Journal: Genome Research

Article Title: A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha

doi: 10.1101/gr.196220.115

Figure Lengend Snippet: LAP2alpha and Lamin A/C associate with eu- and heterochromatin. ( A , B ) Screenshot of Integrative Genomics Viewer (IGV) tracks of Chromosome 11 (mm9) representing log e ratios of ChIP/Input, scale is [−0.4;0.4] ( upper panel); peak regions identified by EDD ( mid panel); MEF LADs (GSE36132) and the RefSeq gene track ( lower panel). Chromatin was sonicated for ( A ) 12 or ( B ) 30 cycles. ChIPs were performed with antibodies against LAP2alpha (8C10-1H11) and lamin A/C (3A6-4C11 and N18). ( C ) MEF LAD overlap. Degree of overlap (% of bp) of EDD peaks with MEF LADs. Error bars indicate the interval that contains 95% of all mean overlaps obtained through random permutation tests. All overlaps are significantly smaller or larger than expected under the null model ( P < 10 −4 ). ( D ) Gene density. Average gene density in EDD/DamID peak regions. Significance of gene density change from 12- to 30-cycle samples was tested using the Wilcoxon rank-sum test. (*) P < 10 −3 . ( E ) Histone marks in lamin-interacting sites. Average log e (ChIP/Input) of H3K4me3, H3K9ac, H3K27me3, and H3K9me3 in regions associated with lamin A/C and lamin B1. Differences between distributions of values from 12- to 30-cycle samples were tested by the Wilcoxon rank-sum test; green asterisk signifies P < 0.05, black asterisk P < 10 −5 , and red asterisk P < 10 −15 . ( F ) Extent of overlap between EDD peaks of LAP2alpha, lamin A/C, and lamin B1 after 12 and 30 cycles of sonication; numbers indicate Mb.

Article Snippet: Lamin A/C (3A6-4C11, Active Motif 39287, hybridoma supernatant, 100 μL for ChIP and Co-IP, 1:500 for WB, 1:100 for IF); lamin A/C (N18, Santa Cruz sc-6215, 10 μg for ChIP and Co-IP, 1:100 for WB, 1:50 for IF); lamin B1 (Proteintech 12987-1-AP, 3 μg for ChIP, 1:1000 for WB, 1:1000 for IF); mouse LAP2alpha (MFPL Monoclonal Antibody Facility, 8C10-1H11, hybridoma supernatant, 50 μL for ChIP and Co-IP, 1:500 for WB, 1:50 for IF; [specificity validated in Supplemental Fig. 3]); LAP2alpha (245.2, Abcam ab5162, 1:1000 for IF); H3K9me3 (Abcam ab8898, 3 μg for ChIP); H3K4me3 (Millipore 07-473, 3 μg for ChIP); H3K9ac (Millipore 06-942, 3 μg for ChIP); H3K27me3 (Millipore 07-449, 3 μg for ChIP); mouse normal IgG (Millipore 12-371, 10 μg for ChIP); rabbit normal IgG (Abcam ab46540, 10 μg for Co-IP).

Techniques: Sonication

Journal: Genome Research

Article Title: A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha

doi: 10.1101/gr.196220.115

Figure Lengend Snippet: LAP2alpha and Lamin A/C overlap in euchromatic regions. ( A ) Venn diagrams of genome-wide overlapping EDD peaks (Mb) of lamin B1 and lamin A/C samples upon 12 and 30 cycles of sonication. Overlaps (hatched) of lamin A/C-3A6 and -N18 with lamin B1 and overlaps of lamin A/C-3A6 with -N18 are highlighted in light blue (12 cycles) and light orange (30 cycles), and the corresponding % of overlap of EDD peaks are depicted as bar graphs. ( B ) Confocal immunofluorescence microscopic images of a mixed culture of Tmpo WT and KO mouse dermal fibroblasts double-stained for LAP2alpha and lamins as indicated. Unstained nuclei in LAP2alpha panels are outlined by a white dashed line. Scale bar, 10 µm. ( C ) Co-IPs for lamin A/C-3A6, lamin A/C-N18, and LAP2alpha were performed in Tmpo WT imMDFs. Normal rabbit IgG was used as a negative control. ( D ) Venn diagrams of genome-wide overlapping EDD peaks (Mb) of LAP2alpha and Lamin A/C samples upon 12 and 30 cycles of sonication. Overlaps (hatched) of LAP2alpha with lamin A/C-3A6 and -N18 are highlighted in light blue (12 cycles) and light orange (30 cycles), and the corresponding % of overlap of EDD peaks are depicted as bar graphs.

Article Snippet: Lamin A/C (3A6-4C11, Active Motif 39287, hybridoma supernatant, 100 μL for ChIP and Co-IP, 1:500 for WB, 1:100 for IF); lamin A/C (N18, Santa Cruz sc-6215, 10 μg for ChIP and Co-IP, 1:100 for WB, 1:50 for IF); lamin B1 (Proteintech 12987-1-AP, 3 μg for ChIP, 1:1000 for WB, 1:1000 for IF); mouse LAP2alpha (MFPL Monoclonal Antibody Facility, 8C10-1H11, hybridoma supernatant, 50 μL for ChIP and Co-IP, 1:500 for WB, 1:50 for IF; [specificity validated in Supplemental Fig. 3]); LAP2alpha (245.2, Abcam ab5162, 1:1000 for IF); H3K9me3 (Abcam ab8898, 3 μg for ChIP); H3K4me3 (Millipore 07-473, 3 μg for ChIP); H3K9ac (Millipore 06-942, 3 μg for ChIP); H3K27me3 (Millipore 07-449, 3 μg for ChIP); mouse normal IgG (Millipore 12-371, 10 μg for ChIP); rabbit normal IgG (Abcam ab46540, 10 μg for Co-IP).

Techniques: Genome Wide, Sonication, Immunofluorescence, Staining, Negative Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Sphingomyelinase-Like Phosphodiesterase 3b Expression Levels Determine Podocyte Injury Phenotypes in Glomerular Disease

doi: 10.1681/ASN.2013111213

Figure Lengend Snippet: In vitro effects of suPAR treatment of human podocytes on SMPDL3b expression levels and β3 integrin activation. (A) Treatment of human podocytes with different concentrations of suPAR (0, 0.5, 1, and 2 μg/ml) for different time spans (3, 6, and 24 hours) indicates that suPAR concentrations and time of exposure to suPAR do not change SMPDL3b mRNA expression levels. (B) Western blot analysis followed by (C) quantitative analysis of normal human podocytes treated with different concentrations of suPAR (0, 0.5, and 1 μg/ml) for 6 and 24 hours reveal significant β3 integrin activation at 24 hours of treatment (*P<0.05). AP5 antibody was used to detect activated β3 integrin and AP3 antibody was used to detect total β3 integrin. (D) Normal human podocytes treated with the sera from patients with DKD (top panel) show a characteristic cytoskeletal remodeling phenotype in the form of cell blebbing. The same cytoskeletal phenotype was observed when SMPDL3b OE podocytes were treated with suPAR (bottom panel) but not when human podocytes expressing endogenous levels of SMPDL3b (middle panel) were exposed to suPAR. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: AP5 and AP3 monoclonal mouse antibodies (Gen-Probe), polyclonal anti-rabbit synaptopodin (Santa Cruz Biotechnology), or active RhoA mouse mAb (New East Biosciences) were used.

Techniques: In Vitro, Expressing, Activation Assay, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Sphingomyelinase-Like Phosphodiesterase 3b Expression Levels Determine Podocyte Injury Phenotypes in Glomerular Disease

doi: 10.1681/ASN.2013111213

Figure Lengend Snippet: Decreased SMPDL3b expression is associated with β3 integrin activation, resulting in a migratory FSGS-like phenotype in podocytes. (A) Human podocytes were incubated for 24 hours with or without suPAR (1 μg/ml). suPAR treatment (S) significantly increased AP5 and phospho-Src protein expression in podocytes, and these changes are more prominent in suPAR-treated human SMPDL3b KD podocytes compared with control podocytes. However, human SMPDL3b OE podocytes are protected from suPAR-induced increases in AP5 (activated β3 integrin) and Src expression (*P<0.05). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Immunofluorescence staining using antibodies for AP5 (green) and AP3 (red) reveals that human SMPDL3b KD podocytes are characterized by activation of β3 integrin at baseline compared with normal podocytes. Furthermore, although β3 integrin can be activated by suPAR in control podocytes, human SMPDL3b OE podocytes are protected from suPAR-induced β3 integrin activation (*P<0.05; **P<0.01). MFI, mean fluorescence intensity. (C) Migration assay showing that suPAR-induced cell migration is significantly increased in control human podocytes and even more accentuated in human SMPDL3b KD podocytes (*P<0.05; **P<0.01). However, suPAR does not increase migration in human SMPDL3b OE podocytes. (D) Rac1 activity is significantly increased in suPAR-treated human SMPDL3b KD podocytes compared with control podocytes and SMPDL3b OE podocytes (*P<0.05, control versus SMPDL3b KD podocytes; #P<0.05, control versus SMPDL3b OE podocytes).

Article Snippet: AP5 and AP3 monoclonal mouse antibodies (Gen-Probe), polyclonal anti-rabbit synaptopodin (Santa Cruz Biotechnology), or active RhoA mouse mAb (New East Biosciences) were used.

Techniques: Expressing, Activation Assay, Incubation, Immunofluorescence, Staining, Fluorescence, Migration, Activity Assay

Journal: The EPMA journal

Article Title: Identifying potential signatures for atherosclerosis in the context of predictive, preventive, and personalized medicine using integrative bioinformatics approaches and machine-learning strategies.

doi: 10.1007/s13167-022-00289-y

Figure Lengend Snippet: Fig. 5 a The diagnostic power of DHRS9, PTPRJ, and combined in atherosclerosis by ROC curve. b–d The expressions of DHRS9 and PTPRJ in GSE100927 (b), GSE43292 (c), and GSE28829 (d). DHRS9, dehydrogenase/reductase 9; PTPRJ, protein tyrosine phosphatase receptor type J

Article Snippet: For immunofluorescence staining, the samples were incubated overnight at 4 °C, and stained with DHRS9 polyclonal antibody (1:300; Cat# 14,560–1-AP; Proteintech), and a Cy3-labeled polyclonal secondary antibody(1:500; Cat# G1223; Servicebio).

Techniques: Diagnostic Assay

Journal: The EPMA journal

Article Title: Identifying potential signatures for atherosclerosis in the context of predictive, preventive, and personalized medicine using integrative bioinformatics approaches and machine-learning strategies.

doi: 10.1007/s13167-022-00289-y

Figure Lengend Snippet: Fig. 6 The multifactor regulatory network based on DHRS9 and PTPRJ. DHRS9, dehydrogenase /reductase 9; PTPRJ protein tyrosine phos- phatase receptor type J

Article Snippet: For immunofluorescence staining, the samples were incubated overnight at 4 °C, and stained with DHRS9 polyclonal antibody (1:300; Cat# 14,560–1-AP; Proteintech), and a Cy3-labeled polyclonal secondary antibody(1:500; Cat# G1223; Servicebio).

Techniques:

Journal: Journal of proteomics

Article Title: Comparative proteome analysis of lung tissue from patients with idiopathic pulmonary fibrosis (IPF), non-specific interstitial pneumonia (NSIP) and organ donors.

doi: 10.1016/j.jprot.2013.04.033

Figure Lengend Snippet: Fig. 3 – Expression analysis of leucine aminopeptidase LAP3 in lung tissue from patients with fibrotic NSIP and IPF in comparison to Control lungs. Representative immunohistochemistry for proSP-C (A–D), LAP3 (E-I), clara cell-protein 10 [CC10] (J), FoxJ1 (K) and cytokeratin-5 [KRT5] (L) in serial sections of fNSIP- and IPF lung tissues, and for LAP3 (M, N) and proSP-C (O, P) in control lungs. (E, F, G, H) Type-II alveolar epithelial cells (AECII) in areas of thickened alveolar septae in fibrotic NSIP lungs (fNSIP; E, F) as well as AECII in regions of dense fibrotic remodelling in IPF lungs (G, H) reveal robust overexpression of LAP3. AECII are indicated by proSP-C staining (A–D) and arrows in A–H. LAP3 immunostaining is also observed in alveolar macrophages (indicated by hashmark in G and H). (I, J, K, L) Ciliated bronchial cells in IPF lungs express also LAP3 (I), as indicated by dashed arrows and immunostaining of serial sections with the marker protein FoxJ1 (K). Clara cells (marked by CC10 staining in Fig. J) do not express LAP3. The same observations can be made in control lungs (not shown) (M, N) AECII of control lungs (as indicated by proSP-C staining in parallel sections O and P) reveal a basal level of LAP3 expression. Original magnification of photomicrographs A, C, E, G: ×200 (bar = 100 μm); original magnification of photomicrographs B, D, F, H, I–L and M–P: ×400 (bar = 50 μm).

Article Snippet: In the following, the primary antibodies used for IHC are listed, including the sources and dilutions: rabbit polyclonal for human proSP-C (1:750, Millipore), rabbit monoclonal for cytokeratin-5 [KRT5] (1:200, abcam), rabbit polyclonal for human PPIA (1:100, Santa Cruz Biotechnology Inc.), mouse monoclonal for human LAP3 (1:100, Santa Cruz B.

Techniques: Expressing, Comparison, Control, Immunohistochemistry, Over Expression, Staining, Immunostaining, Marker