201 002.

Techniques: Purification, Binding Assay, Staining, Mutagenesis, Quantitative RT-PCR, Derivative Assay, Amplification, Activity Assay, Generated, CRISPR, Expressing, Construct, Clone Assay, Stable Transfection

Journal: Cell reports

Article Title: Loss of Human TGS1 Hypermethylase Promotes Increased Telomerase RNA and Telomere Elongation

doi: 10.1016/j.celrep.2020.01.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TMG cap specific (R1131) polyclonal antibody , Synaptic Systems , Cat# 201 002.

Techniques: Recombinant, Sequencing, CRISPR, Labeling, Software, Control

Journal: Cell reports

Article Title: Loss of Human TGS1 Hypermethylase Promotes Increased Telomerase RNA and Telomere Elongation

doi: 10.1016/j.celrep.2020.01.004

Figure Lengend Snippet: (A) Schematic representation of the experiments. hTR was precipitated from nuclear extracts with an anti-TERT antibody, and purified TERT-hTR complexes were subjected to IP using an anti-TMG cap antibody. (B) hTR associated with TERT eluates from TGS1 M1 and two TGS1 -proficient CTR cells was detected by NB. IgG IP is a CTR for nonspecific binding. EtBr-stained rRNA is a loading CTR. Note that hTR is more abundant in TGS1 mutant cells than in CTR cells. (C) RNA IP with an anti-TMG antibody from TERT-hTR complexes. qRT-PCR on eluates indicates that TERT-associated hTR is not hypermethylated. The monomethylated beta-actin RNA is a negative CTR. Error bars represent standard deviations derived from two independent RNA IP experiements. (D) Top: telomeric repeat amplification protocol (TRAP) performed in 33 dilutions on extracts from cells of the indicated genotypes. IC, internal CTR. TGS1 M1 and M2 cells exhibit higher telomerase activities than both TGS1 -proficient CTR cells and TGS1 M1 rescued cells ( M1R ). Bottom: quantification of TRAP activity. Error bars, SEM. a.u., arbitrary units. (E) Mutations in TGS1 (C1, C2) were generated by CRISPR/Cas9 in the UMUC3 bladder tumor cell line. WB with anti-TGS1 or anti-FLAG antibodies shows reduced levels of endogenous TGS1 in mutant cells and the expression of the TGS1-FLAG rescue construct. See also . (F) qRT-PCR showing that TGS1 CRISPR clones stably expressing FLAG-GFP exhibit increased hTR abundance compared to the same clones stably expressing the rescue construct FLAG-TGS1. Data are from three biological replicates, are normalized to GAPDH and relative to the parental cell line (*p < 0.05; **p < 0.01; one-way ANOVA). (G) TGS1 loss induces telomere lengthening in UMUC3 cells. Telomere restriction fragment (TRF) analysis was performed on genomic DNA extracted from TGS1 mutant (C1, C2) and CTR cell lines kept in culture for the indicated time. PD, population doublings. The growth kinetics were similar for all lines (average doubling time: 1.8 days).

Article Snippet: Purified RNA was then subjected to IP with the R1131 anti-TMG cap specific antibody ( ) (Synaptic Systems).

Techniques: Purification, Binding Assay, Staining, Mutagenesis, Quantitative RT-PCR, Derivative Assay, Amplification, Activity Assay, Generated, CRISPR, Expressing, Construct, Clone Assay, Stable Transfection

Journal: Journal of Cell Science

Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology

doi: 10.1242/jcs.211482

Figure Lengend Snippet: SNRPN exhibits similar behaviour to SNRPB in SH-SY5Y cells. (A) SH-SY5Y cells transiently expressing YFP–SNRPN and fixed after 24, 48 and 72 h show variations in distribution of the YFP–SNRPN with time. Immunostaining with Y12 (detecting Sm proteins, red on overlay) and anti-coilin (white on overlay) shows splicing speckles (chevron arrowheads) and cajal bodies (CBs, triangular arrowheads) respectively. Images are deconvolved z -stacks with 0.2 µm spacing. (B) After transient expression, SNRPN initially localises diffusely in the cytoplasm, before localising to speckles at the 48 and 72 h time-points. Results are mean±s.d. from three independent experiments, n =100 cells per experiment. (C) Western blot analysis of snRNPs immunoprecipitated using TMG beads (against the characteristic tri-methyl guanosine Cap of snRNAs, left hand lane) confirms that both YFP–SNRPN (detected with anti-YFP, top row) and mCherry–SNRPN (detected with anti-mCherry, bottom row) are incorporated into snRNPs. (D) mCherry–SNRPN cytoplasmic structures are mobile and stain with the lipophilic dye BODIPY 493. Chevron arrowheads identify mCherry–SNRPN structures stained with BODIPY 493; triangular arrowheads identify BODIPY 493-stained vesicles not containing mCherry–SNRPN. mCherry alone does not accumulate in BODIPY 493-stained vesicles. Cells were imaged approximately every 4 s for 9 min. Images are single deconvolved z -sections. (E) mCherry–SNRPN and GFP–SMN colocalise in cytoplasmic foci in SH-SY5Y cells (chevron arrowheads in left hand panels), whereas YFP alone shows no accumulation in mCherry-SNRPN foci (triangular arrowheads in right hand panels). White signal on the overlay indicates areas of colocalisation. Images are single deconvolved z -sections. (F) Comparison of the percentage of mCherry–SNRPN vesicles per cell colocalising with GFP–SMN to those showing co-incidental overlap with YFP alone confirms the colocalisation. Results are mean±s.d., n =5 ( P <0.0001, unpaired two-tailed t -test). Scale bars: 7 µm.

Article Snippet: To investigate whether the interaction between NCDN and SMN could reflect a previously unidentified role for NCDN in snRNP assembly, splicing snRNPs were affinity purified from whole-cell lysates of SH-SY5Y cells constitutively expressing NCDN–GFP using agarose beads coupled to antibodies against the characteristic tri-methyl guanosine Cap of snRNAs (TMG beads, Millipore) ( A).

Techniques: Expressing, Immunostaining, Western Blot, Immunoprecipitation, Staining, Comparison, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Neurochondrin interacts with the SMN protein suggesting a novel mechanism for spinal muscular atrophy pathology

doi: 10.1242/jcs.211482

Figure Lengend Snippet: NCDN does not co-purify with snRNPs, while NCDN and SMN interact with Rab5 and colocalise with a subset of Rab5 vesicles within neurites of SH-SY5Y cells. (A) Incubation of whole-cell lysate from an SH-SY5Y cell line constitutively expressing NCDN–GFP with agarose beads conjugated to antibodies against the tri-methyl guanosine cap (Me3Gppp) of snRNAs (TMG beads) affinity purifies snRNPs as evidenced by the enrichment of the core snRNP protein SNRPN (detected with anti-SNRPN antibody, bottom row). The enriched snRNP fraction also contains SMN, which is essential for snRNP assembly. NCDN–GFP, however, does not co-enrich with snRNPs. Also shown is the core structure of mature snRNPs consisting of the heptameric Sm protein ring bound at the Sm-binding site of snRNA, as well as the characteristic tri-methyl guanosine Cap of snRNAs (Me 3 Gppp) at the 5′ end. (B) Affinity isolation of mRFP–Rab5 using RFP-Trap from cells co-transfected with plasmids to express mRFP–Rab5 together with NCDN–GFP, GFP–SMN or YFP alone co-enriches both NCDN–GFP (top row, detected with anti-GFP antibody, band is present in RFP-Trap lane but not Sepharose beads lane) and SMN-GFP (second row, detected with anti-GFP antibody, band is present in RFP-Trap lane but not Sepharose beads lane), but not YFP (third row, no band detected in RFP-Trap lane). Endogenous SMN (fourth row, detected with mouse anti-SMN) co-enriches with mRFP–Rab5 in all three samples. Detection of mRFP–Rab5 (bottom row, detected with anti-RFP antibody) confirms substantial enrichment of mRFP–Rab5 in all three samples. (C) Both GFP–SMN and NCDN–GFP partially colocalise with mRFP–Rab5 in a subset of mRFP–Rab5-containing vesicles in co-transfected SH-SY5Y cells (white signal in overlaid images, top row; yellow signal in colocalisation images, bottom row). (D) Enlargement of the boxed areas in C confirms that the colocalisation between SMN or NCDN and Rab5 occurs in punctate structures. Arrowheads identify areas of colocalisation. Colocalisation images were generated by Volocity using automatic thresholds on non-deconvolved z -sections before excluding values below 0.05. Images (excluding the colocalisation images) are single deconvolved z -sections. Scale bars: 7 µm.

Article Snippet: To investigate whether the interaction between NCDN and SMN could reflect a previously unidentified role for NCDN in snRNP assembly, splicing snRNPs were affinity purified from whole-cell lysates of SH-SY5Y cells constitutively expressing NCDN–GFP using agarose beads coupled to antibodies against the characteristic tri-methyl guanosine Cap of snRNAs (TMG beads, Millipore) ( A).

Techniques: Incubation, Expressing, Binding Assay, Isolation, Transfection, Generated