Journal: ACS Omega

Article Title: Modification of Fab Fragments by Dibromopyridazinediones Carrying Mono- and Double-Biotin Functionalities

doi: 10.1021/acsomega.2c04379

Figure Lengend Snippet: Preparation of mono-biotin Fab and double-biotin Fab according to Scheme . The top three images (a–c) show the results for mono-biotin Fab 9 and the bottom three images (d–f) show the results for double-biotin Fab 10 : non-reducing SDS-PAGE with Coomassie staining (a,d), Western blots with Cy3-conjugated antigen (b,e), and Western blots with Cy3-conjugated streptavidin (c,f). Lanes 1, 2, and 3 are starting Fab, reduced Fab, and biotin-labeled Fab, respectively. Lane M shows the molecular size standard with masses given in kDa.

Article Snippet: SDS-PAGE and subsequent Western blotting were performed as described above except that Cy3-ChromPure Mouse IgG, whole molecule (Jackson ImmunoResearch Laboratories, Inc.) (1.0 mg/gel) was used instead of Cy3–streptavidin to detect protein bands exerting anti-mouse IgG activity.

Techniques: SDS Page, Staining, Western Blot, Labeling

Journal: Nature Communications

Article Title: Abl family tyrosine kinases govern IgG extravasation in the skin in a murine pemphigus model

doi: 10.1038/s41467-019-12232-3

Figure Lengend Snippet: Kinetics of homeostatic IgG extravasation in the skin. a Manifestation of hair loss in mice 10 days after intravenous injection of AK18 or AK23 at 10 or 100 μg. b A representative flow cytometry plot and a histogram (the left and middle panels) to detect epidermal AK18 deposition in CD45 + E-cadherin + keratinocytes, and IgG1-MFI (the right panel) in keratinocytes 24 h after intravenous injection of 20 μg AK18 ( n = 4), 20 μg control mouse IgG1 (Ctrl-mIgG1) ( n = 4), or PBS ( n = 3). * P < 0.05 (a one-way ANOVA test). NS not significant. c A plot of epidermal IgG1-MFI against time after 100 μg of AK18 injection ( n = 2 for each time point). d A plot of AK18 injection dose against epidermal ΔIgG1-MFI (the changes in IgG1-MFI from PBS-injected sample) 24 h after AK18 injection ( n = 2 for each injection dose, two-tailed Spearman rank-correlation test). e Immunohistochemical evaluation in the mouse ear 24 h after intravenous injection of AK18 or AK23 at 10 or 100 μg, PBS, and 100 μg of control mouse IgG1 (Ctrl-mIgG1). Yellow arrows represent deposition of mouse IgG (mIgG) in keratinocytes. Blue represents nuclei stained with DAPI. White dotted line represents the border between the epidermis or the hair bulb and the dermis. Scale bar = 20 μm. f Images of ear skin dermis 24 h after intravenous injection of A555-conjugated mIgG or PBS. Scale bar = 100 μm. g The percentage of dermal macrophages positive for the fluorescein evaluated by flow cytometry, 24 h after intravenous injection of A647-conjugated mIgG ( n = 6) or PBS ( n = 4). * P < 0.05 ( t -test). In each figure, the error bars represent the standard deviation of a data set

Article Snippet: To detect the extravasation of nonspecific IgG or dextran in the murine ears by flow cytometry, 100–200 μg of fluorescein (A647)-conjugated mouse IgG (Cat. 015-600-003, Jackson ImmunoResearch, West Grove, PA) and/or 1 mg of FITC-conjugated 150 kDa dextran (Sigma Aldrich) was intravenously injected via tail vein to mice or was intradermally injected to the ears of mice.

Techniques: Injection, Flow Cytometry, Control, Two Tailed Test, Immunohistochemical staining, Staining, Standard Deviation

Journal: Nature Communications

Article Title: Abl family tyrosine kinases govern IgG extravasation in the skin in a murine pemphigus model

doi: 10.1038/s41467-019-12232-3

Figure Lengend Snippet: Homeostatic IgG extravasation in the skin relies on the transcellular pathway and dynamin-dependent endocytic vesicles in BECs. a Time-lapse images of paracellular IgG leakage (15 min after intravenous injection of fluorescein-conjugated IgG via the tail vein) in the ear dermis (the upper panels) exhibited in the rainbow color scale. Scale bar = 150 μm. Kinetics of MFI in the blood vessel area (red) and in the interstitium area (blue) in the dermis are plotted in the lower panels. b Representative flow cytometry plots for the identification of injected AK18 (dotted-line box) in dermal BECs of the mouse ear by flow cytometry. c The % frequency of IgG1-positive BECs 24 h after intravenous AK18 injection ( n = 7) or PBS ( n = 5), compared with isotype-stained control. * P < 0.05 ( t -test). d Electron microscopic images of dermal BECs in the ear skin evaluated in VE-cadherin-CreERT2; D1D2-floxed mice in homeostatic condition. Red arrowheads show intracellular vesicles. Yellow arrows show interendothelial junctions. Scale bar = 500 nm. e, f Epidermal IgG1-MFI ( e ) and the % frequency of IgG1-positive BECs ( f ) 24 h after 100 μg of intravenous AK18 injection or PBS in VE-cadherin-CreERT2; D1D2-floxed mice ( n = 5 for Cre − or n = 4 for Cre + , both ears were evaluated separately in e ). * P < 0.05 ( t -test). g, h The ear thickness changes ( g ) and epidermal IgG1-MFI ( h ), 24 h after 20 μg of intravenous AK18 injection or PBS and topical PMA application to the ears, in VE-cadherin-CreERT2; D1D2-floxed mice ( n = 6 for Cre − and n = 5 for Cre + ). In each figure, the error bars represent the standard deviation of a data set

Article Snippet: To detect the extravasation of nonspecific IgG or dextran in the murine ears by flow cytometry, 100–200 μg of fluorescein (A647)-conjugated mouse IgG (Cat. 015-600-003, Jackson ImmunoResearch, West Grove, PA) and/or 1 mg of FITC-conjugated 150 kDa dextran (Sigma Aldrich) was intravenously injected via tail vein to mice or was intradermally injected to the ears of mice.

Techniques: Injection, Flow Cytometry, Staining, Control, Standard Deviation

Journal: Nature Communications

Article Title: Abl family tyrosine kinases govern IgG extravasation in the skin in a murine pemphigus model

doi: 10.1038/s41467-019-12232-3

Figure Lengend Snippet: Imatinib reduces IgG extravasation in the skin. a IgG1-MFI of ear epidermis 24 h after 5–20 μg of AK18 injection and topical PMA to the ears, pretreated with imatinib (6000 μg body −1 ) or vehicle ( n = 4, respectively). * P < 0.05 ( t -test). b The ear thickness changes 24 h after topical PMA to the ears pretreated imatinib (6000 μg body −1 ) or vehicle ( n = 6, each. Both ears were evaluated separately). c IgG1-MFI of ear epidermis 24 h after 100 μg of AK18 injection, pretreated with imatinib (0–6000 μg body −1 ) or vehicle ( n = 4, respectively). d IgG1-MFI of ear epidermis 24 h after 50–200 μg of AK18 injection pretreated with imatinib (6000 μg body −1 ) ( n = 3 or 4, respectively). * P < 0.05 ( t -test). e Serum AK18 levels 24 h after 100 μg of AK18 injection pretreated with imatinib (6000 μg body −1 ) or vehicle ( n = 3, each). f The left panel shows IgG1-positive cells in BECs 24 h after 100 μg of AK18 injection with imatinib (6000 μg body −1 ) or vehicle pretreatment. The right panel shows % frequency of IgG1-positive cells in BECs ( n = 6). * P < 0.05 ( t -test). g , h The whole mount image of the ear dermis ( g ) and the percentage of dermal macrophages evaluated by flow cytometry ( h ), positive for fluorescein after intravenous (i.v.) or intradermal (i.d.) fluorescein-conjugated mIgG injection, with imatinib (6000 μg body −1 ) or vehicle pretreatment ( n = 6 or 5 for i.v. and n = 6 for i.d., respectively). Scale bar = 100 μm * P < 0.05 ( t -test). In each figure, the error bars represent the standard deviation of a data set

Article Snippet: To detect the extravasation of nonspecific IgG or dextran in the murine ears by flow cytometry, 100–200 μg of fluorescein (A647)-conjugated mouse IgG (Cat. 015-600-003, Jackson ImmunoResearch, West Grove, PA) and/or 1 mg of FITC-conjugated 150 kDa dextran (Sigma Aldrich) was intravenously injected via tail vein to mice or was intradermally injected to the ears of mice.

Techniques: Injection, Flow Cytometry, Standard Deviation