Journal: eLife

Article Title: Induction of osteogenesis by bone-targeted Notch activation

doi: 10.7554/eLife.60183

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , iScript cDNA Synthesis Kit , BIO-RAD , Cat# 170–8891 , .

Techniques: Recombinant, Variant Assay, Plasmid Preparation, Sequencing, Control, Gene Expression, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Amplification, Magnetic Beads, Protease Inhibitor, Western Blot, Software, Staining

Journal: Journal of cell science

Article Title: Dissociation of β2m from MHC class I triggers formation of noncovalent transient heavy chain dimers.

doi: 10.1242/jcs.259498

Figure Lengend Snippet: Fig. 1. MHC I HC–HC association requires dissociation of β2m but no disulfide bonds. (A) Schematic of MHC class I states at the plasma membrane. Dissociation of peptide from the HC–β2m–peptide trimer results in an ‘empty’ HC–β2m heterodimer. Dissociation of β2m then produces free HCs (FHCs), which can form FHC associations (HC–HC dimer and oligomer shown). Other forms such as disulfide-linked dimers are known depending on the allotype (see the text). (B) Schematic representation of the two-hybrid antibody micropattern assay. Cells expressing a class I HC GFP fusion (green) and an N-terminally HA-tagged class I HC (gray) are seeded onto glass slides that are printed with micrometer-sized patterns of fluorescently labeled anti-HA antibodies. Dissociation of β2m generates FHCs of both constructs, which diffuse freely in the plasma membrane and eventually associate with each other to form HC–HC dimers (center) or oligomers (not shown). The HC–HC associations, which contain both HA-tagged and GFP-fused FHCs, localize in the pattern elements and are visible as pattern- shaped GFP fluorescence on the plasma membrane. (C) Representative fluorescence micrograph showing one single STF1 cell expressing both HA-tagged and GFP-fused H-2Kb in phase contrast (left), the purple anti-HA antibody pattern on the glass slide (middle), and green Kb–GFP colocalizing with the antibody pattern (right). The arrows point to the fluorophore observed in the respective panel and emphasize the plane of the image. Scale bar: 20 μm. (D) Interaction (arrow) occurs between HA-tagged Kb and Db–GFP FHCs (37°C) but not between HC–β2m heterodimers (25°C). Scale bars: 20 μm. (E) HC–HC interaction does not involve intracellular disulfide bond formation, since the FHCs (37°C) of HA–Kb and Kb(C332S)–GFP, which lacks the cytosolic cysteine, interact in the micropattern assay (arrow). Scale bars: 20 μm. (F) HA-B*27:05 (B27), but not Kb, forms covalent dimers. HA–Kb (Kb in the label) and B27 molecules were immunoprecipitated from the lysate of transduced STF1 cells with an anti-HA monoclonal antibody, separated by reducing (+ DTT) or nonreducing (– DTT) SDS- PAGE, and monomers (heavy chain) and covalent homodimers as indicated were detected by western blotting with an anti-HA antiserum. * denotes a background band. (G) There is no interaction of Kb–GFP with F pocket-stabilized HA–Kb(Y84C/A139C), a disulfide-stabilized Kb variant with increased β2m affinity. Scale bars: 20 μm. Images in C–G are representative of at least three independent experiments with the exception of F, which was performed twice.

Article Snippet: MHC molecules were visualized on the membranes with polyclonal rabbit anti-HA antibody as primary antibody (1:1000, ab9110, Abcam, Cambridge, UK) and alkaline phosphatase-conjugated anti-rabbitIgG serum from goat as secondary antibody (1706518, Biorad, Munich, Germany).

Techniques: Clinical Proteomics, Membrane, Expressing, Labeling, Construct, Fluorescence, Immunoprecipitation, SDS Page, Western Blot, Variant Assay

Journal:

Article Title: Amino Acid Substitutions at Ambler Position Gly238 in the SHV-1 ?-Lactamase: Exploring Sequence Requirements for Resistance to Penicillins and Cephalosporins

doi: 10.1128/AAC.46.12.3971-3977.2002

Figure Lengend Snippet: Western blot and slot blot of SHV-1 β-lactamase and mutants at the 238 position. The Western blot and slot blot were probed with 1 μg of anti-SHV-1 antibody/ml and horseradish peroxidase-conjugated protein G (7). Single-letter designations for the Gly238 mutant β-lactamases are listed above each slot. SK− represents the strain E. coli DH10B with the vector pBCSK(−) without the SHV β-lactamase. (Inset) Western blot of SHV-1 and Gly238Ala variant of the SHV β-lactamase. Equal amounts of purified β-lactamase were loaded in each lane. Lane 1, SHV-1; lane 2, Gly238Ala. As evaluated by densitometry, SHV-1 and Gly238Ala are nearly equivalent.

Article Snippet: Each of the strains possessing the 19 variants and the wild-type β-lactamase were assayed for in vivo steady-state expression levels by probing them with 1 μg/ml of purified anti-SHV antibody and horseradish peroxidase-conjugated protein G (Bio-Rad) as previously reported ( 7 ).

Techniques: Western Blot, Dot Blot, Mutagenesis, Plasmid Preparation, Variant Assay, Purification

Journal: Journal of Diabetes and Metabolic Disorders

Article Title: A case-control study to determination FBXW7 and Fetuin-A levels in patients with type 2 diabetes in Iraq

doi: 10.1007/s40200-021-00738-x

Figure Lengend Snippet: Clinical data in patients having diabetes mellitus (GI newly diagnosed DM, GII DM with metformin therapy) and control group

Article Snippet: While HbA1c was determined using Bio- Rad VARIANT Hemoglobin A1c programmer.

Techniques: Control, Significance Assay

Journal: Journal of Diabetes and Metabolic Disorders

Article Title: A case-control study to determination FBXW7 and Fetuin-A levels in patients with type 2 diabetes in Iraq

doi: 10.1007/s40200-021-00738-x

Figure Lengend Snippet: Correlation analysis of variables associated with serum FBXW7 protein level in the study populations

Article Snippet: While HbA1c was determined using Bio- Rad VARIANT Hemoglobin A1c programmer.

Techniques: Control