Journal: Chromosoma

Article Title: CTCF is essential for proper mitotic spindle structure and anaphase segregation.

doi: 10.1007/s00412-023-00810-w

Figure Lengend Snippet: Fig. 4 Mitotic spindle structure is perturbed in CTCF knock- downs. A Example images of mitosis in wild-type and CTCF knockdowns c13 and c21 with labeled microtubules (green, α-tubulin) and DNA (magenta, Hoechst). Mitotic spindles were classified as abnormal mitotic spindle if they were tri-/ tetrapolar spindles (yellow) or other abnormal (blue) if DNA was substantially behind the spindle pole or no division was observed. For all images, see Supplemental Figure 1. B Graph of the percentage of abnormal mitotic spindles (WT n = 0/48; CTCF knockdown c13 n = 12/42; CTCF knockdown c21 n = 12/57; CENP-E inhibitor GSK923295 72 h n = 11/17). C Box and whisker plot of average and individual measurement of DNA position relative to the spindle pole. Purple dots represent spindles where DNA is > 1 μm behind the spindle pole (WT 0%, c13 30%, c21 12%, GSK923295 29%). Error bar represents standard error. Statistical tests are Student’s t-tests, significance denoted by *p < 0.05, **p < 0.01, and ***p < 0.001, while no signifi- cance is denoted by ns. Scale bar is 10 μm

Article Snippet: Coverslips were placed on 50-μL drops of the primary antibody mixture consisting of rat anti-alpha tubulin at 1:100 dilution (Novus biological); mouse anti-lamin A/C at 1:100 dilution (Cell Signaling Technology); and PBS with 5% BSA.

Techniques: Labeling, Knockdown, Whisker Assay

Journal: Frontiers in Immunology

Article Title: miR-210 promotes the anti-inflammatory phenotype and M2 polarization in murine macrophages

doi: 10.3389/fimmu.2025.1633163

Figure Lengend Snippet: Effects of miR-210-KO in M0 macrophages. (A) Enrichr dot-plot representation for pathways enriched among differentially expressed genes in miR-210-KO versus WT M0 macrophages. The signed odds ratio (x-axis) indicates over-representation in up-regulated (positive) or down-regulated (negative) genes. (B) Western blot analysis of pro-IL1β levels in M0 macrophages, with α-tubulin used as a loading control. Densitometry analysis performed using TotalLab. Data are presented as the mean ± SEM, n = 3 mice per condition. Statistical significance was determined using Student’s t test, *p< 0.05. (C) ELISA quantification of IL-6, TNF-α and IL-1β in the supernatant of M0 macrophages. Data are presented as mean ± SEM, n = 3 for IL-6 and IL-1β and n=4 for TNF-α.

Article Snippet: The membranes were incubated overnight at 4°C with antibodies against p53 (Abcam, #ab90363, 1:1000), TGF-β (Abcam, #ab215715, 1:1000), GAPDH (Abcam, #ab37168, 1:1000), IL-1β (Abcam, ab9722, 1:1000), αTubulin (R&D Systems, MAB9344 1:1000), β-actin (Sigma-Aldrich, A2228), and MMP-9 antibody (Novus Biologicals, NBP1-57840), prepared in 5% BSA in TBST.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries * ^♦

doi: 10.1074/jbc.M113.499608

Figure Lengend Snippet: Spag protein structure and patterns of gene expression and protein accumulation during Drosophila development. A, schematic structure of the Spag protein and evolutionary conservation of the TPR region in Spag orthologs from yeast to human. Filled boxes represent predicted structural domains and lines unstructured domains. The three TPR motifs forming the TPR region are indicated as red, blue, and green boxes. TPR 3′ is a putative helical domain absent in Tah1. Orange box represents a potential α-helical junction between TPR 3 and TPR 3′. The potential monad binding motif (interPro 025986) overlaps with a predicted structured domain (magenta). Below are aligned the amino acid residues characterizing the three TPR motifs in S. cerevisiae (S.c.), D. melanogaster (D.m.), Danio rero (D.r.), Homo sapiens (H.s.), and Xenopus tropicalis (X.t.). Conserved residues identified in Tah1 for Hsp90 binding are indicated in bold red (21). Blue indicates additional residues conserved in TPR domains and important for Hsp70/Hsp90 binding, as defined previously (23). B, expression pattern of spag mRNA (left panel) and pattern of Spag protein accumulation (right panel) during Drosophila development. RNAs and proteins were extracted from (E) embryos with age in hours after egg laying indicated above the lanes (L1, L2, and L3) first, second, and third instar larvae, (P) pupae, (F) female, and (M) male 3-days old imagos. The Northern blot was hybridized with either 32P-labeled spag or β-tubulin cDNA. The Western blot was probed with anti-Spag or anti-ribosomal P40 protein polyclonal antibodies. The β-tubulin cDNA and anti-P40 antibodies were used for loading control.

Article Snippet: Proteins were detected as follows: Rpb1 detected with mouse monoclonal PB7-C2 antibody; Rpb2 with goat S20 from Santa Cruz Biotechnology; Nop58 with polyclonal antibodies generated from rabbits immunized with an KKLQEVDSLWKEFETPEK peptide ( 14 ); p70 S6K with monoclonal antibody SC-9027 from Santa Cruz Biotechnology; phospho-Thr-398 p70 S6K with monoclonal antibody provided by Cell Signaling Technology (reference 9209); fibrillarin with monoclonal antibody 5821 from Abcam; tubulin with monoclonal 12G10 (Developmental Studies Hybridoma Bank, Iowa City, IA); polyclonal anti-15.5K antibodies from Santa Cruz Biotechnology (SC-86760); polyclonal anti-P40 antibodies have been described previously ( 15 ), and anti-FLAG M2 monoclonal antibody was from Sigma.

Techniques: Gene Expression, Binding Assay, Expressing, Northern Blot, Labeling, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries * ^♦

doi: 10.1074/jbc.M113.499608

Figure Lengend Snippet: Spag and Nufip are required for box C/D sno core protein stabilization. A, Western blot analysis of protein extracts from third instar larvae spagk12101, as compared with wild-type w1118 (Ct), showed a significant diminution in the content of Nop58 (Nop5) and 15.5K (Hoip) but not fibrillarin. Tubulin was used as a loading control. B, this phenomenon was also observed in pupae extracts from animals in which RNAi was induced by Gal4act5C to knock down spag (Gal4act5c/spagRNAi as compared with Gal4act5c/+). C, similar results were observed in pupae extracts from animals in which RNAi was induced against Nufip (C, Gal4act5c/nufipRNAi compared with w1118).

Article Snippet: Proteins were detected as follows: Rpb1 detected with mouse monoclonal PB7-C2 antibody; Rpb2 with goat S20 from Santa Cruz Biotechnology; Nop58 with polyclonal antibodies generated from rabbits immunized with an KKLQEVDSLWKEFETPEK peptide ( 14 ); p70 S6K with monoclonal antibody SC-9027 from Santa Cruz Biotechnology; phospho-Thr-398 p70 S6K with monoclonal antibody provided by Cell Signaling Technology (reference 9209); fibrillarin with monoclonal antibody 5821 from Abcam; tubulin with monoclonal 12G10 (Developmental Studies Hybridoma Bank, Iowa City, IA); polyclonal anti-15.5K antibodies from Santa Cruz Biotechnology (SC-86760); polyclonal anti-P40 antibodies have been described previously ( 15 ), and anti-FLAG M2 monoclonal antibody was from Sigma.

Techniques: Western Blot, Control, Knockdown