nup153 antibody  (Bio-Techne corporation)

Journal: bioRxiv

Article Title: Nuclear translocation of LINE-1 encoded ORF1p alters nuclear envelope integrity and disrupts nucleocytoplasmic transport in neurons

doi: 10.1101/2023.08.10.552479

Figure Lengend Snippet: (A) Co-immunostaining of ORF1p and Lamin B1 reveals their colocalization (yellow). (B) Validation of ORF1p interaction with Lamin B1 by co-immunoprecipitation and Western-blotting. (C) Direct interaction of ORF1p with Lamin B1 was revealed by PLA and quantified in (D), n > 50 neurons were quantified per condition, (3 wells per condition) (E) Co-immunostaining of ORF1p and KPNB1 or NUP153. (F) Validation of a direct interaction between ORF1p and KPNB1 or NUP153 by PLA. The quantification in separate cellular compartments is shown in (G, H), n > 50 neurons were quantified per condition, (3 wells per condition), mean ± SEM, Two-tailed t test (*p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar, 5 µm.

Article Snippet: Stressed and control differentiated LUHMES cells cultured in 24-well plates (150 000 cells/well) were fixed with 4% paraformaldehyde (PFA) for 10 min. Immunofluorescence were performed overnight at 4°C after a 20 min permeabilization in 0,2% Triton X100 and incubation in blocking buffer (10 % bovine serum albumin BSA) for 1 h with the following antibodies: ORF1p 1:500 (Millipore, MABC1152 clone 4H1), TH 1:500 (Millipore, AB9702), Tuj1 1:1000 (Abcam, ab18207), Lamin B1 1:1000 (Abcam, ab16048), KPNB1 1:500 (GTX, 133733), NUP153 1:1000 (Novus biological, NB100-93329), H4K20me3 1:1000 (Abcam, ab9053), HP1 1:1000 (Cell Signaling, #2623S), TDP-43 1:600 (Proteintech, 107-82-2AP), HA 1:1000 (Roche, 11867423001), RANGAP1 1:500 (Santa Cruz, sc-28322), ssDNA 1:500 (ENZO, (F7-26) ALX 804-192) diluted in the blocking buffer, overnight at 4°C.

Techniques: Immunostaining, Biomarker Discovery, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Nuclear translocation of LINE-1 encoded ORF1p alters nuclear envelope integrity and disrupts nucleocytoplasmic transport in neurons

doi: 10.1101/2023.08.10.552479

Figure Lengend Snippet: (A) Immunostaining of KPNB1 in oxidative stress conditions and quantification of the intensity levels, the number and the volume of dots in the cytoplasm. (B) Immunostaining of NPC protein RANgap1 in oxidative stress conditions and quantification of the intensity levels, the number and the volume of dots in the cytoplasm. (C) Immunostaining of TDP-43 in oxidative stress conditions and quantification of the intensity levels, the number and the volume of dots in the cytoplasm. (D) Immunostaining of NUP153 in oxidative stress conditions and quantification of the intensity levels in the nucleoplasm and the cytoplasm. n > 100 neurons were quantified per condition, a representative experiment (3 wells per condition) of 3 independent experiments, mean ± SEM, Two-tailed t test (*p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar, 5 µm.

Article Snippet: Stressed and control differentiated LUHMES cells cultured in 24-well plates (150 000 cells/well) were fixed with 4% paraformaldehyde (PFA) for 10 min. Immunofluorescence were performed overnight at 4°C after a 20 min permeabilization in 0,2% Triton X100 and incubation in blocking buffer (10 % bovine serum albumin BSA) for 1 h with the following antibodies: ORF1p 1:500 (Millipore, MABC1152 clone 4H1), TH 1:500 (Millipore, AB9702), Tuj1 1:1000 (Abcam, ab18207), Lamin B1 1:1000 (Abcam, ab16048), KPNB1 1:500 (GTX, 133733), NUP153 1:1000 (Novus biological, NB100-93329), H4K20me3 1:1000 (Abcam, ab9053), HP1 1:1000 (Cell Signaling, #2623S), TDP-43 1:600 (Proteintech, 107-82-2AP), HA 1:1000 (Roche, 11867423001), RANGAP1 1:500 (Santa Cruz, sc-28322), ssDNA 1:500 (ENZO, (F7-26) ALX 804-192) diluted in the blocking buffer, overnight at 4°C.

Techniques: Immunostaining, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Nuclear pore protein TPR associates with lamin B1 and affects nuclear lamina organization and nuclear pore distribution

doi: 10.1007/s00018-019-03037-0

Figure Lengend Snippet: A distribution of NPCs is affected in TPR-depleted HeLa cells (shTPR HeLa cell line). a–c The distribution of NPCs in control HeLa cells as viewed by immunostaining with anti-Nup62 antibody and wide-field Leica DM6000 microscope. a Homogenous distribution. b Small NPC-free zones. c Large NPC-free zones comprehend more than 1/10 of nuclear surface. d The ratio of homogenous distribution, small and large NPC-free zones in control wild-type S-G2 cells (wt), in cells stably expressing shRNA targeting non-coding regions (shNC) and TPR (shTPR cells). In TPR-depleted cells, the number of nuclei with NPC-free zones increased significantly (χ2 = 11.41, df = 2, P < 0.001, Chi squared test), **P < 0.01, ***P < 0.001, n.s. not significant. e The ratio of homogenous, small and large NPC-free zones in control wild type cells (wt) and in cells stably expressing shRNA targeting non-coding regions (shNC) and TPR (shTPR) synchronized in G1/S and in G2 phase of the cell cycle. The number of NPC-free zones significantly increased in TPR-depleted cells (***P < 0.001, n.s. not significant). f–i Recruitment of nucleoporins Nup107, Nup133 and central FG-Nups into NPC-free zones is impaired in TPR-depleted cells. f The ratio of distribution of NPC-free zones in shNC and shTPR cells stained with Nup153 antibody (χ2 = 7.08, df = 2, P = 0.029). g The ratio of homogenous distribution, small and large NPC-free zones in shNC and shTPR cells transfected with GFP–Nup107 construct (χ2 = 9.713, df = 2, P = 0.008). h The ratio of homogenous distribution, small and large NPC-free zones in shNC and shTPR cells overexpressing GFP–Nup133 (χ2 = 23.855, df = 2, P < 0.001). i The ratio of distribution of NPC-free zones in shNC and shTPR cells stained with antibody recognizing central FG-Nups (χ2 = 138.5, df = 2, P < 0.0001)

Article Snippet: Antibodies Primary antibodies were used as follows:. anti-TPR mouse monoclonal (nTPR, ab58344, Abcam, Cambridge, UK, 1:300), anti-TPR rabbit polyclonal (cTPR, ab84516, Abcam, Cambridge, UK, 1:300), anti-lamin B1 rabbit polyclonal IgG (ab16048, Abcam, Cambridge, UK, 1:100), anti-lamin A/C mouse monoclonal (SAB4200236, Sigma, Sigma-Aldrich, St. Louis, USA, 1:100), anti-Nup 62 rat monoclonal IgG (ab188413, Abcam, Cambridge, UK, 1:500); anti-Nup153 rabbit polyclonal antibody (NB100-93329, Novus Biologicals, 1:200), Nuclear pore complex proteins (ab24609, Abcam Cambridge, UK, 1:200), anti-GFP mouse monoclonal (ab1218, Abcam, Cambridge, UK, 1:200, anti-GFP rabbit polyclonal (1828014, Molecular probes at Thermofisher Scientific, Waltham, MA USA, 1:200), anti-PCNA human polyclonal, anti-tubulin α (N-terminal structural domain, TU-01, aa 65–79, 1:100) mouse monoclonal was kindly provided by Dr. Pavel Draber (Institute of Molecular Genetics of the ASCR, v.v.i., Prague, Czech Republic).

Techniques: Control, Immunostaining, Microscopy, Stable Transfection, Expressing, shRNA, Staining, Transfection, Construct