rabbit anti trf2  (Novus Biologicals)

Journal: iScience

Article Title: Human SKI component SKIV2L regulates telomeric DNA-RNA hybrids and prevents telomere fragility

doi: 10.1016/j.isci.2024.111096

Figure Lengend Snippet: SKIV2L of the hSKI complex is present at telomeres in G2 (A) Subcellular fractionation assay in asynchronous HeLa1.3 cells. (B) Proteomics of isolated chromatin segments analysis showing the binding of hSKI to telomeres throughout the cell cycle. Tables are listing the number of unique peptide numbers isolated, including the fold change values of unique peptides normalized to the asynchronous values (top panel) and the relative LFQ intensity values identified by PICh. AS: asynchronous. Scr: scramble. (C) WB of hSKI (SKIV2L, TTC37, and WDR61) and SKIV2L2 in shCtr (Control) or shSKIV2L HeLa1.3 cells. (D) Immunofluorescence of pre-extracted cells showing co-localization of SKIV2L and TTC37 with TRF2 in asynchronous (AS) and G2-synchronized cells. % of total number of SKIV2L or TTC37 foci colocalizing with TRF2 are normalized to shCtr AS samples (means ± SEM, n = 2 independent experiments, scale bar 15 μm). t test ∗ p < 0.05, ∗∗∗ p < 0.001. (E) Proximity ligation assay of SKIV2L-TRF2, showing increasing number of foci in G2 synchronized cells (median, Q1 and Q3, 707 (AS) and 638 (G2) cells scored per condition, 3–4 independent experiments, scale bar 10 μm). Mann-Whitney U test ∗∗∗∗ p < 0.0001. (F) ChIP-dot blot of SKIV2L and TRF1 in AS and G2-synchronized shCtr and shSKIV2L cells (means ± SEM, n = 3). t test ∗ p < 0.05, ∗∗∗∗ p < 0.0001. See also Figure S1 .

Article Snippet: For S9.6-TRF2 PLA, cells were blocked for 3 h (1 h at 37°C and 2 h at 4°C) in blocking buffer (BSA 2% (w/v) in PBS), incubated with the mouse anti-DNA-RNA hybrid S9.6 antibody (1/2000, Kerafast ENH001) and rabbit anti-TRF2 antibody (1/1000, Novus NB110-57130) in blocking buffer overnight at 4°C followed by 3 washes with PBS-Tween 0.1% (v/v).

Techniques: Fractionation, Isolation, Binding Assay, Control, Immunofluorescence, Proximity Ligation Assay, MANN-WHITNEY, Dot Blot

Journal: iScience

Article Title: Human SKI component SKIV2L regulates telomeric DNA-RNA hybrids and prevents telomere fragility

doi: 10.1016/j.isci.2024.111096

Figure Lengend Snippet: SKIV2L and TTC37 prevents telomere fragility (A and B) Telomere FISH analysis in SKIV2L- and TTC37-depleted HeLa1.3 and HT1080-ST cells using siRNAs: % of telomere fragility (yellow) and loss (purple) per metaphase in siCtr, siSKIV2L and siTTC37 (means ± SD, n > 25 metaphases, 2 independent experiments, scale bar, 10 μm). t test ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001. (C) Telomere FISH analysis in shCtr (Control) or shSKIV2L HEK293 cells expressing GFP or SKIV2L (means ± SD, n > 25 metaphases, 2 independent experiments, scale bar, 10 μm). Details are as in (A). (D) Telomere FISH analysis in TTC37-depleted HEK293 cells using siRNAs (means ± SD, n = 25 metaphases). Details are as in (A). (E) Telomere FISH analysis in HeLa1.3 cells using shRNAs: shCtr and shSKIV2L treated with DMSO (−, control) or APH (+) ( n > 45 metaphases, 2 independent experiments). Details are as in (A). (F) IF of pre-extracted cells showing co-localization of pS1981 ATM autophosphorylation with TRF2 (telomeres) in shCtr and SKIV2L-depleted (shSKIV2L) HeLa1.3 cells. Quantification of the percentage of cells with co-localization foci and mean number of foci per nucleus is depicted (means ± SEM, n = 200 cells, 2 independent experiments, scale bar 15 μm). t test ∗∗∗ p < 0.001. (G) IF-FISH of pre-extracted cells showing co-localization of 53BP1 with telomeres in asynchronous (AS) or G2-synchronized control (shCtr) and SKIV2L-depleted (shSKIV2L) HeLa1.3 cells. Quantification of the percentage of cells with co-localization foci is depicted (means ± SEM, n > 400 cells, 3 independent experiments, scale bar 15 μm). t test ∗ p < 0.05. See also Figure S2 .

Article Snippet: For S9.6-TRF2 PLA, cells were blocked for 3 h (1 h at 37°C and 2 h at 4°C) in blocking buffer (BSA 2% (w/v) in PBS), incubated with the mouse anti-DNA-RNA hybrid S9.6 antibody (1/2000, Kerafast ENH001) and rabbit anti-TRF2 antibody (1/1000, Novus NB110-57130) in blocking buffer overnight at 4°C followed by 3 washes with PBS-Tween 0.1% (v/v).

Techniques: Control, Expressing

Journal: iScience

Article Title: Human SKI component SKIV2L regulates telomeric DNA-RNA hybrids and prevents telomere fragility

doi: 10.1016/j.isci.2024.111096

Figure Lengend Snippet: SKIV2L regulates telomeric DNA-RNA hybrids in cellulo to prevent telomere fragility (A) Proximity ligation assay (PLA) showing co-localization of SKIV2L and TRF2 in asynchronous (AS) and in G2-synchronized HeLa1.3 cells with and without RNase H1 (RNH1) overexpression (median, Q1 and Q3, at least 600 cells scored per condition, 4 independent experiments, scale bar 10 μm). Mann-Whitney U test ∗∗∗∗ p < 0.0001. (B) S9.6 IF in HeLa1.3 cells treated with RNAse III, with and without RNase H1 (RNH1) overexpression (median ± interquartile range, 360 cells scored per condition, 4 independent experiments, scale bar, 10 μm). Mann-Whitney U test ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (C) PLA showing the co-localization of DNA-RNA hybrids (S9.6) and TRF2 in HeLa1.3 cells pre-extracted and treated with RNAse III (median, Q1 and Q3, at least 400 cells scored per condition, 2 independent experiments, scale bar 10 μm). Mann-Whitney U test ∗∗∗∗ p < 0.0001. (D) DRIP showing the levels of DNA-RNA hybrids at telomeres in AS and G2-synchronized HeLa1.3 cells, RNH, RNase H treatment (means ± SEM, n = 4). (E) DRIP-qPCR assay of G2-synchronized HEK293 cells overexpressing GFP or SKIV2L at 10q, 13q, 20q, and 22q subtelomeric regions. RNH, RNase H treatment (means ± SEM, n = 5). Percent input values were normalized to the GFP overexpressing condition. (F) Model proposing the function of hSKI at telomeres. Telomeric DNA-RNA hybrid accumulation in late S/G2 phase drives hSKI recruitment to telomeres to regulate physiological DNA-RNA hybrid levels, prevent telomere replication stress and ensure telomere stability. See also Figure S5 .

Article Snippet: For S9.6-TRF2 PLA, cells were blocked for 3 h (1 h at 37°C and 2 h at 4°C) in blocking buffer (BSA 2% (w/v) in PBS), incubated with the mouse anti-DNA-RNA hybrid S9.6 antibody (1/2000, Kerafast ENH001) and rabbit anti-TRF2 antibody (1/1000, Novus NB110-57130) in blocking buffer overnight at 4°C followed by 3 washes with PBS-Tween 0.1% (v/v).

Techniques: Proximity Ligation Assay, Over Expression, MANN-WHITNEY

Journal: iScience

Article Title: Human SKI component SKIV2L regulates telomeric DNA-RNA hybrids and prevents telomere fragility

doi: 10.1016/j.isci.2024.111096

Figure Lengend Snippet:

Article Snippet: For S9.6-TRF2 PLA, cells were blocked for 3 h (1 h at 37°C and 2 h at 4°C) in blocking buffer (BSA 2% (w/v) in PBS), incubated with the mouse anti-DNA-RNA hybrid S9.6 antibody (1/2000, Kerafast ENH001) and rabbit anti-TRF2 antibody (1/1000, Novus NB110-57130) in blocking buffer overnight at 4°C followed by 3 washes with PBS-Tween 0.1% (v/v).

Techniques: Virus, Recombinant, Protease Inhibitor, Reverse Transcription, Blocking Assay, Mass Spectrometry, SYBR Green Assay, Flow Cytometry, Imaging, Mutagenesis, Cell Cycle Assay, shRNA, Software