rabbit anti dkc1  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: Impaired phase separation and nucleolar functions in hiPSC models of SNORD118 -related ribosomopathies

doi: 10.1016/j.isci.2024.110430

Figure Lengend Snippet: Nucleolar morphology and LLPS impairment in SNORD118 mutant NPCs (A) Confocal image of immunofluorescent staining of p-eIF2α staining in NPCs. Scale bars, 1 μm. (B) Quantification of the fold change of p-eIF2α intensity. (C) Representative images of NPM1 (green) and FBL (red) staining in control and S NORD118 ∗ 5C>G homozygous mutant NPCs. Scale bar, 1 μm. (D–F) Quantification of the volume of the nucleolus, the area of the nucleolus, and the nucleolar circularity. Three individual cell lines have been used for analysis, and about 800 cells in each cell line were analyzed. (G) Representative images of GFP fluorescence recovery after photobleaching (FRAP) analysis. Red arrowheads represent spots with photo bleach. Scale bar, 1 μm. (H) Line plots showing the dynamics of GFP recovery in each group. (I) Quantification of the percentage of GFP intensity recovery in the 60 s of each group, n (control) = 39, n ( SNORD118 ∗ 5C>G ) = 39 cells. (J) Diagram of EGFP-DKC1 vector. (K) EGFP-DKC1 co-stained with FBL and NPM1 in NPCs. Scale bar, 1 μm. (L) Representative images of FRAP analysis of EGFP-DKC1. Red arrowheads represent spots with photo bleach. Scale bar, 1 μm. (M) Line plots showing the dynamics of EGFP recovery in each group. (N) Quantification of the percentage of EGFP intensity recovery in the 60 s of each group, n = 42-39 cells. All data are represented as mean ± SEM calculated by Student’s t test. Ns represents not significant, ∗ p < 0.05, ∗∗∗ p < 0.001.

Article Snippet: Rabbit-anti DKC1 , Thermo Fisher Scientific , Cat# PA5-28922; RRID: AB_2546398.

Techniques: Mutagenesis, Staining, Control, Fluorescence, Plasmid Preparation

Journal: iScience

Article Title: Impaired phase separation and nucleolar functions in hiPSC models of SNORD118 -related ribosomopathies

doi: 10.1016/j.isci.2024.110430

Figure Lengend Snippet: Ribosome biogenesis and FBL mRNA translation are compromised in mutant NPCs (A) Western blot (WB) analysis of FBL protein levels. (B) Quantification of WB results. N = 3. (C) RNA screen tape analysis for the 28S and 18S rRNA biogenesis. (D) Quantification of 28S/18S ratio. N = 9 samples from 3 hPSC-derived NPCs. (E) OPP assay of control and SNORD118 ∗ 5C>G mutant NPCs following treatment with DMSO or ActD (100 ng/mL) for 24 h. Scale bar, 20 μm. (F) Fold change of OPP intensity. N = 3. (G) Interaction profiles of snoRNA U8 with protein FBL, DKC1, and β-Actin in control and mutant NPCs. (H) Quantification of U8-binding proteins. N = 3. (I) Representative polysome profiles of Control and SNORD118 ∗ 5C>G mutant NPCs. (J) Fold change of assembled 80S ribosome peak area. N = 3. (K) Fold change of translating FBL mRNA from polysomes, as determined by qPCR. N = 3. All data are represented as mean ± SEM calculated by Student’s t test. The ns, no signification, ∗ p < 0.05.

Article Snippet: Rabbit-anti DKC1 , Thermo Fisher Scientific , Cat# PA5-28922; RRID: AB_2546398.

Techniques: Mutagenesis, Western Blot, Derivative Assay, Control, Binding Assay

Journal: iScience

Article Title: Impaired phase separation and nucleolar functions in hiPSC models of SNORD118 -related ribosomopathies

doi: 10.1016/j.isci.2024.110430

Figure Lengend Snippet: FBL rescues nucleolar morphology and LLPS defects of mutant NPCs in a phase separation motif-dependent manner (A) Diagram of wild type (FBL), IDR-depleted (FBL-C) mutant, and chimeric FBL, in which the intrinsic IDR is replaced by another LLPS motif from an unrelated protein. (B) Immunostaining of different EGFP-FBL isoforms for their co-localization with DKC1 and NPM1 in NPCs. Scale bar, 1 μm. (C) Representative images of NPM1 staining in control and SNORD118 ∗ 5C>G NPCs with or without the expression of FBL, FBL-C, or chimeric FBL. Scale bar, 5 μm. (D) Quantification of the percentage of intact and disrupted nucleolar morphology. (E) Representative images of GFP fluorescence recovery after photobleaching (FRAP) analysis. White arrowheads represent spots with photo bleach. Control and SNORD118 ∗ 5C>G NPCs were labeled with GFP-tagged NPM1, while the rest of the NPCs were visualized using various forms of EGFP-fused FBL. Scale bar, 2 μm. (F) Line plots showing the dynamics of GFP recovery in each group. (G) Quantification of the percentage of GFP intensity recovery in the 60 s of each group, n (control) = 39, n ( SNORD118 ∗ 5C>G ) = 39, n (FBL) = 35, n (FBL-C) = 38, n (Chimeric FBL) = 37 cells. All data are represented as mean ± SEM calculated by Student’s t test, ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns, not significant.

Article Snippet: Rabbit-anti DKC1 , Thermo Fisher Scientific , Cat# PA5-28922; RRID: AB_2546398.

Techniques: Mutagenesis, Immunostaining, Staining, Control, Expressing, Fluorescence, Labeling

Journal: iScience

Article Title: Impaired phase separation and nucleolar functions in hiPSC models of SNORD118 -related ribosomopathies

doi: 10.1016/j.isci.2024.110430

Figure Lengend Snippet:

Article Snippet: Rabbit-anti DKC1 , Thermo Fisher Scientific , Cat# PA5-28922; RRID: AB_2546398.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Modification, Software