Journal:

Article Title: Upregulation of CD40 Expression on Endothelial Cells Infected with Human Cytomegalovirus

doi: 10.1128/JVI.76.24.12803-12812.2002

Figure Lengend Snippet: Immunoblot analysis of CD40 in whole-cell lysates. Cell monolayers were infected with HCMV TB40E at an MOI of 1 (inf.) or mock infected (noninfected [n.i.]). Cell lysates were prepared at the indicated times and analyzed by immunoblotting. (A) CD40 protein levels were detected with a polyclonal rabbit serum directed against human CD40. (B) Blots from panel A were stripped and reprobed with a rabbit serum directed against calreticulin. (C) Signal intensities from panels A and B were densitometrically quantified; data represent the ratio of CD40 to calreticulin (y axis) versus time p.i. (x axis). (D) Protein lysates from infected or noninfected cells were treated with N-glycosidase F (+) or left untreated (−) prior to immunoblot analysis with anti-CD40 antibodies. Molecular masses are given in kilodaltons.

Article Snippet: The following monoclonal antibodies or polyclonal sera were used: mouse IgG1-fluorescein isothiocyanate (FITC)-conjugated anti-human CD54 (Calbiochem, La Jolla, Calif.), anti-human major histocompatibility complex class I (MHC-I) (BD PharMingen, San Diego, Calif.), mouse IgG1-FITC anti-human CD40 (Dianova, Hamburg, Germany), mouse IgG1-FITC anti-human CD62E (Calbiochem), antibodies G28.5 and Ro1 (anti-human CD40) (gifts from H. Engelmann, Institute for Immunology, University of Munich, Munich, Germany) ( 39 ), polyclonal rabbit anti-CD40 serum (Santa Cruz Biotechnology), rabbit antiserum against human calreticulin (Dianova), biotin-conjugated rabbit anti-mouse antibodies, and biotinylated swine anti-rabbit antibodies (Dako, Hamburg, Germany).

Techniques: Western Blot, Infection

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Induction of apoptosis in 70Z/3 cells treated with 3CPA at 250 μ M and 500 μ M . ( A ) Cytochrome c release into the cytosol after exposure to 3CPA for indicated time periods measured by Western blot analysis. ( B ) Pro-caspase-9 processing determined in cytosolic fractions after exposure to 3CPA. Actin was used as an internal standard of the protein load and the results shown are representatives of two separate experiments. ( C ) Caspase inhibitors reduce 3CPA-induced apoptosis in 70Z/3 cells. Apoptosis, measured as AnnexinV + 7AAD − cells, was determined after exposure to 3CPA at 250 μ M (left) and 500 μ M (right) for 24 h. The pan-caspase inhibitor ZVAD-fmk (50 μ M , n =4), the caspase-9 inhibitor LEHD-fmk (50 μ M , n =3), or the caspase-8 inhibitor IETD-fmk (50 μ M , n =3) were added 1 h before treatment with 3CPA. The level of apoptosis is expressed relative to the level in cell cultures treated with the inhibitor only. Results show mean±s.d., and (*) indicates a statistical difference compared to 3CPA only (paired t -test, P <0.05).

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Western Blot

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Over-expression of Bcl-2 in 70Z/3 cells inhibits 3CPA-induced apoptosis. ( A ) FACS-analysis of 70Z/3 (upper panel) and 70Z/3 Bcl-2+ (lower panel) cells after exposure to 3CPA (500 μ M ) for 18 h. 7AAD negative (viable) cells were gated (gate A) and analysed for Annexin V + expression (% of gated cells). ( B ) Levels of Bcl-2 and cytochrome c expression were determined by Western blot analysis in mitochondrial enriched fractions after 18 h incubation with 3CPA at the indicated concentrations (30 μg protein per lane). ( C ) Pro-caspase-9 processing into active caspase-9 was determined in the cytosolic fraction after 18 h incubation with 3CPA at the indicated concentrations. Actin was used as an internal standard to control the amount of cytosolic protein loaded in each lane. Results shown are representatives of two independent experiments.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Over Expression, Expressing, Western Blot, Incubation

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: N-substituted benzamides induce cytochrome c release and activation of caspase-9 in HL60 cells. ( A ) Cytochrome c release induced by MCA (500 μ M ) and 3CPA (250 μ M ) in HL60 cells after 24 and 48 h, measured by Western blot analysis of mitochondria or cytosol fractions (30 μg protein/lane). Cells were incubated with etoposide for 6 and 18 h at 100 μ M as a positive control. ( B ) Activation of caspase-9 after treatment of HL60 cells with 3CPA at 250 and 500 μ M for 12 and 18 h (48 μg protein per lane). One representative experiment out of two is shown.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Activation Assay, Western Blot, Incubation, Positive Control

Journal: Nucleic Acids Research

Article Title: Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

doi: 10.1093/nar/gkn895

Figure Lengend Snippet: Photo-cross-linking reactions between hRPA and Oligo 31S and identification of complexes by immunoblotting. ( A ) 32 P-labeled Oligo 31S (2 nM) were mixed with the indicated amounts of hRPA then irradiated at 15°C for 45 min. SDS-PAGE (10%) were used to separate multiple cross-linked species. Cross-linked species (arrows) were detected and quantified by the phosphorimager—Molecular Dynamic. ( B ) After electrophoresis and transfer onto a nitrocellulose membrane, DNA was removed by incubation of the membrane with DNase I. ECL western blotting used anti-RPA1 (AbRPA1), anti-RPA2 (AbRPA2) and anti-RPA3 (AbRPA3) antibodies to reveal the cross-linked RPA1, RPA2 and RPA3 subunits, respectively. The immunoblot shown here identifies each subunit implicated in photo-cross-linking with Oligo 31S 29 and 10 nM of hRPA (lanes 1). For comparison, free hRPA (10 nM) was loaded (lanes 2).

Article Snippet: Rabbit anti-RPA1 polyclonal antibodies were obtained from Chemicom International.

Techniques: Western Blot, Labeling, Irradiation, SDS Page, Electrophoresis, Membrane, Incubation, Comparison

Journal: Nucleic Acids Research

Article Title: Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

doi: 10.1093/nar/gkn895

Figure Lengend Snippet: Quantification of cross-linked species. The relative amounts of cross-linked species involving RPA1 (red, long dashed line), RPA2 (blue, medium dashed line) and RPA3 (green, short dashed line) subunits were quantified for each hRPA concentration from the hRPA-Oligo 31S gel ( A). Error bar is ±20%. The results obtained for 2 nM of hRPA were not reported since no cross-links were observed except for Oligo 31S 29 . To correlate the nature of the complexes with the presence of the subunit on the DNA, the relative yield of each complex was taken from data in B. The same scales are used for each graph, but to simplify the figure only scales for the Oligo 31S 3 graph are shown.

Article Snippet: Rabbit anti-RPA1 polyclonal antibodies were obtained from Chemicom International.

Techniques: Concentration Assay

Journal: Nucleic Acids Research

Article Title: Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

doi: 10.1093/nar/gkn895

Figure Lengend Snippet: Schematic models of the 30, 13–22 and 8–10 nt binding modes. The s 4 T substituted positions are represented by dashed lines. RPA1 is represented by a red bubble, RPA2 by a blue bubble and RPA3 by a green bubble.

Article Snippet: Rabbit anti-RPA1 polyclonal antibodies were obtained from Chemicom International.

Techniques: Binding Assay

Journal: The Journal of Liquid Biopsy

Article Title: Circulating epithelial cell as viral infection and tissue origin marker in patients with severe COVID-19

doi: 10.1016/j.jlb.2023.100005

Figure Lengend Snippet: Antibodies and fluorophores included in the study design, channels used for detection and cell lines used as controls.

Article Snippet: The hAELVi cell line was used as a positive control for RAGE protein (Rabbit polyclonal anti-RAGE-AF750, bs-0177R-A750, Bioss Inc), a specific lung tissue marker.

Techniques: Staining

Journal: bioRxiv

Article Title: Single-cell transcriptional landscape of temporal neutrophil response to burn wound in larval zebrafish

doi: 10.1101/2024.04.01.587641

Figure Lengend Snippet: (A) Schematic view of validating signatures identified from zebrafish myeloid subsets in human burn patient expression profile. (B) Expression level distribution of Immature_Neuts signatures (top panel) and lgals3bpb_Mac1 (bottom panel) by patient TBSA shown as raincloud plots. Paired T-tests were performed between the Low and the Very High TBSA groups. (C) Expression distribution of GPR84 and GYG1 in patients by TBSA level. Spearman correlation (ρ) is calculated between gene expression and TBSA level. (D) Representative flow plots (left) and quantifications of frequencies shown as percentages (right) of GPR84+ neutrophils in burn (n = 6) and healthy (n = 4) blood. Paired samples t-test, p = 0.022.

Article Snippet: Protein-targeted staining was done with the following antibodies in Cell Staining Buffer: Alexa Fluor 700-conjugated anti-human CD16 at 1:400, PE/Fire-640-conjugated anti-human CD66b at 1:400, PerCP-conjugated anti-human CD3 at 1:200, PerCP-conjugated anti-human CD19 at 1:200, PerCP-conjugated anti-human CD56 at 1:400, PerCP-conjugated anti-human CD203c at 1:100 (BioLegend), BUV395-conjugated mouse anti-human CD45 at 1:100 (BD Biosciences), and PE-conjugated GPR84 at 1:200 (Alomone Labs).

Techniques: Expressing, Gene Expression