goat anti mouse igg1 cross adsorbed secondary antibody  (Thermo Fisher)


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    Name:
    Goat anti Mouse IgG1 Cross Adsorbed Secondary Antibody
    Description:
    Goat anti Mouse IgG1 Cross Adsorbed Secondary Antibody for Western Blot IF ICC IHC Flow IP
    Catalog Number:
    31236
    Price:
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    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher goat anti mouse igg1 cross adsorbed secondary antibody
    TonEBP interacts with METTL3 and m6A methylase. ( A ) The TonEBP interactome includes METTL3 and R-loop-related proteins. ( B ) HEK293T cell lysates were immunoprecipitated with normal serum (Serum) or anti-TonEBP antibody (TonEBP). Precipitates and cell lysates were blotted for TonEBP and METTL3. ( C ) Cell lysates were immunoprecipitated with normal rabbit <t>IgG</t> (IgG) or anti-METTL3 IgG (METTL3). ( D ) Domain structures of human TonEBP (WT), and deletion proteins ΔRHD and Yc1. ( E ) Domain structures of human METTL3 (WT) and deletion proteins 1–380, 1–200 and 381–580. ( F ) Cells were transfected with plasmids expressing Flag-METTL3 together with Myc-tagged TonEBP (WT), ΔRHD, or Yc1. After 24 h, cell lysates were prepared and immunoprecipitated using anti-FLAG antibody. ( G ) Cells were transfected with plasmids expressing Myc-Yc1 together with Flag-tagged METTL3 (WT), 1–380, 1–200 or 381–500 and immunoprecipitation was performed with Myc antibody 24 h later. ( H ) Amino-acid sequence alignment of the highly conserved and charged regions of TonEBP from seven species. 3M is a mutant Yc1 in which R, E and R were all exchanged to A shown in red and 5M is another mutant where K, R, and the three Ks were all replaced by A shown in green. ( I ) Cells were transfected with a plasmid expressing Flag-tagged Yc1, 3M or 5M. Cell lysates were prepared and immunoprecipitated with anti-FLAG antibody after 24 h incubation.
    Goat anti Mouse IgG1 Cross Adsorbed Secondary Antibody for Western Blot IF ICC IHC Flow IP
    https://www.bioz.com/result/goat anti mouse igg1 cross adsorbed secondary antibody/product/Thermo Fisher
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    goat anti mouse igg1 cross adsorbed secondary antibody - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution"

    Article Title: TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa1162

    TonEBP interacts with METTL3 and m6A methylase. ( A ) The TonEBP interactome includes METTL3 and R-loop-related proteins. ( B ) HEK293T cell lysates were immunoprecipitated with normal serum (Serum) or anti-TonEBP antibody (TonEBP). Precipitates and cell lysates were blotted for TonEBP and METTL3. ( C ) Cell lysates were immunoprecipitated with normal rabbit IgG (IgG) or anti-METTL3 IgG (METTL3). ( D ) Domain structures of human TonEBP (WT), and deletion proteins ΔRHD and Yc1. ( E ) Domain structures of human METTL3 (WT) and deletion proteins 1–380, 1–200 and 381–580. ( F ) Cells were transfected with plasmids expressing Flag-METTL3 together with Myc-tagged TonEBP (WT), ΔRHD, or Yc1. After 24 h, cell lysates were prepared and immunoprecipitated using anti-FLAG antibody. ( G ) Cells were transfected with plasmids expressing Myc-Yc1 together with Flag-tagged METTL3 (WT), 1–380, 1–200 or 381–500 and immunoprecipitation was performed with Myc antibody 24 h later. ( H ) Amino-acid sequence alignment of the highly conserved and charged regions of TonEBP from seven species. 3M is a mutant Yc1 in which R, E and R were all exchanged to A shown in red and 5M is another mutant where K, R, and the three Ks were all replaced by A shown in green. ( I ) Cells were transfected with a plasmid expressing Flag-tagged Yc1, 3M or 5M. Cell lysates were prepared and immunoprecipitated with anti-FLAG antibody after 24 h incubation.
    Figure Legend Snippet: TonEBP interacts with METTL3 and m6A methylase. ( A ) The TonEBP interactome includes METTL3 and R-loop-related proteins. ( B ) HEK293T cell lysates were immunoprecipitated with normal serum (Serum) or anti-TonEBP antibody (TonEBP). Precipitates and cell lysates were blotted for TonEBP and METTL3. ( C ) Cell lysates were immunoprecipitated with normal rabbit IgG (IgG) or anti-METTL3 IgG (METTL3). ( D ) Domain structures of human TonEBP (WT), and deletion proteins ΔRHD and Yc1. ( E ) Domain structures of human METTL3 (WT) and deletion proteins 1–380, 1–200 and 381–580. ( F ) Cells were transfected with plasmids expressing Flag-METTL3 together with Myc-tagged TonEBP (WT), ΔRHD, or Yc1. After 24 h, cell lysates were prepared and immunoprecipitated using anti-FLAG antibody. ( G ) Cells were transfected with plasmids expressing Myc-Yc1 together with Flag-tagged METTL3 (WT), 1–380, 1–200 or 381–500 and immunoprecipitation was performed with Myc antibody 24 h later. ( H ) Amino-acid sequence alignment of the highly conserved and charged regions of TonEBP from seven species. 3M is a mutant Yc1 in which R, E and R were all exchanged to A shown in red and 5M is another mutant where K, R, and the three Ks were all replaced by A shown in green. ( I ) Cells were transfected with a plasmid expressing Flag-tagged Yc1, 3M or 5M. Cell lysates were prepared and immunoprecipitated with anti-FLAG antibody after 24 h incubation.

    Techniques Used: Immunoprecipitation, Transfection, Expressing, Sequencing, Mutagenesis, Plasmid Preparation, Incubation

    2) Product Images from "Chromatin-remodelling factor Brg1 regulates myocardial proliferation and regeneration in zebrafish"

    Article Title: Chromatin-remodelling factor Brg1 regulates myocardial proliferation and regeneration in zebrafish

    Journal: Nature Communications

    doi: 10.1038/ncomms13787

    Brg1 represses cdkn1c expression by increasing the level of DNA methylation in its promoter region. ( a ) Methylation patterns of 10 individual CpG sites in the cdkn1c promoter of Tg( hsp70 :dn-xBrg1) and wild-type sibling hearts after daily heat shock from 5 to 14 d.p.a. Upper panel, schematic of 10 CpG island sites (set A) of the cdkn1c promoter region and transcription start site (TSS); lower panels, cdkn1c methylation patterns of wild-type sibling (sibling) and dn-xBrg1 transgenic (tg) hearts, with open circles for ‘unmethylated’ and filled circles for ‘methylated’ CpG islands. Methylated DNA sequences were obtained by bisulfite sequencing. Note decreased methylation of ckkn1c promoter in dn-xBrg1 transgenic hearts ( b ). ( c ) cdkn1c promoter methylation of 10 individual CpG sites (set A) of mock, 3 d.p.a. and 5 d.p.a. wild-type hearts. The percentages of unmethylated (white) and methylated (black) DNA from a and b are shown in b and d . ( e ) Left panel, ChIP assays with anti-Brg1 antibody. Right panel, quantitation of Brg1 immunoprecipitated cdkn1c promoter in wild-type mock, 3 d.p.a. and 5 d.p.a. hearts. Data are presented as Brg1 enrichment relative to control IgG. The 335 bp DNA fragment within the cdkn1c promoter region (−1,625 to −1,290 bp) was amplified from immunoprecipitated DNA of mock, 3 d.p.a. and 5 d.p.a. hearts by anti-Brg1 antibody or control IgG. ( f ) Immunoprecipitation by anti-Myc antibody in 293T cells over-expressing Brg1 and Myc-tagged Dnmt3ab. ( g ) Upper panel, immunoprecipitation of Brg1 and Myc-Dnmt3ab by Myc antibody or control IgG antibody in H9C2 cells over-expressing Brg1 and Myc-tagged Dnmt3ab. Lower panel, immunoprecipitation of dn-xBrg1-Flag and Myc-Dnmt3ab by Myc antibody or IgG antibody in H9C2 cells over-expressing dn-xBrg1-Flag and Myc-Dnmt3ab. ( h ) Luciferase reporter assays showed that over-expression of zebrafish brg1 and dnmt3ab synergistically suppressed the transcription of cdkn1c in 293T cells. 293T cells were transfected/infected with the indicated adenoviral constructs and luciferase reporter constructs, and those cells were then collected and measured for luciferase activity at 24 h after transfection/infection. Equal amounts of adenovirus were used for each group. Firefly luciferase activity was normalized by Renilla luciferase activity (* P
    Figure Legend Snippet: Brg1 represses cdkn1c expression by increasing the level of DNA methylation in its promoter region. ( a ) Methylation patterns of 10 individual CpG sites in the cdkn1c promoter of Tg( hsp70 :dn-xBrg1) and wild-type sibling hearts after daily heat shock from 5 to 14 d.p.a. Upper panel, schematic of 10 CpG island sites (set A) of the cdkn1c promoter region and transcription start site (TSS); lower panels, cdkn1c methylation patterns of wild-type sibling (sibling) and dn-xBrg1 transgenic (tg) hearts, with open circles for ‘unmethylated’ and filled circles for ‘methylated’ CpG islands. Methylated DNA sequences were obtained by bisulfite sequencing. Note decreased methylation of ckkn1c promoter in dn-xBrg1 transgenic hearts ( b ). ( c ) cdkn1c promoter methylation of 10 individual CpG sites (set A) of mock, 3 d.p.a. and 5 d.p.a. wild-type hearts. The percentages of unmethylated (white) and methylated (black) DNA from a and b are shown in b and d . ( e ) Left panel, ChIP assays with anti-Brg1 antibody. Right panel, quantitation of Brg1 immunoprecipitated cdkn1c promoter in wild-type mock, 3 d.p.a. and 5 d.p.a. hearts. Data are presented as Brg1 enrichment relative to control IgG. The 335 bp DNA fragment within the cdkn1c promoter region (−1,625 to −1,290 bp) was amplified from immunoprecipitated DNA of mock, 3 d.p.a. and 5 d.p.a. hearts by anti-Brg1 antibody or control IgG. ( f ) Immunoprecipitation by anti-Myc antibody in 293T cells over-expressing Brg1 and Myc-tagged Dnmt3ab. ( g ) Upper panel, immunoprecipitation of Brg1 and Myc-Dnmt3ab by Myc antibody or control IgG antibody in H9C2 cells over-expressing Brg1 and Myc-tagged Dnmt3ab. Lower panel, immunoprecipitation of dn-xBrg1-Flag and Myc-Dnmt3ab by Myc antibody or IgG antibody in H9C2 cells over-expressing dn-xBrg1-Flag and Myc-Dnmt3ab. ( h ) Luciferase reporter assays showed that over-expression of zebrafish brg1 and dnmt3ab synergistically suppressed the transcription of cdkn1c in 293T cells. 293T cells were transfected/infected with the indicated adenoviral constructs and luciferase reporter constructs, and those cells were then collected and measured for luciferase activity at 24 h after transfection/infection. Equal amounts of adenovirus were used for each group. Firefly luciferase activity was normalized by Renilla luciferase activity (* P

    Techniques Used: Expressing, DNA Methylation Assay, Methylation, Transgenic Assay, Methylation Sequencing, Chromatin Immunoprecipitation, Quantitation Assay, Immunoprecipitation, Amplification, Luciferase, Over Expression, Transfection, Infection, Construct, Activity Assay

    3) Product Images from "Lupus auto-antibodies act as positive allosteric modulators at NMDA receptors and induce spatial memory deficits"

    Article Title: Lupus auto-antibodies act as positive allosteric modulators at NMDA receptors and induce spatial memory deficits

    Journal: bioRxiv

    doi: 10.1101/791715

    Differential sensitivity of N2A- and N2B-containing NMDARs to DNRAbs. ( A B ) Moderate pathophysiological levels of DNRAbs (10 μg/mL) potentiate glutamate-activated currents in N2A-containing, but not in N2B-containing NMDARs. Upper panels , Whole-cell currents from HEK293 cells expressing human NMDAR subunits, either hGluN1/hGluN2A ( A ) or hGluN1/hGluN2B ( B ). Currents were elicited by a 2.5 s application of glutamate (1 mM) in the continuous presence of glycine (0.1 mM) (holding potential, −70 mV). Lower panels , control antibody B1 (IgG1, gray circles) or human-derived DNRAb G11 (green circles) were added 75 s after a baseline recording of 5 sweeps and were included in the bath throughout the remaining period. Current amplitudes for individual recordings were normalized to its baseline. Values are mean ± SEM (hN2A+B1, n = 6; hN2A+G11, n = 6; hN2B+B1, n = 5; hN2B+G11, n = 5). Example traces in upper panels show the +G11 recordings for the initial sweep during baseline (no antibody present) or for the last sweep during steady-state (in antibody). ( C D ) Peak current amplitudes in N2A-containing NMDAR are more strongly potentiated than those in N2B-containing receptors. Bar graphs (mean ± SEM with dots indicating individual values) (from left to right for hN1/hN2A, n = 6, 6, 6, 6, 5, 5; and for hN1/hN2B, n = 5, 5, 6, 5) showing normalized steady-state peak current amplitudes either for control antibody (B1) or DNRAbs (G11). Significance of DNRAb values are measured relative to their respective control (* p
    Figure Legend Snippet: Differential sensitivity of N2A- and N2B-containing NMDARs to DNRAbs. ( A B ) Moderate pathophysiological levels of DNRAbs (10 μg/mL) potentiate glutamate-activated currents in N2A-containing, but not in N2B-containing NMDARs. Upper panels , Whole-cell currents from HEK293 cells expressing human NMDAR subunits, either hGluN1/hGluN2A ( A ) or hGluN1/hGluN2B ( B ). Currents were elicited by a 2.5 s application of glutamate (1 mM) in the continuous presence of glycine (0.1 mM) (holding potential, −70 mV). Lower panels , control antibody B1 (IgG1, gray circles) or human-derived DNRAb G11 (green circles) were added 75 s after a baseline recording of 5 sweeps and were included in the bath throughout the remaining period. Current amplitudes for individual recordings were normalized to its baseline. Values are mean ± SEM (hN2A+B1, n = 6; hN2A+G11, n = 6; hN2B+B1, n = 5; hN2B+G11, n = 5). Example traces in upper panels show the +G11 recordings for the initial sweep during baseline (no antibody present) or for the last sweep during steady-state (in antibody). ( C D ) Peak current amplitudes in N2A-containing NMDAR are more strongly potentiated than those in N2B-containing receptors. Bar graphs (mean ± SEM with dots indicating individual values) (from left to right for hN1/hN2A, n = 6, 6, 6, 6, 5, 5; and for hN1/hN2B, n = 5, 5, 6, 5) showing normalized steady-state peak current amplitudes either for control antibody (B1) or DNRAbs (G11). Significance of DNRAb values are measured relative to their respective control (* p

    Techniques Used: Expressing, Derivative Assay

    4) Product Images from "A Formulated TLR7/8 Agonist is a Flexible, Highly Potent and Effective Adjuvant for Pandemic Influenza Vaccines"

    Article Title: A Formulated TLR7/8 Agonist is a Flexible, Highly Potent and Effective Adjuvant for Pandemic Influenza Vaccines

    Journal: Scientific Reports

    doi: 10.1038/srep46426

    HA Subtype Binding Profile Analysis of Sera From 3M-052 Immunized Mice. Mice were immunized twice with split H5N1 vaccine (SP-H5, A/VN/1203/04, Sanofi Pasteur) 28 days apart (D0, D28) in combination with adjuvants as indicated. Adjuvant dose was 1 or 2 μg 3M-052 in SE or liposomes, respectively. Sera was collected from mice (n = 7/group) at D63, and analyzed using a high density HA array. IgG1 and IgG2 subtype antibodies were simultaneously detected using a two-color microarray scanner ( A ). Global binding profiles of serum from mice immunized with different adjuvants were determined by plotting background subtracted median fluorescence values for all HA proteins in the array. The ability of 3M-052 to significantly increase antibody titer, indicated by an increase in median fluorescence, was determined by comparison of fluorescence levels observed within a formulation (SE, Liposomes) with and without 3M-052, and calculation of P-values corrected for false discovery rate (FDR) using the Benjamini Hochenberg method (BHp-value) ( B ). BH p-values
    Figure Legend Snippet: HA Subtype Binding Profile Analysis of Sera From 3M-052 Immunized Mice. Mice were immunized twice with split H5N1 vaccine (SP-H5, A/VN/1203/04, Sanofi Pasteur) 28 days apart (D0, D28) in combination with adjuvants as indicated. Adjuvant dose was 1 or 2 μg 3M-052 in SE or liposomes, respectively. Sera was collected from mice (n = 7/group) at D63, and analyzed using a high density HA array. IgG1 and IgG2 subtype antibodies were simultaneously detected using a two-color microarray scanner ( A ). Global binding profiles of serum from mice immunized with different adjuvants were determined by plotting background subtracted median fluorescence values for all HA proteins in the array. The ability of 3M-052 to significantly increase antibody titer, indicated by an increase in median fluorescence, was determined by comparison of fluorescence levels observed within a formulation (SE, Liposomes) with and without 3M-052, and calculation of P-values corrected for false discovery rate (FDR) using the Benjamini Hochenberg method (BHp-value) ( B ). BH p-values

    Techniques Used: Binding Assay, Mouse Assay, Microarray, Fluorescence

    H5N1 Cross-Clade Antibody Binding Analysis of Sera From Mice Immunized With 3M-052 Adjuvants. Mice were immunized twice with split H5N1 vaccine (A/VN/1203/04, Sanofi Pasteur) 28 days apart (D0, D28) in combination with adjuvants as indicated. Adjuvant dose was 1 or 2 μg 3M-052 in SE or liposomes, respectively. Sera was collected from mice at D63 (n = 7/group), and analyzed using a high density HA array. Specific binding of both IgG1 and IgG2c antibodies to H5N1 proteins from different clades is shown. Specific binding levels are determined by normalization of array data to determine the fold over control (FOC) binding for each H5N1 protein. Log2 transformed FOC data for both SE and liposomal formulations with or without 3M-052 are presented for both IgG1 and IgG2c antibody subtypes with error bars representing standard error of them mean (SEM). Significant increases in specific binding resulting from the inclusion of 3M-052 are determined by calculation of Benjamini-Hochenberg FDR corrected P-values (Q = 5%), with BH p-vales
    Figure Legend Snippet: H5N1 Cross-Clade Antibody Binding Analysis of Sera From Mice Immunized With 3M-052 Adjuvants. Mice were immunized twice with split H5N1 vaccine (A/VN/1203/04, Sanofi Pasteur) 28 days apart (D0, D28) in combination with adjuvants as indicated. Adjuvant dose was 1 or 2 μg 3M-052 in SE or liposomes, respectively. Sera was collected from mice at D63 (n = 7/group), and analyzed using a high density HA array. Specific binding of both IgG1 and IgG2c antibodies to H5N1 proteins from different clades is shown. Specific binding levels are determined by normalization of array data to determine the fold over control (FOC) binding for each H5N1 protein. Log2 transformed FOC data for both SE and liposomal formulations with or without 3M-052 are presented for both IgG1 and IgG2c antibody subtypes with error bars representing standard error of them mean (SEM). Significant increases in specific binding resulting from the inclusion of 3M-052 are determined by calculation of Benjamini-Hochenberg FDR corrected P-values (Q = 5%), with BH p-vales

    Techniques Used: Binding Assay, Mouse Assay, Transformation Assay

    5) Product Images from "PRMT5 is essential for B cell development and germinal center dynamics"

    Article Title: PRMT5 is essential for B cell development and germinal center dynamics

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07884-6

    Antibody response and GC defects caused by Prmt5 deficiency. a – i Cγ1-cre (Ctrl) and Prmt5 F/F Cγ1-cre (F/F) mice were used throughout. a Total anti-NP IgG1 in the serum of mice, measured by ELISA 14 days after NP-CGG immunization. Mean ± s.d. OD values for serial dilutions are plotted for n mice from three experiments. b Representative pictures of ELISPOT for NP-specific IgG1 antibody secreting cells (ASC) at day 14 post-immunization. The number of ASC of individual mice (symbols) and means (bars) are plotted. c Anti-NP IgG1 in the serum of mice at various times post-immunization. Mean ± s.d. values for n mice at each time point from two experiments are plotted. d Total levels of antibody isotypes in the serum of n non-immunized mice. e Mean + s.d. number of lymphocytes per spleen at day 14 post-immunization, enumerated by flow cytometry for n mice from two experiments. f Representative flow cytometry plots (gated on B220 + ) of splenic GC B cell proportions at 14 days post-immunization with NP-CGG. The number of GC B cells per spleen for individual mice (symbol) and medians (bars) from three experiments are plotted. g As in f , for MLN of mice infected with H. polygyrus for 14 days. Data from two experiments are plotted. h Mean + s.d. number of lymphocytes per MLN in the mice from g . i Representative IF in MLN from mice infected with H. polygyrus from g , stained for the indicated antigens. Scale bar, 100 µm. GC numbers per MLN scored in individual mice (symbols) are plotted to the right. p -Values throughout are by an unpaired, two-tailed Student's t test
    Figure Legend Snippet: Antibody response and GC defects caused by Prmt5 deficiency. a – i Cγ1-cre (Ctrl) and Prmt5 F/F Cγ1-cre (F/F) mice were used throughout. a Total anti-NP IgG1 in the serum of mice, measured by ELISA 14 days after NP-CGG immunization. Mean ± s.d. OD values for serial dilutions are plotted for n mice from three experiments. b Representative pictures of ELISPOT for NP-specific IgG1 antibody secreting cells (ASC) at day 14 post-immunization. The number of ASC of individual mice (symbols) and means (bars) are plotted. c Anti-NP IgG1 in the serum of mice at various times post-immunization. Mean ± s.d. values for n mice at each time point from two experiments are plotted. d Total levels of antibody isotypes in the serum of n non-immunized mice. e Mean + s.d. number of lymphocytes per spleen at day 14 post-immunization, enumerated by flow cytometry for n mice from two experiments. f Representative flow cytometry plots (gated on B220 + ) of splenic GC B cell proportions at 14 days post-immunization with NP-CGG. The number of GC B cells per spleen for individual mice (symbol) and medians (bars) from three experiments are plotted. g As in f , for MLN of mice infected with H. polygyrus for 14 days. Data from two experiments are plotted. h Mean + s.d. number of lymphocytes per MLN in the mice from g . i Representative IF in MLN from mice infected with H. polygyrus from g , stained for the indicated antigens. Scale bar, 100 µm. GC numbers per MLN scored in individual mice (symbols) are plotted to the right. p -Values throughout are by an unpaired, two-tailed Student's t test

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Flow Cytometry, Cytometry, Infection, Staining, Two Tailed Test

    6) Product Images from "Alveolar macrophage functions during the transition phase to active immunity in calves"

    Article Title: Alveolar macrophage functions during the transition phase to active immunity in calves

    Journal: Journal of Animal Science

    doi: 10.1093/jas/sky261

    Immunoglobulin from healthy calves. (A) BAL sample; (B) blood serum. Means ± SEM from 10 calves during the 3 to 6 mo of life. *Statistical difference for IgG1 BAL (T6 to T1, T2, and T3, P = 0.005).
    Figure Legend Snippet: Immunoglobulin from healthy calves. (A) BAL sample; (B) blood serum. Means ± SEM from 10 calves during the 3 to 6 mo of life. *Statistical difference for IgG1 BAL (T6 to T1, T2, and T3, P = 0.005).

    Techniques Used:

    7) Product Images from "Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles"

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles

    Journal: The EMBO Journal

    doi: 10.15252/embj.2019102539

    Tollip and Parkin are in complex in a CUE domain‐dependent manner, but independent of autophagy and Tom1 HeLa cells expressing mycBioID–Tollip and HA‐Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 h in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole‐cell extracts and pulldowns from each condition were then separated by SDS–PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID–Tollip was used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions. HeLa cells expressing mycBioID alone and HA‐Parkin were used to confirm the specificity of the Tollip–Parkin interaction. HeLa cells coexpressing GFP‐Tollip and HA‐Parkin were either left untreated or treated with AO for 2 h prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP, and Western blot analysis was performed using antibodies against Parkin and GFP. HeLa cells expressing mycBioID–Tollip containing the CUE domain mutation (CUEmut) and HA‐Parkin were subjected to biotinylation and streptavidin pulldowns, followed by Western blot analysis. HeLa wild type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID–Tollip and HA‐Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins. Source data are available online for this figure.
    Figure Legend Snippet: Tollip and Parkin are in complex in a CUE domain‐dependent manner, but independent of autophagy and Tom1 HeLa cells expressing mycBioID–Tollip and HA‐Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 h in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole‐cell extracts and pulldowns from each condition were then separated by SDS–PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID–Tollip was used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions. HeLa cells expressing mycBioID alone and HA‐Parkin were used to confirm the specificity of the Tollip–Parkin interaction. HeLa cells coexpressing GFP‐Tollip and HA‐Parkin were either left untreated or treated with AO for 2 h prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP, and Western blot analysis was performed using antibodies against Parkin and GFP. HeLa cells expressing mycBioID–Tollip containing the CUE domain mutation (CUEmut) and HA‐Parkin were subjected to biotinylation and streptavidin pulldowns, followed by Western blot analysis. HeLa wild type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID–Tollip and HA‐Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins. Source data are available online for this figure.

    Techniques Used: Expressing, SDS Page, Cell Culture, Negative Control, Lysis, Western Blot, Mutagenesis

    8) Product Images from "Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation"

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201609073

    Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.
    Figure Legend Snippet: Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.

    Techniques Used: In Vivo, In Vitro, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Immunofluorescence, Staining, Construct, Functional Assay, Mutagenesis

    9) Product Images from "Rotavirus capsid VP6 protein acts as an adjuvant in vivo for norovirus virus-like particles in a combination vaccine"

    Article Title: Rotavirus capsid VP6 protein acts as an adjuvant in vivo for norovirus virus-like particles in a combination vaccine

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.1080/21645515.2015.1099772

    NoV GII.4 (A, B) and GI.3 (C, D) genotype-specific serum IgG antibody responses induced with 0.3 μg or 3 μg of GII.4 or GI.3 VLPs alone or with 0.3 μg doses in a combination with 10 μg of RV VP6. Control (Ctrl) mice received carrier (PBS) only. OD 490nm values of GII.4- (A) and GI.3-specific (C) antibodies in 1:100 diluted sera of individual mice are shown with the horizontal line representing the mean OD 490nm value of the experimental group. GII.4- (B) and GI.3-specific (D) end-point titers of the groups of mice expressed as the geometric mean titers of the reciprocal of the highest sample dilution giving a positive reading. Error bars represent 95% confidence intervals, CIs. Groups were compared by Mann-Whitney U-test or Fisher's exact test and p values determined.
    Figure Legend Snippet: NoV GII.4 (A, B) and GI.3 (C, D) genotype-specific serum IgG antibody responses induced with 0.3 μg or 3 μg of GII.4 or GI.3 VLPs alone or with 0.3 μg doses in a combination with 10 μg of RV VP6. Control (Ctrl) mice received carrier (PBS) only. OD 490nm values of GII.4- (A) and GI.3-specific (C) antibodies in 1:100 diluted sera of individual mice are shown with the horizontal line representing the mean OD 490nm value of the experimental group. GII.4- (B) and GI.3-specific (D) end-point titers of the groups of mice expressed as the geometric mean titers of the reciprocal of the highest sample dilution giving a positive reading. Error bars represent 95% confidence intervals, CIs. Groups were compared by Mann-Whitney U-test or Fisher's exact test and p values determined.

    Techniques Used: Mouse Assay, MANN-WHITNEY

    RV VP6-specific IgG antibody responses in sera of mice immunized with 0.3 μg of GII.4 (A) or GI.3 (B) VLPs in a combination with 10 μg VP6. End-point titration curves of each immunized mice and mean curve of the control (Ctrl) mice are shown.
    Figure Legend Snippet: RV VP6-specific IgG antibody responses in sera of mice immunized with 0.3 μg of GII.4 (A) or GI.3 (B) VLPs in a combination with 10 μg VP6. End-point titration curves of each immunized mice and mean curve of the control (Ctrl) mice are shown.

    Techniques Used: Mouse Assay, Titration

    Cross-reactivity of serum IgG antibodies to NoV GII.4 NO (A), GII.4 Sydney (B) and GI.1 (C) in mice immunized with 0.3 μg of GII.4 or GI.3 VLPs alone or in a combination with 10 μg of RV VP6. Control (Ctrl) mice received carrier only (PBS). Mean ODs of the experimetal groups with standard errors are shown.
    Figure Legend Snippet: Cross-reactivity of serum IgG antibodies to NoV GII.4 NO (A), GII.4 Sydney (B) and GI.1 (C) in mice immunized with 0.3 μg of GII.4 or GI.3 VLPs alone or in a combination with 10 μg of RV VP6. Control (Ctrl) mice received carrier only (PBS). Mean ODs of the experimetal groups with standard errors are shown.

    Techniques Used: Mouse Assay

    10) Product Images from "An antibody to RGMa promotes regeneration of cochlear synapses after noise exposure"

    Article Title: An antibody to RGMa promotes regeneration of cochlear synapses after noise exposure

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-81294-5

    Threshold recovery 3 weeks after noise trauma. ( A ) DPOAE and ABR thresholds in unexposed (No noise, n = 4), noise-exposed and anti-RGMa treated (RGMa Ab, n = 15), and noise-exposed and IgG-treated (IgG, n = 13) ears before noise trauma. ( B , C ) Post-noise and final DPOAE and ABR threshold shifts are defined relative to the mean thresholds for the same animals, measured respectively 24 h after the noise exposure, and before sacrifice i.e. 3 weeks after noise exposure. Temporary threshold shifts (TTS) are apparent at higher frequencies, post-noise, in all groups, without correlation to treatment. Error bars indicate SEM.
    Figure Legend Snippet: Threshold recovery 3 weeks after noise trauma. ( A ) DPOAE and ABR thresholds in unexposed (No noise, n = 4), noise-exposed and anti-RGMa treated (RGMa Ab, n = 15), and noise-exposed and IgG-treated (IgG, n = 13) ears before noise trauma. ( B , C ) Post-noise and final DPOAE and ABR threshold shifts are defined relative to the mean thresholds for the same animals, measured respectively 24 h after the noise exposure, and before sacrifice i.e. 3 weeks after noise exposure. Temporary threshold shifts (TTS) are apparent at higher frequencies, post-noise, in all groups, without correlation to treatment. Error bars indicate SEM.

    Techniques Used:

    Recovery of wave-I amplitude in anti-RGMa treated group. ( A ) Mean amplitude of wave-I in ABR 3 weeks after noise trauma i.e. 2 weeks after treatment by anti-RGMa or IgG, for each intensity of acoustic stimulation (dB SPL). There is no difference between the anti-RGMa (RGMa Ab) and control (IgG) groups at 11 kHz, where there is no synaptopathy. ( B ) Wave-I amplitude at 32 kHz increased 3 weeks after noise trauma in the anti-RGMa (RGMa Ab) as compared to the IgG group, indicating an effect of the antibody treatment. Statistical significance: * p
    Figure Legend Snippet: Recovery of wave-I amplitude in anti-RGMa treated group. ( A ) Mean amplitude of wave-I in ABR 3 weeks after noise trauma i.e. 2 weeks after treatment by anti-RGMa or IgG, for each intensity of acoustic stimulation (dB SPL). There is no difference between the anti-RGMa (RGMa Ab) and control (IgG) groups at 11 kHz, where there is no synaptopathy. ( B ) Wave-I amplitude at 32 kHz increased 3 weeks after noise trauma in the anti-RGMa (RGMa Ab) as compared to the IgG group, indicating an effect of the antibody treatment. Statistical significance: * p

    Techniques Used:

    Number of ribbon synapses on inner hair cells are increased in anti-RGMa treated group. ( A ) Analysis of mean number of ribbon synapses in inner hair cells at 32 kHz shows a significant increase in the anti-RGMa treated group (11.7 ± 3.27; n = 10) compared to the IgG treated group (8.3 ± 1.52; n = 12). Red and green lines represent the mean number of synapses in each group. Statistical significance *** p
    Figure Legend Snippet: Number of ribbon synapses on inner hair cells are increased in anti-RGMa treated group. ( A ) Analysis of mean number of ribbon synapses in inner hair cells at 32 kHz shows a significant increase in the anti-RGMa treated group (11.7 ± 3.27; n = 10) compared to the IgG treated group (8.3 ± 1.52; n = 12). Red and green lines represent the mean number of synapses in each group. Statistical significance *** p

    Techniques Used:

    11) Product Images from "Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17–36"

    Article Title: Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17–36

    Journal: Viruses

    doi: 10.3390/v9110336

    Immunofluorescence detection of anti-L2 mAb binding to L2 protein expressed in 293TT cells. Cells were transfected with sheLL plasmids for HPV16, 18, 35, 39, 45, 59 and fixed/permeabilized 48 h later. Anti-L2 mAbs were added to the cells and recognition of the L2 protein was detected with a goat anti-mouse immunoglobulin G (IgG) secondary antibody conjugated to Alexafluor 488.
    Figure Legend Snippet: Immunofluorescence detection of anti-L2 mAb binding to L2 protein expressed in 293TT cells. Cells were transfected with sheLL plasmids for HPV16, 18, 35, 39, 45, 59 and fixed/permeabilized 48 h later. Anti-L2 mAbs were added to the cells and recognition of the L2 protein was detected with a goat anti-mouse immunoglobulin G (IgG) secondary antibody conjugated to Alexafluor 488.

    Techniques Used: Immunofluorescence, Binding Assay, Transfection

    12) Product Images from "Atoxic Derivative of Botulinum Neurotoxin A as a Prototype Molecular Vehicle for Targeted Delivery to the Neuronal Cytoplasm"

    Article Title: Atoxic Derivative of Botulinum Neurotoxin A as a Prototype Molecular Vehicle for Targeted Delivery to the Neuronal Cytoplasm

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085517

    Intraneuronal persistence of LC ad . E19 rat hippocampal neurons were cultured in maintenance medium for 10°C to 50 nM BoNT/A ad . After incubation, cells were washed twice with maintenance medium to remove residual BoNT/A ad and chased with the fresh medium for 1 to 11 days. Panel A: Western blot analysis of LC ad (mAb F1-40). GAPDH was used as a loading control. Panel B: Flow cytometric quantification of the LC ad signal at different days of chase. Panel C: Immunostaining for tau (red, anti- tau mouse monoclonal IgG 2b , Cat # 610672, BD Biosciences) and LC ad (white). Scale is 10 µm.
    Figure Legend Snippet: Intraneuronal persistence of LC ad . E19 rat hippocampal neurons were cultured in maintenance medium for 10°C to 50 nM BoNT/A ad . After incubation, cells were washed twice with maintenance medium to remove residual BoNT/A ad and chased with the fresh medium for 1 to 11 days. Panel A: Western blot analysis of LC ad (mAb F1-40). GAPDH was used as a loading control. Panel B: Flow cytometric quantification of the LC ad signal at different days of chase. Panel C: Immunostaining for tau (red, anti- tau mouse monoclonal IgG 2b , Cat # 610672, BD Biosciences) and LC ad (white). Scale is 10 µm.

    Techniques Used: Cell Culture, Incubation, Western Blot, Flow Cytometry, Immunostaining

    13) Product Images from "Alveolar macrophage functions during the transition phase to active immunity in calves"

    Article Title: Alveolar macrophage functions during the transition phase to active immunity in calves

    Journal: Journal of Animal Science

    doi: 10.1093/jas/sky261

    Immunoglobulin from healthy calves. (A) BAL sample; (B) blood serum. Means ± SEM from 10 calves during the 3 to 6 mo of life. *Statistical difference for IgG1 BAL (T6 to T1, T2, and T3, P = 0.005).
    Figure Legend Snippet: Immunoglobulin from healthy calves. (A) BAL sample; (B) blood serum. Means ± SEM from 10 calves during the 3 to 6 mo of life. *Statistical difference for IgG1 BAL (T6 to T1, T2, and T3, P = 0.005).

    Techniques Used:

    14) Product Images from "Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation"

    Article Title: Novel Non-phosphorylated Serine 9/21 GSK3β/α Antibodies: Expanding the Tools for Studying GSK3 Regulation

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2016.00123

    12B2 and 15C2 are specific for nonphospho-S GSK3 in brain lysates of human, mouse, and rat, and both effectively immunoprecipitate GSK3 from cell lysates. (A) Protein sequence alignments for GSK3β (amino acids 1–25) and GSK3α (amino acids 13–37) from human, mouse and rat (Uniprot IDs in parentheses). (B,C) Blots of lysates from human, mouse and rat cortical tissue and GAPDH was used as a loading control (40 μg/lane total protein loaded; experiment repeated three times). (B) 12B2 (red) specifically labeled GSK3β, not GSK3α, in lysates and total GSK3α/β (green) was used to identify both isoforms. (C) 15C2 (red) labeled both GSK3β and GSK3α in lysates and total GSK3α/β (green) was used to identify both isoforms. (D–F) The 12B2 (D) , 15C2 (E) , or control mouse IgG ( F , Ms IgG) were used to immunoprecipitate GSK3 enzymes from HEK293T cell lysates. The starting lysate (Input) was incubated with magnetic beads coated with 12B2 (D) , 15C2 (E) , or Ms IgG control (F) antibodies. 12B2 pulled down only GSK3β (12B2-IP), 15C2 pulled down both GSK3α and β (15C2-IP) and Ms IgG did not pull down GSK3α or β (MsIgG-IP). The post-IP lysates were also run for comparisons to the input samples. These experiments were performed three independent times.
    Figure Legend Snippet: 12B2 and 15C2 are specific for nonphospho-S GSK3 in brain lysates of human, mouse, and rat, and both effectively immunoprecipitate GSK3 from cell lysates. (A) Protein sequence alignments for GSK3β (amino acids 1–25) and GSK3α (amino acids 13–37) from human, mouse and rat (Uniprot IDs in parentheses). (B,C) Blots of lysates from human, mouse and rat cortical tissue and GAPDH was used as a loading control (40 μg/lane total protein loaded; experiment repeated three times). (B) 12B2 (red) specifically labeled GSK3β, not GSK3α, in lysates and total GSK3α/β (green) was used to identify both isoforms. (C) 15C2 (red) labeled both GSK3β and GSK3α in lysates and total GSK3α/β (green) was used to identify both isoforms. (D–F) The 12B2 (D) , 15C2 (E) , or control mouse IgG ( F , Ms IgG) were used to immunoprecipitate GSK3 enzymes from HEK293T cell lysates. The starting lysate (Input) was incubated with magnetic beads coated with 12B2 (D) , 15C2 (E) , or Ms IgG control (F) antibodies. 12B2 pulled down only GSK3β (12B2-IP), 15C2 pulled down both GSK3α and β (15C2-IP) and Ms IgG did not pull down GSK3α or β (MsIgG-IP). The post-IP lysates were also run for comparisons to the input samples. These experiments were performed three independent times.

    Techniques Used: Sequencing, Labeling, Mass Spectrometry, Incubation, Magnetic Beads

    15) Product Images from "An Antibody to RGMa Promotes Regeneration of Cochlear Synapses after Noise Exposure"

    Article Title: An Antibody to RGMa Promotes Regeneration of Cochlear Synapses after Noise Exposure

    Journal: bioRxiv

    doi: 10.1101/2020.07.01.183269

    Number of ribbon synapses on inner hair cells are increased in anti-RGMa treated group. (A) Analysis of mean number of ribbon synapses in inner hair cells at 32 kHz shows a significant increase in the anti-RGMa treated group (11.7 ±3.27; n=10) compared to the IgG treated group (8.3 ±1.52; n=12). Red and green lines represent the mean number of synapses in each group. Statistical significance ***: p
    Figure Legend Snippet: Number of ribbon synapses on inner hair cells are increased in anti-RGMa treated group. (A) Analysis of mean number of ribbon synapses in inner hair cells at 32 kHz shows a significant increase in the anti-RGMa treated group (11.7 ±3.27; n=10) compared to the IgG treated group (8.3 ±1.52; n=12). Red and green lines represent the mean number of synapses in each group. Statistical significance ***: p

    Techniques Used:

    Recovery of wave 1 amplitude in anti-RGMa treated group. (A) Mean amplitude of wave-I in ABR 3 weeks after noise trauma i.e. 2 weeks after treatment by anti-RGMa or IgG, for each intensity of acoustic stimulation (dB SPL). There is no difference between the anti-RGMa (RGMa Ab) and control (IgG) groups at 11 kHz, where there is no synaptopathy. (B) Wave-I amplitude at 32 kHz increased 3 weeks after noise trauma in the anti-RGMa (RGMa Ab) as compared to the IgG group, indicating an effect of the antibody treatment. Statistical significance: *: p
    Figure Legend Snippet: Recovery of wave 1 amplitude in anti-RGMa treated group. (A) Mean amplitude of wave-I in ABR 3 weeks after noise trauma i.e. 2 weeks after treatment by anti-RGMa or IgG, for each intensity of acoustic stimulation (dB SPL). There is no difference between the anti-RGMa (RGMa Ab) and control (IgG) groups at 11 kHz, where there is no synaptopathy. (B) Wave-I amplitude at 32 kHz increased 3 weeks after noise trauma in the anti-RGMa (RGMa Ab) as compared to the IgG group, indicating an effect of the antibody treatment. Statistical significance: *: p

    Techniques Used:

    Threshold recovery 3 weeks after noise trauma. (A) DPOAE and ABR thresholds in unexposed (No noise, n=4), noise-exposed and anti-RGMa treated (RGMa Ab, n=15), and noise-exposed and IgG-treated (IgG, n=13) before noise trauma. (B, C) Post-noise and final DPOAE and ABR threshold shifts are defined relative to the mean thresholds for the same animals, measured respectively 24 h after the noise exposure, and before sacrifice i.e. 3 weeks after noise exposure. Temporary threshold shifts (TTS) are apparent at higher frequencies, post-noise, in all groups, without correlation to treatment. Error bars indicate SEM.
    Figure Legend Snippet: Threshold recovery 3 weeks after noise trauma. (A) DPOAE and ABR thresholds in unexposed (No noise, n=4), noise-exposed and anti-RGMa treated (RGMa Ab, n=15), and noise-exposed and IgG-treated (IgG, n=13) before noise trauma. (B, C) Post-noise and final DPOAE and ABR threshold shifts are defined relative to the mean thresholds for the same animals, measured respectively 24 h after the noise exposure, and before sacrifice i.e. 3 weeks after noise exposure. Temporary threshold shifts (TTS) are apparent at higher frequencies, post-noise, in all groups, without correlation to treatment. Error bars indicate SEM.

    Techniques Used:

    16) Product Images from "Endocytic Adaptor Protein HIP1R Controls Intracellular Trafficking of Epidermal Growth Factor Receptor in Neuronal Dendritic Development"

    Article Title: Endocytic Adaptor Protein HIP1R Controls Intracellular Trafficking of Epidermal Growth Factor Receptor in Neuronal Dendritic Development

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00447

    HIP1R interacts with EGFR via amino acid 633–822 fragment. (A,B) EGFR interacts with HIP1R in cultured hippocampal neurons harvested at DIV 6–8. (A) Neuron lysates were immunoprecipitated with an anti-EGFR antibody. Lysates (input) and precipitates (Immunoprecipitation, IP: IgG and IP: EGFR) were blotted with anti-EGFR, anti-HIP1R and anti-actin antibodies. (B) Neurons electroporated with GFP-HIP1R or GFP-control were immunoprecipitated with anti-GFP antibody. (C) EGFR interacts with HIP1R in hippocampus tissue dissected from 6-week-old male mice. (D) GST-pull down of EGFR using purified GST-HIP1R 1–632 , GST-HIP1R 633–822 and GST-HIP1R 823–1068 in cultured hippocampal neurons harvested at DIV 6–8.
    Figure Legend Snippet: HIP1R interacts with EGFR via amino acid 633–822 fragment. (A,B) EGFR interacts with HIP1R in cultured hippocampal neurons harvested at DIV 6–8. (A) Neuron lysates were immunoprecipitated with an anti-EGFR antibody. Lysates (input) and precipitates (Immunoprecipitation, IP: IgG and IP: EGFR) were blotted with anti-EGFR, anti-HIP1R and anti-actin antibodies. (B) Neurons electroporated with GFP-HIP1R or GFP-control were immunoprecipitated with anti-GFP antibody. (C) EGFR interacts with HIP1R in hippocampus tissue dissected from 6-week-old male mice. (D) GST-pull down of EGFR using purified GST-HIP1R 1–632 , GST-HIP1R 633–822 and GST-HIP1R 823–1068 in cultured hippocampal neurons harvested at DIV 6–8.

    Techniques Used: Cell Culture, Immunoprecipitation, Mouse Assay, Purification

    17) Product Images from "Loss of MYBBP1A Induces Cancer Stem Cell Activity in Renal Cancer"

    Article Title: Loss of MYBBP1A Induces Cancer Stem Cell Activity in Renal Cancer

    Journal: Cancers

    doi: 10.3390/cancers11020235

    Characterization of MYBBP1A expression and MYBBP1A binding to c-MYB in renal carcinoma cell lines. ( A ) Measurement of MYBBP1A, c-MYB, p53 and pVHL levels in renal carcinoma cell lines A498, 786-O, ACHN and CaKi-1 by western blot. Cells were cultured in 4500 mg/L glucose media. ( B ) Measurement of MYBBP1A, c-MYB, p53, acetyl-p53 and pVHL levels in A498, 786-O, ACHN and CaKi-1 by western blot. Cells were cultured in 1000 mg/L or 100 mg/L glucose media. ( C ) MYBBP1A and c-MYB co-localize in the nucleolus of renal carcinoma cell lines. Cells were stained using DAPI (nuclear control), MYBBP1A and c-MYB antibodies. Scale Bar: 8 µM. ( D ) Co-immunoprecipitation of c-MYB and MYBBP1A in renal carcinoma cells. Protein extracts from 786-O and A498 were subject to immunoprecipitation with c-MYB or normal IgG antibodies. The resultant immunoprecipitates were then analyzed by WB with c-MYB and MYBBP1A antibodies.
    Figure Legend Snippet: Characterization of MYBBP1A expression and MYBBP1A binding to c-MYB in renal carcinoma cell lines. ( A ) Measurement of MYBBP1A, c-MYB, p53 and pVHL levels in renal carcinoma cell lines A498, 786-O, ACHN and CaKi-1 by western blot. Cells were cultured in 4500 mg/L glucose media. ( B ) Measurement of MYBBP1A, c-MYB, p53, acetyl-p53 and pVHL levels in A498, 786-O, ACHN and CaKi-1 by western blot. Cells were cultured in 1000 mg/L or 100 mg/L glucose media. ( C ) MYBBP1A and c-MYB co-localize in the nucleolus of renal carcinoma cell lines. Cells were stained using DAPI (nuclear control), MYBBP1A and c-MYB antibodies. Scale Bar: 8 µM. ( D ) Co-immunoprecipitation of c-MYB and MYBBP1A in renal carcinoma cells. Protein extracts from 786-O and A498 were subject to immunoprecipitation with c-MYB or normal IgG antibodies. The resultant immunoprecipitates were then analyzed by WB with c-MYB and MYBBP1A antibodies.

    Techniques Used: Expressing, Binding Assay, Western Blot, Cell Culture, Staining, Immunoprecipitation

    18) Product Images from "Alveolar macrophage functions during the transition phase to active immunity in calves"

    Article Title: Alveolar macrophage functions during the transition phase to active immunity in calves

    Journal: Journal of Animal Science

    doi: 10.1093/jas/sky261

    Immunoglobulin from healthy calves. (A) BAL sample; (B) blood serum. Means ± SEM from 10 calves during the 3 to 6 mo of life. *Statistical difference for IgG1 BAL (T6 to T1, T2, and T3, P = 0.005).
    Figure Legend Snippet: Immunoglobulin from healthy calves. (A) BAL sample; (B) blood serum. Means ± SEM from 10 calves during the 3 to 6 mo of life. *Statistical difference for IgG1 BAL (T6 to T1, T2, and T3, P = 0.005).

    Techniques Used:

    19) Product Images from "Alveolar macrophage functions during the transition phase to active immunity in calves"

    Article Title: Alveolar macrophage functions during the transition phase to active immunity in calves

    Journal: Journal of Animal Science

    doi: 10.1093/jas/sky261

    Immunoglobulin from healthy calves. (A) BAL sample; (B) blood serum. Means ± SEM from 10 calves during the 3 to 6 mo of life. *Statistical difference for IgG1 BAL (T6 to T1, T2, and T3, P = 0.005).
    Figure Legend Snippet: Immunoglobulin from healthy calves. (A) BAL sample; (B) blood serum. Means ± SEM from 10 calves during the 3 to 6 mo of life. *Statistical difference for IgG1 BAL (T6 to T1, T2, and T3, P = 0.005).

    Techniques Used:

    20) Product Images from "Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity"

    Article Title: Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity

    Journal: Nature Communications

    doi: 10.1038/s41467-020-20251-8

    RBD-CLP vaccines induce high antigen-specific antibody titers in mice. Serum samples were obtained before vaccination (pre-bleed) and two weeks after primary (1st bleed) and boost (2nd bleed) vaccination, respectively. ELISA results are depicted both as raw serum dilution curves ( a , c ) as well as the area under curve (AUC) and the geometric mean/SD ( b , d ). a Serum dilution curves and b geometric mean titer/SD (RBDc 1 st bleed: GMT = 0.289; RBDc 2 nd bleed: GMT = 0.4369; RBDc-CLP 1 st bleed: GMT = 2.252; RBDc-CLP 2 nd bleed: GMT = 8.515) of total anti-SARS-CoV-2 spike (aa35-1227) IgG antibodies detected in sera from BALB/c mice immunized intramuscularly with soluble RBDc (prime 2 µg/boost 2 µg) ( n = 4) or CLP-displayed RBDc (RBDc-CLP) (prime 1 µg/boost 1 µg) ( n = 4). c Serum dilution curves and d geometric mean titer/SD (RBDn 1 st bleed: GMT = 0.2578; RBDn 2 nd bleed: GMT = 2.727; RBDn-CLP 1 st bleed: GMT = 4.427; RBDn-CLP 2 nd bleed: GMT = 12.44) of total anti-SARS-CoV-2 spike (aa35-1227) IgG antibodies detected in sera from Balb/c mice immunized intramuscularly with soluble RBDn (prime 5 µg/boost 5 µg) ( n = 4) or CLP-displayed RBDn (RBDn-CLP) (prime 6.5 µg/boost
    Figure Legend Snippet: RBD-CLP vaccines induce high antigen-specific antibody titers in mice. Serum samples were obtained before vaccination (pre-bleed) and two weeks after primary (1st bleed) and boost (2nd bleed) vaccination, respectively. ELISA results are depicted both as raw serum dilution curves ( a , c ) as well as the area under curve (AUC) and the geometric mean/SD ( b , d ). a Serum dilution curves and b geometric mean titer/SD (RBDc 1 st bleed: GMT = 0.289; RBDc 2 nd bleed: GMT = 0.4369; RBDc-CLP 1 st bleed: GMT = 2.252; RBDc-CLP 2 nd bleed: GMT = 8.515) of total anti-SARS-CoV-2 spike (aa35-1227) IgG antibodies detected in sera from BALB/c mice immunized intramuscularly with soluble RBDc (prime 2 µg/boost 2 µg) ( n = 4) or CLP-displayed RBDc (RBDc-CLP) (prime 1 µg/boost 1 µg) ( n = 4). c Serum dilution curves and d geometric mean titer/SD (RBDn 1 st bleed: GMT = 0.2578; RBDn 2 nd bleed: GMT = 2.727; RBDn-CLP 1 st bleed: GMT = 4.427; RBDn-CLP 2 nd bleed: GMT = 12.44) of total anti-SARS-CoV-2 spike (aa35-1227) IgG antibodies detected in sera from Balb/c mice immunized intramuscularly with soluble RBDn (prime 5 µg/boost 5 µg) ( n = 4) or CLP-displayed RBDn (RBDn-CLP) (prime 6.5 µg/boost

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    21) Product Images from "Differential distribution of the sodium‐activated potassium channels slick and slack in mouse brain"

    Article Title: Differential distribution of the sodium‐activated potassium channels slick and slack in mouse brain

    Journal: The Journal of Comparative Neurology

    doi: 10.1002/cne.23934

    Slick and Slack channel mRNA expression and immunoreactivity in the mouse brain (Bregma approximately –1.7 mm). A: Strong mRNA signal for the Slick channel was detected through in situ hybridization in pyramidal layer of hippocampus and in granule cell layer of the dentate gyrus. E: Slick channel immunolabeling revealed only very weak signal in these areas. In contrast, intense Slick channel immunolabeling was observed in the strata oriens, radiatum, and lucidum as well as in the molecular layer of the dentate gyrus. Slick channel immunoreactivity was particularly strong in ventromedial hypothalamic nucleus. Moderate to strong immunolabeling for the Slick channel was detected in amygdaloid nuclei. C: Moderate to strong Slack channel mRNA signal in in situ hybridization in the amygdala and in hippocampus. G: Slack channel immunoreactivity was comparably low in these areas. B,D: Representative autoradiographs of in situ hybridization experiments with sense probes of Slick and Slack channel mRNA, respectively. F: Experiments using a nonimmune IgG of the same subtype (IgG1). H: Experiments in which the primary antibody was omitted served as controls for immunohistochemical experiments. Scale bar = 1,000 μm.
    Figure Legend Snippet: Slick and Slack channel mRNA expression and immunoreactivity in the mouse brain (Bregma approximately –1.7 mm). A: Strong mRNA signal for the Slick channel was detected through in situ hybridization in pyramidal layer of hippocampus and in granule cell layer of the dentate gyrus. E: Slick channel immunolabeling revealed only very weak signal in these areas. In contrast, intense Slick channel immunolabeling was observed in the strata oriens, radiatum, and lucidum as well as in the molecular layer of the dentate gyrus. Slick channel immunoreactivity was particularly strong in ventromedial hypothalamic nucleus. Moderate to strong immunolabeling for the Slick channel was detected in amygdaloid nuclei. C: Moderate to strong Slack channel mRNA signal in in situ hybridization in the amygdala and in hippocampus. G: Slack channel immunoreactivity was comparably low in these areas. B,D: Representative autoradiographs of in situ hybridization experiments with sense probes of Slick and Slack channel mRNA, respectively. F: Experiments using a nonimmune IgG of the same subtype (IgG1). H: Experiments in which the primary antibody was omitted served as controls for immunohistochemical experiments. Scale bar = 1,000 μm.

    Techniques Used: Expressing, In Situ Hybridization, Immunolabeling, Immunohistochemistry

    22) Product Images from "Herpes Simplex Virus 1-Induced Blood-Brain Barrier Damage Involves Apoptosis Associated With GM130-Mediated Golgi Stress"

    Article Title: Herpes Simplex Virus 1-Induced Blood-Brain Barrier Damage Involves Apoptosis Associated With GM130-Mediated Golgi Stress

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2020.00002

    Golgi apparatus (GA) disruption and cleaved-caspase 3 activations in Bend.3 cells infected with HSV-1. Bend.3 cells were mock-infected (mock) or infected with HSV-1 (V) at an MOI of 3 and then harvested at 12 hours post-infection (hpi), 24 hpi, 36 hpi, and 48 hpi. (A) GA disruption and cleaved-caspase 3 activations were detected by immunofluorescence analysis. Mock-infected or HSV-1 infected cells were fixed, permeabilized, and incubated with antibodies to GM130 and cleaved-caspase 3, followed by incubation with Alexa Fluor 594-conjugated IgG (red channel) and Alexa Fluor 647-conjugated IgG (cyan channel). Infected cells expressed green fluorescent protein (GFP; green channel). DAPI was used to stain nuclei (blue channel). Merged channels generated the fifth image shown in each row. Scale bar, 10 μm. (B) GA disruption was determined by transmission electron microscopy. GA regions (red box) were captured at a magnification of 2,500× in mock-infected (upper) and HSV-1 infected (lower) cells. Scale bar, 2 μm. Highly ordered Golgi stacks are observed in mock-infected cells at a magnification of 5,000× (black arrowheads). The ultrastructure of the GA in HSV-1-infected cells gradually varied from organized stacks to discontinued stacks and dilated-elongated Golgi cisternae (red arrowheads). Representative images are shown from three independent experiments. The asterisk indicates the nucleus. Scale bar, 1 μm.
    Figure Legend Snippet: Golgi apparatus (GA) disruption and cleaved-caspase 3 activations in Bend.3 cells infected with HSV-1. Bend.3 cells were mock-infected (mock) or infected with HSV-1 (V) at an MOI of 3 and then harvested at 12 hours post-infection (hpi), 24 hpi, 36 hpi, and 48 hpi. (A) GA disruption and cleaved-caspase 3 activations were detected by immunofluorescence analysis. Mock-infected or HSV-1 infected cells were fixed, permeabilized, and incubated with antibodies to GM130 and cleaved-caspase 3, followed by incubation with Alexa Fluor 594-conjugated IgG (red channel) and Alexa Fluor 647-conjugated IgG (cyan channel). Infected cells expressed green fluorescent protein (GFP; green channel). DAPI was used to stain nuclei (blue channel). Merged channels generated the fifth image shown in each row. Scale bar, 10 μm. (B) GA disruption was determined by transmission electron microscopy. GA regions (red box) were captured at a magnification of 2,500× in mock-infected (upper) and HSV-1 infected (lower) cells. Scale bar, 2 μm. Highly ordered Golgi stacks are observed in mock-infected cells at a magnification of 5,000× (black arrowheads). The ultrastructure of the GA in HSV-1-infected cells gradually varied from organized stacks to discontinued stacks and dilated-elongated Golgi cisternae (red arrowheads). Representative images are shown from three independent experiments. The asterisk indicates the nucleus. Scale bar, 1 μm.

    Techniques Used: Infection, Immunofluorescence, Incubation, Staining, Generated, Transmission Assay, Electron Microscopy

    23) Product Images from "Lupus auto-antibodies act as positive allosteric modulators at NMDA receptors and induce spatial memory deficits"

    Article Title: Lupus auto-antibodies act as positive allosteric modulators at NMDA receptors and induce spatial memory deficits

    Journal: bioRxiv

    doi: 10.1101/791715

    Differential sensitivity of N2A- and N2B-containing NMDARs to DNRAbs. ( A B ) Moderate pathophysiological levels of DNRAbs (10 μg/mL) potentiate glutamate-activated currents in N2A-containing, but not in N2B-containing NMDARs. Upper panels , Whole-cell currents from HEK293 cells expressing human NMDAR subunits, either hGluN1/hGluN2A ( A ) or hGluN1/hGluN2B ( B ). Currents were elicited by a 2.5 s application of glutamate (1 mM) in the continuous presence of glycine (0.1 mM) (holding potential, −70 mV). Lower panels , control antibody B1 (IgG1, gray circles) or human-derived DNRAb G11 (green circles) were added 75 s after a baseline recording of 5 sweeps and were included in the bath throughout the remaining period. Current amplitudes for individual recordings were normalized to its baseline. Values are mean ± SEM (hN2A+B1, n = 6; hN2A+G11, n = 6; hN2B+B1, n = 5; hN2B+G11, n = 5). Example traces in upper panels show the +G11 recordings for the initial sweep during baseline (no antibody present) or for the last sweep during steady-state (in antibody). ( C D ) Peak current amplitudes in N2A-containing NMDAR are more strongly potentiated than those in N2B-containing receptors. Bar graphs (mean ± SEM with dots indicating individual values) (from left to right for hN1/hN2A, n = 6, 6, 6, 6, 5, 5; and for hN1/hN2B, n = 5, 5, 6, 5) showing normalized steady-state peak current amplitudes either for control antibody (B1) or DNRAbs (G11). Significance of DNRAb values are measured relative to their respective control (* p
    Figure Legend Snippet: Differential sensitivity of N2A- and N2B-containing NMDARs to DNRAbs. ( A B ) Moderate pathophysiological levels of DNRAbs (10 μg/mL) potentiate glutamate-activated currents in N2A-containing, but not in N2B-containing NMDARs. Upper panels , Whole-cell currents from HEK293 cells expressing human NMDAR subunits, either hGluN1/hGluN2A ( A ) or hGluN1/hGluN2B ( B ). Currents were elicited by a 2.5 s application of glutamate (1 mM) in the continuous presence of glycine (0.1 mM) (holding potential, −70 mV). Lower panels , control antibody B1 (IgG1, gray circles) or human-derived DNRAb G11 (green circles) were added 75 s after a baseline recording of 5 sweeps and were included in the bath throughout the remaining period. Current amplitudes for individual recordings were normalized to its baseline. Values are mean ± SEM (hN2A+B1, n = 6; hN2A+G11, n = 6; hN2B+B1, n = 5; hN2B+G11, n = 5). Example traces in upper panels show the +G11 recordings for the initial sweep during baseline (no antibody present) or for the last sweep during steady-state (in antibody). ( C D ) Peak current amplitudes in N2A-containing NMDAR are more strongly potentiated than those in N2B-containing receptors. Bar graphs (mean ± SEM with dots indicating individual values) (from left to right for hN1/hN2A, n = 6, 6, 6, 6, 5, 5; and for hN1/hN2B, n = 5, 5, 6, 5) showing normalized steady-state peak current amplitudes either for control antibody (B1) or DNRAbs (G11). Significance of DNRAb values are measured relative to their respective control (* p

    Techniques Used: Expressing, Derivative Assay

    24) Product Images from "Endocytic Adaptor Protein HIP1R Controls Intracellular Trafficking of Epidermal Growth Factor Receptor in Neuronal Dendritic Development"

    Article Title: Endocytic Adaptor Protein HIP1R Controls Intracellular Trafficking of Epidermal Growth Factor Receptor in Neuronal Dendritic Development

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00447

    HIP1R interacts with EGFR via amino acid 633–822 fragment. (A,B) EGFR interacts with HIP1R in cultured hippocampal neurons harvested at DIV 6–8. (A) Neuron lysates were immunoprecipitated with an anti-EGFR antibody. Lysates (input) and precipitates (Immunoprecipitation, IP: IgG and IP: EGFR) were blotted with anti-EGFR, anti-HIP1R and anti-actin antibodies. (B) Neurons electroporated with GFP-HIP1R or GFP-control were immunoprecipitated with anti-GFP antibody. (C) EGFR interacts with HIP1R in hippocampus tissue dissected from 6-week-old male mice. (D) GST-pull down of EGFR using purified GST-HIP1R 1–632 , GST-HIP1R 633–822 and GST-HIP1R 823–1068 in cultured hippocampal neurons harvested at DIV 6–8.
    Figure Legend Snippet: HIP1R interacts with EGFR via amino acid 633–822 fragment. (A,B) EGFR interacts with HIP1R in cultured hippocampal neurons harvested at DIV 6–8. (A) Neuron lysates were immunoprecipitated with an anti-EGFR antibody. Lysates (input) and precipitates (Immunoprecipitation, IP: IgG and IP: EGFR) were blotted with anti-EGFR, anti-HIP1R and anti-actin antibodies. (B) Neurons electroporated with GFP-HIP1R or GFP-control were immunoprecipitated with anti-GFP antibody. (C) EGFR interacts with HIP1R in hippocampus tissue dissected from 6-week-old male mice. (D) GST-pull down of EGFR using purified GST-HIP1R 1–632 , GST-HIP1R 633–822 and GST-HIP1R 823–1068 in cultured hippocampal neurons harvested at DIV 6–8.

    Techniques Used: Cell Culture, Immunoprecipitation, Mouse Assay, Purification

    25) Product Images from "Lupus auto-antibodies act as positive allosteric modulators at NMDA receptors and induce spatial memory deficits"

    Article Title: Lupus auto-antibodies act as positive allosteric modulators at NMDA receptors and induce spatial memory deficits

    Journal: bioRxiv

    doi: 10.1101/791715

    Differential sensitivity of N2A- and N2B-containing NMDARs to DNRAbs. ( A B ) Moderate pathophysiological levels of DNRAbs (10 μg/mL) potentiate glutamate-activated currents in N2A-containing, but not in N2B-containing NMDARs. Upper panels , Whole-cell currents from HEK293 cells expressing human NMDAR subunits, either hGluN1/hGluN2A ( A ) or hGluN1/hGluN2B ( B ). Currents were elicited by a 2.5 s application of glutamate (1 mM) in the continuous presence of glycine (0.1 mM) (holding potential, −70 mV). Lower panels , control antibody B1 (IgG1, gray circles) or human-derived DNRAb G11 (green circles) were added 75 s after a baseline recording of 5 sweeps and were included in the bath throughout the remaining period. Current amplitudes for individual recordings were normalized to its baseline. Values are mean ± SEM (hN2A+B1, n = 6; hN2A+G11, n = 6; hN2B+B1, n = 5; hN2B+G11, n = 5). Example traces in upper panels show the +G11 recordings for the initial sweep during baseline (no antibody present) or for the last sweep during steady-state (in antibody). ( C D ) Peak current amplitudes in N2A-containing NMDAR are more strongly potentiated than those in N2B-containing receptors. Bar graphs (mean ± SEM with dots indicating individual values) (from left to right for hN1/hN2A, n = 6, 6, 6, 6, 5, 5; and for hN1/hN2B, n = 5, 5, 6, 5) showing normalized steady-state peak current amplitudes either for control antibody (B1) or DNRAbs (G11). Significance of DNRAb values are measured relative to their respective control (* p
    Figure Legend Snippet: Differential sensitivity of N2A- and N2B-containing NMDARs to DNRAbs. ( A B ) Moderate pathophysiological levels of DNRAbs (10 μg/mL) potentiate glutamate-activated currents in N2A-containing, but not in N2B-containing NMDARs. Upper panels , Whole-cell currents from HEK293 cells expressing human NMDAR subunits, either hGluN1/hGluN2A ( A ) or hGluN1/hGluN2B ( B ). Currents were elicited by a 2.5 s application of glutamate (1 mM) in the continuous presence of glycine (0.1 mM) (holding potential, −70 mV). Lower panels , control antibody B1 (IgG1, gray circles) or human-derived DNRAb G11 (green circles) were added 75 s after a baseline recording of 5 sweeps and were included in the bath throughout the remaining period. Current amplitudes for individual recordings were normalized to its baseline. Values are mean ± SEM (hN2A+B1, n = 6; hN2A+G11, n = 6; hN2B+B1, n = 5; hN2B+G11, n = 5). Example traces in upper panels show the +G11 recordings for the initial sweep during baseline (no antibody present) or for the last sweep during steady-state (in antibody). ( C D ) Peak current amplitudes in N2A-containing NMDAR are more strongly potentiated than those in N2B-containing receptors. Bar graphs (mean ± SEM with dots indicating individual values) (from left to right for hN1/hN2A, n = 6, 6, 6, 6, 5, 5; and for hN1/hN2B, n = 5, 5, 6, 5) showing normalized steady-state peak current amplitudes either for control antibody (B1) or DNRAbs (G11). Significance of DNRAb values are measured relative to their respective control (* p

    Techniques Used: Expressing, Derivative Assay

    26) Product Images from "PRMT5 is essential for B cell development and germinal center dynamics"

    Article Title: PRMT5 is essential for B cell development and germinal center dynamics

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07884-6

    Antibody response and GC defects caused by Prmt5 deficiency. a – i Cγ1-cre (Ctrl) and Prmt5 F/F Cγ1-cre (F/F) mice were used throughout. a Total anti-NP IgG1 in the serum of mice, measured by ELISA 14 days after NP-CGG immunization. Mean ± s.d. OD values for serial dilutions are plotted for n mice from three experiments. b Representative pictures of ELISPOT for NP-specific IgG1 antibody secreting cells (ASC) at day 14 post-immunization. The number of ASC of individual mice (symbols) and means (bars) are plotted. c Anti-NP IgG1 in the serum of mice at various times post-immunization. Mean ± s.d. values for n mice at each time point from two experiments are plotted. d Total levels of antibody isotypes in the serum of n non-immunized mice. e Mean + s.d. number of lymphocytes per spleen at day 14 post-immunization, enumerated by flow cytometry for n mice from two experiments. f Representative flow cytometry plots (gated on B220 + ) of splenic GC B cell proportions at 14 days post-immunization with NP-CGG. The number of GC B cells per spleen for individual mice (symbol) and medians (bars) from three experiments are plotted. g As in f , for MLN of mice infected with H. polygyrus for 14 days. Data from two experiments are plotted. h Mean + s.d. number of lymphocytes per MLN in the mice from g . i Representative IF in MLN from mice infected with H. polygyrus from g , stained for the indicated antigens. Scale bar, 100 µm. GC numbers per MLN scored in individual mice (symbols) are plotted to the right. p -Values throughout are by an unpaired, two-tailed Student's t test
    Figure Legend Snippet: Antibody response and GC defects caused by Prmt5 deficiency. a – i Cγ1-cre (Ctrl) and Prmt5 F/F Cγ1-cre (F/F) mice were used throughout. a Total anti-NP IgG1 in the serum of mice, measured by ELISA 14 days after NP-CGG immunization. Mean ± s.d. OD values for serial dilutions are plotted for n mice from three experiments. b Representative pictures of ELISPOT for NP-specific IgG1 antibody secreting cells (ASC) at day 14 post-immunization. The number of ASC of individual mice (symbols) and means (bars) are plotted. c Anti-NP IgG1 in the serum of mice at various times post-immunization. Mean ± s.d. values for n mice at each time point from two experiments are plotted. d Total levels of antibody isotypes in the serum of n non-immunized mice. e Mean + s.d. number of lymphocytes per spleen at day 14 post-immunization, enumerated by flow cytometry for n mice from two experiments. f Representative flow cytometry plots (gated on B220 + ) of splenic GC B cell proportions at 14 days post-immunization with NP-CGG. The number of GC B cells per spleen for individual mice (symbol) and medians (bars) from three experiments are plotted. g As in f , for MLN of mice infected with H. polygyrus for 14 days. Data from two experiments are plotted. h Mean + s.d. number of lymphocytes per MLN in the mice from g . i Representative IF in MLN from mice infected with H. polygyrus from g , stained for the indicated antigens. Scale bar, 100 µm. GC numbers per MLN scored in individual mice (symbols) are plotted to the right. p -Values throughout are by an unpaired, two-tailed Student's t test

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Flow Cytometry, Cytometry, Infection, Staining, Two Tailed Test

    27) Product Images from "Atoxic Derivative of Botulinum Neurotoxin A as a Prototype Molecular Vehicle for Targeted Delivery to the Neuronal Cytoplasm"

    Article Title: Atoxic Derivative of Botulinum Neurotoxin A as a Prototype Molecular Vehicle for Targeted Delivery to the Neuronal Cytoplasm

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085517

    Intraneuronal persistence of LC ad . E19 rat hippocampal neurons were cultured in maintenance medium for 10°C to 50 nM BoNT/A ad . After incubation, cells were washed twice with maintenance medium to remove residual BoNT/A ad and chased with the fresh medium for 1 to 11 days. Panel A: Western blot analysis of LC ad (mAb F1-40). GAPDH was used as a loading control. Panel B: Flow cytometric quantification of the LC ad signal at different days of chase. Panel C: Immunostaining for tau (red, anti- tau mouse monoclonal IgG 2b , Cat # 610672, BD Biosciences) and LC ad (white). Scale is 10 µm.
    Figure Legend Snippet: Intraneuronal persistence of LC ad . E19 rat hippocampal neurons were cultured in maintenance medium for 10°C to 50 nM BoNT/A ad . After incubation, cells were washed twice with maintenance medium to remove residual BoNT/A ad and chased with the fresh medium for 1 to 11 days. Panel A: Western blot analysis of LC ad (mAb F1-40). GAPDH was used as a loading control. Panel B: Flow cytometric quantification of the LC ad signal at different days of chase. Panel C: Immunostaining for tau (red, anti- tau mouse monoclonal IgG 2b , Cat # 610672, BD Biosciences) and LC ad (white). Scale is 10 µm.

    Techniques Used: Cell Culture, Incubation, Western Blot, Flow Cytometry, Immunostaining

    28) Product Images from "Preferred endocytosis of amyloid precursor protein from cholesterol-enriched lipid raft microdomains"

    Article Title: Preferred endocytosis of amyloid precursor protein from cholesterol-enriched lipid raft microdomains

    Journal: bioRxiv

    doi: 10.1101/2020.06.26.172874

    Cellular cholesterol levels altered the accumulation of APP in early endosomes. CHO PS1 WT and PS1 ΔE9 cells were incubated with APP antibody at 4°C and transferred to 37°C for indicated times to allow internalization of the labeled surface APP. Then, cells were fixed and surface APP was stained with anti-mouse IgG secondary antibody to eliminate remaining surface APP signal. Following permeabilization, cells were labeled with EEA1 antibody to label early endosomes. Subsequently, anti-mouse Alexa647 (red)- or anti-rabbit Aexa488 (green)-conjugated secondary antibodies were used to label internalized APP and early endosomes, respectively. A. Representative confocal image showing APP localization at each time. Data are representative of four independent experiments. Scare bars correspond to 10 μm. B. Fluorescence intensities of APP and EEA1 were measured using Image J. The co-efficiency of APP and early endosomes was determined with Image J (n=4). Statistical analysis was analyzed by one-way ANOVA: *** indicates P
    Figure Legend Snippet: Cellular cholesterol levels altered the accumulation of APP in early endosomes. CHO PS1 WT and PS1 ΔE9 cells were incubated with APP antibody at 4°C and transferred to 37°C for indicated times to allow internalization of the labeled surface APP. Then, cells were fixed and surface APP was stained with anti-mouse IgG secondary antibody to eliminate remaining surface APP signal. Following permeabilization, cells were labeled with EEA1 antibody to label early endosomes. Subsequently, anti-mouse Alexa647 (red)- or anti-rabbit Aexa488 (green)-conjugated secondary antibodies were used to label internalized APP and early endosomes, respectively. A. Representative confocal image showing APP localization at each time. Data are representative of four independent experiments. Scare bars correspond to 10 μm. B. Fluorescence intensities of APP and EEA1 were measured using Image J. The co-efficiency of APP and early endosomes was determined with Image J (n=4). Statistical analysis was analyzed by one-way ANOVA: *** indicates P

    Techniques Used: Incubation, Labeling, Staining, Fluorescence

    29) Product Images from "Differential distribution of the sodium‐activated potassium channels slick and slack in mouse brain"

    Article Title: Differential distribution of the sodium‐activated potassium channels slick and slack in mouse brain

    Journal: The Journal of Comparative Neurology

    doi: 10.1002/cne.23934

    Slick and Slack channel mRNA expression and immunoreactivity in the mouse brain (Bregma approximately –1.7 mm). A: Strong mRNA signal for the Slick channel was detected through in situ hybridization in pyramidal layer of hippocampus and in granule cell layer of the dentate gyrus. E: Slick channel immunolabeling revealed only very weak signal in these areas. In contrast, intense Slick channel immunolabeling was observed in the strata oriens, radiatum, and lucidum as well as in the molecular layer of the dentate gyrus. Slick channel immunoreactivity was particularly strong in ventromedial hypothalamic nucleus. Moderate to strong immunolabeling for the Slick channel was detected in amygdaloid nuclei. C: Moderate to strong Slack channel mRNA signal in in situ hybridization in the amygdala and in hippocampus. G: Slack channel immunoreactivity was comparably low in these areas. B,D: Representative autoradiographs of in situ hybridization experiments with sense probes of Slick and Slack channel mRNA, respectively. F: Experiments using a nonimmune IgG of the same subtype (IgG1). H: Experiments in which the primary antibody was omitted served as controls for immunohistochemical experiments. Scale bar = 1,000 μm.
    Figure Legend Snippet: Slick and Slack channel mRNA expression and immunoreactivity in the mouse brain (Bregma approximately –1.7 mm). A: Strong mRNA signal for the Slick channel was detected through in situ hybridization in pyramidal layer of hippocampus and in granule cell layer of the dentate gyrus. E: Slick channel immunolabeling revealed only very weak signal in these areas. In contrast, intense Slick channel immunolabeling was observed in the strata oriens, radiatum, and lucidum as well as in the molecular layer of the dentate gyrus. Slick channel immunoreactivity was particularly strong in ventromedial hypothalamic nucleus. Moderate to strong immunolabeling for the Slick channel was detected in amygdaloid nuclei. C: Moderate to strong Slack channel mRNA signal in in situ hybridization in the amygdala and in hippocampus. G: Slack channel immunoreactivity was comparably low in these areas. B,D: Representative autoradiographs of in situ hybridization experiments with sense probes of Slick and Slack channel mRNA, respectively. F: Experiments using a nonimmune IgG of the same subtype (IgG1). H: Experiments in which the primary antibody was omitted served as controls for immunohistochemical experiments. Scale bar = 1,000 μm.

    Techniques Used: Expressing, In Situ Hybridization, Immunolabeling, Immunohistochemistry

    30) Product Images from "Type I and Type III Interferons Differ in Their Adjuvant Activities for Influenza Vaccines"

    Article Title: Type I and Type III Interferons Differ in Their Adjuvant Activities for Influenza Vaccines

    Journal: Journal of Virology

    doi: 10.1128/JVI.01262-19

    IFN-α enhances antibody production after intraperitoneal immunization with influenza subunit vaccines. (A) Mx1-WT mice were immunized by the intraperitoneal route with 1 μg CTA3M2e-DD (M2e) in the presence or absence of 1 μg IFN-αB/D. Ten days later, a single booster immunization was performed. M2e-specific IgG serum levels were determined by ELISA 10 days after the booster immunization. Each symbol represents an individual animal. Data are expressed as mean ± SEM. **, P
    Figure Legend Snippet: IFN-α enhances antibody production after intraperitoneal immunization with influenza subunit vaccines. (A) Mx1-WT mice were immunized by the intraperitoneal route with 1 μg CTA3M2e-DD (M2e) in the presence or absence of 1 μg IFN-αB/D. Ten days later, a single booster immunization was performed. M2e-specific IgG serum levels were determined by ELISA 10 days after the booster immunization. Each symbol represents an individual animal. Data are expressed as mean ± SEM. **, P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Different impacts of type I and type III IFN receptor deficiencies on antibody production after infection with live attenuated influenza virus. Mx1-WT (open symbols), Mx1- Ifnar1 –/– (closed symbols), and Mx1- Ifnlr1 –/– (gray symbols) mice were infected with 10 5 PFU of NS1-deficient influenza virus (hvPR8-△NS1) by the intranasal route. Levels of influenza virus (Flu)-specific IgG in serum (A, C) and Flu-specific IgA in BAL fluid (B, D) were determined by ELISA on day 21 postinfection. Each symbol represents an individual animal. Data are expressed as mean ± SEM. *, P
    Figure Legend Snippet: Different impacts of type I and type III IFN receptor deficiencies on antibody production after infection with live attenuated influenza virus. Mx1-WT (open symbols), Mx1- Ifnar1 –/– (closed symbols), and Mx1- Ifnlr1 –/– (gray symbols) mice were infected with 10 5 PFU of NS1-deficient influenza virus (hvPR8-△NS1) by the intranasal route. Levels of influenza virus (Flu)-specific IgG in serum (A, C) and Flu-specific IgA in BAL fluid (B, D) were determined by ELISA on day 21 postinfection. Each symbol represents an individual animal. Data are expressed as mean ± SEM. *, P

    Techniques Used: Infection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Exogenous IFN-α stimulates IgG and IgA production after intranasal immunization with M2e fusion construct irrespective of TSLP receptor expression. (A, B) WT mice were immunized by intranasal application of 1 μg CTA1-3M2e-DD (M2e) in the presence or absence of 1 μg IFN-αB/D or 1 μg IFN-λ2. Ten days after a single booster immunization, blood (A) and BAL fluid (B) was analyzed for M2e-specific IgG and IgA by ELISA. Each symbol represents an individual animal. Data are expressed as mean ± SEM. **, P
    Figure Legend Snippet: Exogenous IFN-α stimulates IgG and IgA production after intranasal immunization with M2e fusion construct irrespective of TSLP receptor expression. (A, B) WT mice were immunized by intranasal application of 1 μg CTA1-3M2e-DD (M2e) in the presence or absence of 1 μg IFN-αB/D or 1 μg IFN-λ2. Ten days after a single booster immunization, blood (A) and BAL fluid (B) was analyzed for M2e-specific IgG and IgA by ELISA. Each symbol represents an individual animal. Data are expressed as mean ± SEM. **, P

    Techniques Used: Construct, Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay

    31) Product Images from "Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine"

    Article Title: Development of a Synthetic Poxvirus-Based SARS-CoV-2 Vaccine

    Journal: bioRxiv

    doi: 10.1101/2020.07.01.183236

    Humoral immune responses stimulated by sMVA-CoV2 vectors. Balb/c mice immunized twice in a three week interval with 5×10 7 PFU of the single and double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific humoral immune responses A-B) Binding antibodies. S, RBD, and N-specific binding antibodies induced by the vaccine vectors were evaluated after the first (A) and second (B) immunization by ELISA. Dashed lines in A and B indicate median binding antibody endpoint titers measured in convalescent human sera (Figure S4). One-way ANOVA with Tukey’s multiple comparison test was used to evaluate differences between binding antibody end-point titers. C) IgG2a/IgG1 isotype ratio. S-, RBD-, and N-specific binding antibodies of the IgG2a and IgG1 isotype were measured after the second immunization using 1:10,000 serum dilution, and absorbance reading was used to calculate IgG2a/IgG1 antibody ratio. One-way ANOVA with Dunnett’s multiple comparison test was used to compare each group mean IgG2a/IgG1 ratio to a ratio of 1 (balanced Th1/Th2 response). D-G) NAb responses. SARS-CoV-2-specific NAb (NT90 titer) induced by the vaccine vectors were measured after the first (D, F) and second (E, G) immunization against SARS-CoV-2 pseudovirus (pv) (D-E) or infectious SARS-CoV-2 virus (F-G) in pooled sera of immunized mice. Shown is the average NT90 measured in duplicate (D-E) or triplicate (F-G) infection. N/A=failed quality control of the samples. Dotted lines indicate lowest antibody dilution included in the analysis. H) SARS-CoV-2/SARS-CoV-2pv correlation analysis. Correlation analysis of NT90 measured in mouse sera after one and two immunizations using infectious SARS-CoV-2 virus and SARS-CoV-2pv. Pearson correlation coefficient (r) was calculated in H. *p
    Figure Legend Snippet: Humoral immune responses stimulated by sMVA-CoV2 vectors. Balb/c mice immunized twice in a three week interval with 5×10 7 PFU of the single and double recombinant sMVA-CoV2 vectors derived with FPV HP1.441 (sMVA-S/N hp and sMVA-N/S hp) or TROVAC (sMVA-S/N tv, sMVA-N/S tv, sMVA-S tv, sMVA-N tv) were evaluated for SARS-CoV-2-specific humoral immune responses A-B) Binding antibodies. S, RBD, and N-specific binding antibodies induced by the vaccine vectors were evaluated after the first (A) and second (B) immunization by ELISA. Dashed lines in A and B indicate median binding antibody endpoint titers measured in convalescent human sera (Figure S4). One-way ANOVA with Tukey’s multiple comparison test was used to evaluate differences between binding antibody end-point titers. C) IgG2a/IgG1 isotype ratio. S-, RBD-, and N-specific binding antibodies of the IgG2a and IgG1 isotype were measured after the second immunization using 1:10,000 serum dilution, and absorbance reading was used to calculate IgG2a/IgG1 antibody ratio. One-way ANOVA with Dunnett’s multiple comparison test was used to compare each group mean IgG2a/IgG1 ratio to a ratio of 1 (balanced Th1/Th2 response). D-G) NAb responses. SARS-CoV-2-specific NAb (NT90 titer) induced by the vaccine vectors were measured after the first (D, F) and second (E, G) immunization against SARS-CoV-2 pseudovirus (pv) (D-E) or infectious SARS-CoV-2 virus (F-G) in pooled sera of immunized mice. Shown is the average NT90 measured in duplicate (D-E) or triplicate (F-G) infection. N/A=failed quality control of the samples. Dotted lines indicate lowest antibody dilution included in the analysis. H) SARS-CoV-2/SARS-CoV-2pv correlation analysis. Correlation analysis of NT90 measured in mouse sera after one and two immunizations using infectious SARS-CoV-2 virus and SARS-CoV-2pv. Pearson correlation coefficient (r) was calculated in H. *p

    Techniques Used: Mouse Assay, Recombinant, Derivative Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Infection

    sMVA in vivo immunogenicity. sMVA derived either with FPV HP1.441 (sMVA hp) or TROVAC from two independent virus reconstitution (sMVA tv1 and sMVA tv2) was compared by in vitro analysis with wtMVA. C57BL/6 mice were immunized twice at three week interval with low (1×10 7 PFU) or high (5×10 7 PFU) dose of sMVA or wtMVA. Mock-immunized mice were used as controls A) Binding antibodies. MVA-specific binding antibodies (IgG titer) stimulated by sMVA or wtMVA were measured after the first and second immunization by ELISA. B) NAb responses. MVA-specific NAb titers induced by sMVA or wtMVA were measured after the booster immunization against recombinant wtMVA expressing a GFP marker. C-D) T cell responses. MVA-specific IFN γ , TNFα, IL-4, and IL-10-secreting CD8+ (C) and CD4+ (D) T cell responses induced by sMVA or wtMVA after two immunizations were measured by flow cytometry following ex vivo antigen stimulation using B8R immunodominant peptides. Differences between groups were evaluated using one-way ANOVA with Tukey’s multiple comparison test. ns = not significant.
    Figure Legend Snippet: sMVA in vivo immunogenicity. sMVA derived either with FPV HP1.441 (sMVA hp) or TROVAC from two independent virus reconstitution (sMVA tv1 and sMVA tv2) was compared by in vitro analysis with wtMVA. C57BL/6 mice were immunized twice at three week interval with low (1×10 7 PFU) or high (5×10 7 PFU) dose of sMVA or wtMVA. Mock-immunized mice were used as controls A) Binding antibodies. MVA-specific binding antibodies (IgG titer) stimulated by sMVA or wtMVA were measured after the first and second immunization by ELISA. B) NAb responses. MVA-specific NAb titers induced by sMVA or wtMVA were measured after the booster immunization against recombinant wtMVA expressing a GFP marker. C-D) T cell responses. MVA-specific IFN γ , TNFα, IL-4, and IL-10-secreting CD8+ (C) and CD4+ (D) T cell responses induced by sMVA or wtMVA after two immunizations were measured by flow cytometry following ex vivo antigen stimulation using B8R immunodominant peptides. Differences between groups were evaluated using one-way ANOVA with Tukey’s multiple comparison test. ns = not significant.

    Techniques Used: In Vivo, Derivative Assay, In Vitro, Mouse Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Marker, Flow Cytometry, Ex Vivo

    32) Product Images from "Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles"

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles

    Journal: The EMBO Journal

    doi: 10.15252/embj.2019102539

    Tollip and Parkin are in complex in a CUE domain‐dependent manner, but independent of autophagy and Tom1 HeLa cells expressing mycBioID–Tollip and HA‐Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 h in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole‐cell extracts and pulldowns from each condition were then separated by SDS–PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID–Tollip was used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions. HeLa cells expressing mycBioID alone and HA‐Parkin were used to confirm the specificity of the Tollip–Parkin interaction. HeLa cells coexpressing GFP‐Tollip and HA‐Parkin were either left untreated or treated with AO for 2 h prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP, and Western blot analysis was performed using antibodies against Parkin and GFP. HeLa cells expressing mycBioID–Tollip containing the CUE domain mutation (CUEmut) and HA‐Parkin were subjected to biotinylation and streptavidin pulldowns, followed by Western blot analysis. HeLa wild type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID–Tollip and HA‐Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins. Source data are available online for this figure.
    Figure Legend Snippet: Tollip and Parkin are in complex in a CUE domain‐dependent manner, but independent of autophagy and Tom1 HeLa cells expressing mycBioID–Tollip and HA‐Parkin were either left untreated or treated with 5 μM antimycin A/10 μM oligomycin (AO) or AO/100 μM bafilomycin A (BfnA1) for 6 h in the presence of biotin. Cells were lysed and streptavidin pulldowns performed overnight to isolate biotinylated proteins. Proteins in whole‐cell extracts and pulldowns from each condition were then separated by SDS–PAGE and membranes probed with specific antibodies. Immunoblotting for Tom1 and mycBioID–Tollip was used as positive controls. Cells cultured in media lacking biotin were used as a negative control to assess background levels. Biotinylation of Parkin suggested Tollip specifically interacted with Parkin under these conditions. HeLa cells expressing mycBioID alone and HA‐Parkin were used to confirm the specificity of the Tollip–Parkin interaction. HeLa cells coexpressing GFP‐Tollip and HA‐Parkin were either left untreated or treated with AO for 2 h prior to lysis. Cell lysates were subjected to either a normal rabbit IgG IP or HA IP, and Western blot analysis was performed using antibodies against Parkin and GFP. HeLa cells expressing mycBioID–Tollip containing the CUE domain mutation (CUEmut) and HA‐Parkin were subjected to biotinylation and streptavidin pulldowns, followed by Western blot analysis. HeLa wild type (WT), Tom1 KO and ATG5 KO cells expressing mycBioID–Tollip and HA‐Parkin underwent the same conditions as those above and immunoblotted for the indicated proteins. Source data are available online for this figure.

    Techniques Used: Expressing, SDS Page, Cell Culture, Negative Control, Lysis, Western Blot, Mutagenesis

    33) Product Images from "Lupus auto-antibodies act as positive allosteric modulators at NMDA receptors and induce spatial memory deficits"

    Article Title: Lupus auto-antibodies act as positive allosteric modulators at NMDA receptors and induce spatial memory deficits

    Journal: bioRxiv

    doi: 10.1101/791715

    Differential sensitivity of N2A- and N2B-containing NMDARs to DNRAbs. ( A B ) Moderate pathophysiological levels of DNRAbs (10 μg/mL) potentiate glutamate-activated currents in N2A-containing, but not in N2B-containing NMDARs. Upper panels , Whole-cell currents from HEK293 cells expressing human NMDAR subunits, either hGluN1/hGluN2A ( A ) or hGluN1/hGluN2B ( B ). Currents were elicited by a 2.5 s application of glutamate (1 mM) in the continuous presence of glycine (0.1 mM) (holding potential, −70 mV). Lower panels , control antibody B1 (IgG1, gray circles) or human-derived DNRAb G11 (green circles) were added 75 s after a baseline recording of 5 sweeps and were included in the bath throughout the remaining period. Current amplitudes for individual recordings were normalized to its baseline. Values are mean ± SEM (hN2A+B1, n = 6; hN2A+G11, n = 6; hN2B+B1, n = 5; hN2B+G11, n = 5). Example traces in upper panels show the +G11 recordings for the initial sweep during baseline (no antibody present) or for the last sweep during steady-state (in antibody). ( C D ) Peak current amplitudes in N2A-containing NMDAR are more strongly potentiated than those in N2B-containing receptors. Bar graphs (mean ± SEM with dots indicating individual values) (from left to right for hN1/hN2A, n = 6, 6, 6, 6, 5, 5; and for hN1/hN2B, n = 5, 5, 6, 5) showing normalized steady-state peak current amplitudes either for control antibody (B1) or DNRAbs (G11). Significance of DNRAb values are measured relative to their respective control (* p
    Figure Legend Snippet: Differential sensitivity of N2A- and N2B-containing NMDARs to DNRAbs. ( A B ) Moderate pathophysiological levels of DNRAbs (10 μg/mL) potentiate glutamate-activated currents in N2A-containing, but not in N2B-containing NMDARs. Upper panels , Whole-cell currents from HEK293 cells expressing human NMDAR subunits, either hGluN1/hGluN2A ( A ) or hGluN1/hGluN2B ( B ). Currents were elicited by a 2.5 s application of glutamate (1 mM) in the continuous presence of glycine (0.1 mM) (holding potential, −70 mV). Lower panels , control antibody B1 (IgG1, gray circles) or human-derived DNRAb G11 (green circles) were added 75 s after a baseline recording of 5 sweeps and were included in the bath throughout the remaining period. Current amplitudes for individual recordings were normalized to its baseline. Values are mean ± SEM (hN2A+B1, n = 6; hN2A+G11, n = 6; hN2B+B1, n = 5; hN2B+G11, n = 5). Example traces in upper panels show the +G11 recordings for the initial sweep during baseline (no antibody present) or for the last sweep during steady-state (in antibody). ( C D ) Peak current amplitudes in N2A-containing NMDAR are more strongly potentiated than those in N2B-containing receptors. Bar graphs (mean ± SEM with dots indicating individual values) (from left to right for hN1/hN2A, n = 6, 6, 6, 6, 5, 5; and for hN1/hN2B, n = 5, 5, 6, 5) showing normalized steady-state peak current amplitudes either for control antibody (B1) or DNRAbs (G11). Significance of DNRAb values are measured relative to their respective control (* p

    Techniques Used: Expressing, Derivative Assay

    34) Product Images from "Bacterial superglue enables easy development of efficient virus-like particle based vaccines"

    Article Title: Bacterial superglue enables easy development of efficient virus-like particle based vaccines

    Journal: Journal of Nanobiotechnology

    doi: 10.1186/s12951-016-0181-1

    Antigen-specific IgG levels in mice after immunization with soluble or spy-VLP displayed malaria antigens. ( a , upper panels ) Antigen-specific IgG levels (OD Elisa) in serum from mice (n = 5 per group) immunized with a Pfs25 2xSpyTag-VLP vaccine ( filled circles ) or with a control vaccine consisting of soluble Pfs25 mixed with untagged AP205 VLPs ( open squares ). Both vaccines were formulated using aluminum hydroxide adjuvant (Statens Serum Institut, Copenhagen, Denmark). Mice were immunized on days 0, 21 and 42 and serum was collected on the indicated days after first immunization. Differences in median endpoint titers between vaccination groups were analyzed using Mann–Whitney Rank Sum test; day 14 (P
    Figure Legend Snippet: Antigen-specific IgG levels in mice after immunization with soluble or spy-VLP displayed malaria antigens. ( a , upper panels ) Antigen-specific IgG levels (OD Elisa) in serum from mice (n = 5 per group) immunized with a Pfs25 2xSpyTag-VLP vaccine ( filled circles ) or with a control vaccine consisting of soluble Pfs25 mixed with untagged AP205 VLPs ( open squares ). Both vaccines were formulated using aluminum hydroxide adjuvant (Statens Serum Institut, Copenhagen, Denmark). Mice were immunized on days 0, 21 and 42 and serum was collected on the indicated days after first immunization. Differences in median endpoint titers between vaccination groups were analyzed using Mann–Whitney Rank Sum test; day 14 (P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

    Breakage of self-tolerance. IgG autoantibody responses measured by standard ELISA. a , b C57BL/6 mice (n = 10 per group) were immunized with a PD-L1 ( a ) or CTLA-4 ( b ) SpyCatcher-VLP vaccine ( filled circles ) or with a control vaccine (n = 3 per group) consisting of similar amounts of spy-antigen mixed with untagged AP205 VLPs ( open squares ). Both vaccines were formulated using aluminum hydroxide adjuvant (Statens Serum Institut, Copenhagen, Denmark). Mice were immunized with a dose of 5 µg antigen on days 0, 21 and 42 and serum was collected on day 56 after first immunization. Median endpoint titers were compared for the PD-L1 vaccination groups (P = 0.01) and the CTLA-4 vaccination groups (P = 0.01) using Mann–Whitney Rank Sum test. c , d BALB/c mice (n = 4) were immunized with an IL-5 SpyTag-VLP vaccine or a control vaccine (soluble IL-5 + untagged AP205 VLP) (n = 5) which were both formulated with aluminum hydroxide (Statens Serum Institut, Copenhagen, Denmark). Mice were immunized on days 0, 21 and 42 with antigen doses of 5, 2.5 and 2.5 µg, respectively, and serum was collected on days 56 ( c ) and 112 ( d ). Median endpoint titers for the two vaccination groups were compared using Mann–Whitney Rank Sum test; day 56 (P = 0.2) and day 122 (P = 0.2)
    Figure Legend Snippet: Breakage of self-tolerance. IgG autoantibody responses measured by standard ELISA. a , b C57BL/6 mice (n = 10 per group) were immunized with a PD-L1 ( a ) or CTLA-4 ( b ) SpyCatcher-VLP vaccine ( filled circles ) or with a control vaccine (n = 3 per group) consisting of similar amounts of spy-antigen mixed with untagged AP205 VLPs ( open squares ). Both vaccines were formulated using aluminum hydroxide adjuvant (Statens Serum Institut, Copenhagen, Denmark). Mice were immunized with a dose of 5 µg antigen on days 0, 21 and 42 and serum was collected on day 56 after first immunization. Median endpoint titers were compared for the PD-L1 vaccination groups (P = 0.01) and the CTLA-4 vaccination groups (P = 0.01) using Mann–Whitney Rank Sum test. c , d BALB/c mice (n = 4) were immunized with an IL-5 SpyTag-VLP vaccine or a control vaccine (soluble IL-5 + untagged AP205 VLP) (n = 5) which were both formulated with aluminum hydroxide (Statens Serum Institut, Copenhagen, Denmark). Mice were immunized on days 0, 21 and 42 with antigen doses of 5, 2.5 and 2.5 µg, respectively, and serum was collected on days 56 ( c ) and 112 ( d ). Median endpoint titers for the two vaccination groups were compared using Mann–Whitney Rank Sum test; day 56 (P = 0.2) and day 122 (P = 0.2)

    Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, MANN-WHITNEY

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    Binding Assay:

    Article Title: Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis
    Article Snippet: Sera were diluted 1:20 in PBS and incubated for 60 minutes at room temperature. .. After washing with PBS, binding of IgG1 or IgM on brain tissue was detected using IgG1 and IgM specific secondary antibodies (Invitrogen, A21121 and A21042; 5 ug/ml) conjugated to Alexa Fluor 488. ..

    Incubation:

    Article Title: A Formulated TLR7/8 Agonist is a Flexible, Highly Potent and Effective Adjuvant for Pandemic Influenza Vaccines
    Article Snippet: For post-vaccination immune analysis, arrays were initially blocked with PBS + 1% Fetal Bovine Serum + 0.1% Tween-20, washed three times with Protein Array Wash Buffer (ArrayIt), and incubated with 300 μL of a 1:100 dilution of post-immunization mouse serum for 1 hour with shaking. .. Following primary incubation, arrays were washed 5 times with wash buffer, and incubated with fluorophore conjugated secondary antibodies for IgG2c (Jackson Immunoresearch, Part #: 115-495-208) and IgG1 (Life Technologies, Part#: A21123) at a 1:2000 dilution. .. Following a 30 minute incubation at room temperature, arrays were rinsed with Rinse Buffer (Arrayit), and analyzed using a Molecular Dynamics 400B array scanner.

    Article Title: PRMT5 is essential for B cell development and germinal center dynamics
    Article Snippet: ELISPOT Purified splenocytes or BM cells were added at different dilutions to a 96-well 0.45-μm PVDF membrane (Millipore, cat#MSIPS4W10) previously coated overnight at 4 °C with 2 μg/mL NP20 BSA and blocked with complete RPMI cell culture media for 2 h at 37 °C. .. Plates with cells were incubated in a humid chamber 12 h at 37 °C, 5% CO2 , then washed 6× with PBS 0.01% Tween-20, followed by incubation with goat anti-mouse IgG1-HRP (A10551, Life Technologies, 1/2000) diluted in culture media for 2 h at RT. .. Plates were washed and AEC substrate (3′ amino-9-ethylcarbazole; BD Bioscience) was added to reveal the spots.

    Western Blot:

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation
    Article Snippet: Antibodies and reagents The following primary antibodies were used at indicated concentrations for Western blot (WB), immunofluorescence (IF), or FACS: mouse anti-Smurf1 (clone 1D7; ab117552; Abcam), WB, 1:1,000; IF, 1:100; mouse anti-Talin (clone 8D4; sc-59881; Santa Cruz Biotechnology, Inc.), WB, 1:1,000; rabbit anti-paxillin (clone Y113; ab32084; Abcam), WB, 1:5,000; IF, 1:200; rabbit anti–β1 integrin (clone EPR1040Y; ab134179; Abcam), WB, 1:500; FACS, 1:100; rat anti–β1 integrin (clone KMI6; ab95623; Abcam), WB, 1:1,000; FACS, 1:200; mouse anti–β3 integrin (clone VI-PL2; ab110131; Abcam), WB, 1:1,000; FACS, 1:200; rabbit anti–Kindlin-2 (K3269; Sigma-Aldrich), WB, 1:1,000; mouse anti-Flag (clone M2; Sigma-Aldrich), WB, 1:2,000; mouse anti-GFP (clone GSN149; G1546; Sigma-Aldrich), WB, 1:2,000; rabbit anti-Myc (SAB4301136; Sigma-Aldrich), WB, 1:2,000; mouse anti-HA (clone HA-7; H9658; Sigma-Aldrich), WB, 1:5,000; mouse anti–Kindlin-2 (clone 3A3; Mab2617; EMD Millipore), WB, 1:1,000; IF, 1:200; goat anti–Kindlin-2 (clone Y-15; sc-30854; Santa Cruz Biotechnology, Inc.), IF, 1:100; rabbit anti-ubiquitin (3933; Cell Signaling Technology), WB, 1:1,000; rat anti–active-integrin β1 9EG7 (clone 9EG7; 553715; BD), IF, 1:200; FACS, 1:200; ligand-mimetic anti–integrin aIIbβ3 mAb PAC-1 (340507; BD); and mouse anti-actin (clone 2Q1055; sc-58673; Santa Cruz Biotechnology, Inc.), WB, 1:2,000. .. Secondary antibodies were goat anti–mouse HRP and goat anti–rabbit HRP (both Santa Cruz Biotechnology, Inc.), WB, 1:5,000; donkey anti-mouse Alexa Fluor 488 ( A21202 ); donkey anti-rabbit Alexa Fluor 488 ( A21206 ); donkey anti-goat Alexa Fluor 488 ( A11055 ); donkey anti-mouse Alexa Fluor 568 ( A10037 ); donkey anti-rabbit Alexa Fluor 568 ( A10042 ); goat anti–mouse Alexa Fluor 633 ( A21126 ); goat anti–rat IgM Alexa Fluor 647 ( A21248 ; all Invitrogen), FACS, 1:300; IF, 1:400. .. Proteasome inhibitor MG132 (SML1135) and protein synthesis inhibitor cycloheximide (C7698) were purchased from Sigma-Aldrich.

    FACS:

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation
    Article Snippet: Antibodies and reagents The following primary antibodies were used at indicated concentrations for Western blot (WB), immunofluorescence (IF), or FACS: mouse anti-Smurf1 (clone 1D7; ab117552; Abcam), WB, 1:1,000; IF, 1:100; mouse anti-Talin (clone 8D4; sc-59881; Santa Cruz Biotechnology, Inc.), WB, 1:1,000; rabbit anti-paxillin (clone Y113; ab32084; Abcam), WB, 1:5,000; IF, 1:200; rabbit anti–β1 integrin (clone EPR1040Y; ab134179; Abcam), WB, 1:500; FACS, 1:100; rat anti–β1 integrin (clone KMI6; ab95623; Abcam), WB, 1:1,000; FACS, 1:200; mouse anti–β3 integrin (clone VI-PL2; ab110131; Abcam), WB, 1:1,000; FACS, 1:200; rabbit anti–Kindlin-2 (K3269; Sigma-Aldrich), WB, 1:1,000; mouse anti-Flag (clone M2; Sigma-Aldrich), WB, 1:2,000; mouse anti-GFP (clone GSN149; G1546; Sigma-Aldrich), WB, 1:2,000; rabbit anti-Myc (SAB4301136; Sigma-Aldrich), WB, 1:2,000; mouse anti-HA (clone HA-7; H9658; Sigma-Aldrich), WB, 1:5,000; mouse anti–Kindlin-2 (clone 3A3; Mab2617; EMD Millipore), WB, 1:1,000; IF, 1:200; goat anti–Kindlin-2 (clone Y-15; sc-30854; Santa Cruz Biotechnology, Inc.), IF, 1:100; rabbit anti-ubiquitin (3933; Cell Signaling Technology), WB, 1:1,000; rat anti–active-integrin β1 9EG7 (clone 9EG7; 553715; BD), IF, 1:200; FACS, 1:200; ligand-mimetic anti–integrin aIIbβ3 mAb PAC-1 (340507; BD); and mouse anti-actin (clone 2Q1055; sc-58673; Santa Cruz Biotechnology, Inc.), WB, 1:2,000. .. Secondary antibodies were goat anti–mouse HRP and goat anti–rabbit HRP (both Santa Cruz Biotechnology, Inc.), WB, 1:5,000; donkey anti-mouse Alexa Fluor 488 ( A21202 ); donkey anti-rabbit Alexa Fluor 488 ( A21206 ); donkey anti-goat Alexa Fluor 488 ( A11055 ); donkey anti-mouse Alexa Fluor 568 ( A10037 ); donkey anti-rabbit Alexa Fluor 568 ( A10042 ); goat anti–mouse Alexa Fluor 633 ( A21126 ); goat anti–rat IgM Alexa Fluor 647 ( A21248 ; all Invitrogen), FACS, 1:300; IF, 1:400. .. Proteasome inhibitor MG132 (SML1135) and protein synthesis inhibitor cycloheximide (C7698) were purchased from Sigma-Aldrich.

    other:

    Article Title: Tollip coordinates Parkin‐dependent trafficking of mitochondrial‐derived vesicles
    Article Snippet: These were Alexa Fluor anti‐rabbit 488 (A11034), anti‐mouse 488 (A11029), anti‐mouse IgG1 488 (A21121), anti‐mouse 568 (A11031), anti‐rabbit 568 (A11036), anti‐mouse IgG2b 568 (A21144), anti‐mouse IgG2b 594 (A21145), anti‐rabbit 633 (A21070) and anti‐mouse IgG1 647 (A21240).

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  • 97
    Thermo Fisher texas redx conjugated goat anti mouse igg
    Validation of external S. aureus labeling. Adherent GM-MФs were either treated with cytochalasin D (to block uptake) or control prior to and during incubation with GFP- S. aureus RN6390 and incubated with lysostaphin (to degrade external S. aureus , eliminating external bacteria) or control following incubation with S. aureus . External S. aureus were labeled with an <t>IgG</t> 3 monoclonal mouse anti- S. aureus primary antibody and <t>Texas-RedX-conjugated</t> goat anti-mouse secondary antibody. (A) No treatment. (B) Treatment with cytochalasin D. (C) Treatment with lysostaphin. (D) Treatment with both cytochalasin D and lysostaphin. (Original magnification, 200X).
    Texas Redx Conjugated Goat Anti Mouse Igg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alexa 488 conjugated secondary anti goat antibodies
    Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (10 9 pfu). ( Left ) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. * , residual CD44 staining in nonepithelial lamina propria. ( Right ) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with <t>Alexa</t> 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.
    Alexa 488 Conjugated Secondary Anti Goat Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse fitc
    US27 is found in close proximity to <t>CXCR4.</t> (A) Immunofluorescence microscopy of HEK293-US27 or HEK293-US28 cells stained with anti-FLAG (green) and anti-CXCR4 (red) followed by goat anti-mouse <t>FITC</t> and donkey anti-goat TRITC secondary antibodies, respectively. Nuclei were visualized with DAPI (blue). Bar, 10 μm. (B) HEK293-US27 or HEK293-US28 cells were stained with anti-FLAG and anti-CXCR4 followed by oligonucleotide-conjugated secondary antibodies and amplification using the DuoLink kit for the proximity ligation assay (PLA). Red PLA spots indicate receptors in close proximity. (C) NuFF cells were infected with AD169-GFP (MOI = 1, 72 hpi) and stained with antibodies against CXCR4 and US27 (left) or CXCR4 and US28 (right) followed by DuoLink PLA. Infected cells are shown in green, and red indicates PLA spots. Images were captured using a Zeiss LSM700 laser scanning confocal microscope, and representative images are shown. For PLA (B, C), images are maximum-intensity projections from z-stacks.
    Goat Anti Mouse Fitc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher cross adsorbed goat anti mouse igg conjugated to alexa fluor 568
    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to <t>Alexa</t> Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa <t>Fluor</t> 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.
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    Validation of external S. aureus labeling. Adherent GM-MФs were either treated with cytochalasin D (to block uptake) or control prior to and during incubation with GFP- S. aureus RN6390 and incubated with lysostaphin (to degrade external S. aureus , eliminating external bacteria) or control following incubation with S. aureus . External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. (A) No treatment. (B) Treatment with cytochalasin D. (C) Treatment with lysostaphin. (D) Treatment with both cytochalasin D and lysostaphin. (Original magnification, 200X).

    Journal: PLoS ONE

    Article Title: Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

    doi: 10.1371/journal.pone.0006209

    Figure Lengend Snippet: Validation of external S. aureus labeling. Adherent GM-MФs were either treated with cytochalasin D (to block uptake) or control prior to and during incubation with GFP- S. aureus RN6390 and incubated with lysostaphin (to degrade external S. aureus , eliminating external bacteria) or control following incubation with S. aureus . External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. (A) No treatment. (B) Treatment with cytochalasin D. (C) Treatment with lysostaphin. (D) Treatment with both cytochalasin D and lysostaphin. (Original magnification, 200X).

    Article Snippet: To achieve red fluorescent labeling of external bacteria cells were incubated for 15 minutes with PBS/4% FBS (blocking buffer), incubated for 40 minutes with 10 µg/ml mouse monoclonal IgG3 anti-S. aureus antibody in blocking buffer, washed three times with blocking buffer, incubated for 20 minutes with 40 µg/ml Texas-RedX-conjugated goat anti-mouse IgG (Life Technologies) in blocking buffer, and washed twice with blocking buffer.

    Techniques: Labeling, Blocking Assay, Incubation

    Scanning cytometry fluorescence imaging with GFP-S. aureus RN6390. Adherent GM-MФs were incubated with unopsonized GFP- S. aureus RN6390. External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. Collapsed confocal stack images were acquired by scanning cytometry. (A) CellTracker Blue and Hoechst channel (cells). (B) GFP channel (all bacteria). (C) Texas Red channel (external bacteria). (D) Composite image. (Original magnification, 200X).

    Journal: PLoS ONE

    Article Title: Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

    doi: 10.1371/journal.pone.0006209

    Figure Lengend Snippet: Scanning cytometry fluorescence imaging with GFP-S. aureus RN6390. Adherent GM-MФs were incubated with unopsonized GFP- S. aureus RN6390. External S. aureus were labeled with an IgG 3 monoclonal mouse anti- S. aureus primary antibody and Texas-RedX-conjugated goat anti-mouse secondary antibody. Collapsed confocal stack images were acquired by scanning cytometry. (A) CellTracker Blue and Hoechst channel (cells). (B) GFP channel (all bacteria). (C) Texas Red channel (external bacteria). (D) Composite image. (Original magnification, 200X).

    Article Snippet: To achieve red fluorescent labeling of external bacteria cells were incubated for 15 minutes with PBS/4% FBS (blocking buffer), incubated for 40 minutes with 10 µg/ml mouse monoclonal IgG3 anti-S. aureus antibody in blocking buffer, washed three times with blocking buffer, incubated for 20 minutes with 40 µg/ml Texas-RedX-conjugated goat anti-mouse IgG (Life Technologies) in blocking buffer, and washed twice with blocking buffer.

    Techniques: Cytometry, Fluorescence, Imaging, Incubation, Labeling

    Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (10 9 pfu). ( Left ) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. * , residual CD44 staining in nonepithelial lamina propria. ( Right ) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with Alexa 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1

    doi: 10.1073/pnas.2536800100

    Figure Lengend Snippet: Expression analysis of Wnt/β-catenin target genes, CD44 and EphB2, in the gastrointestinal tract of Ad Dkk1- or Ad Fc-treated adult C57BL/6 mice (12–16 weeks old). Organs were harvested 2 days after Ad Dkk1 i.v injection (10 9 pfu). ( Left ) Ad Dkk1 repression of CD44 expression in proliferative zones of all levels of the gastrointestinal epithelium. Arrowheads indicate the absence of CD44 immunoreactivity in proliferative compartments of the intestinal epithelium in Ad Dkk1 animals. * , residual CD44 staining in nonepithelial lamina propria. ( Right ) Ad Dkk1 repression of EphB2 in small intestine and colon. Repression was weaker in ascending colon and no repression was observed in stomach. EphB2 immunofluorescence was performed with Alexa 488 detection of EphB2 immunoreactivity (green) and Hoechst 33342 nuclear counterstain (blue). Stomach (st), duodenum (du), jejunum (je), ileum (il), cecum (ce), ascending colon (ac), and descending colon (dc) are shown.

    Article Snippet: Stainings were visualized with Alexa 488-conjugated secondary anti-goat antibodies (Molecular Probes) and nuclei were counterstained with Hoechst 33342 (Molecular Probes).

    Techniques: Expressing, Mouse Assay, Injection, Staining, Immunofluorescence

    US27 is found in close proximity to CXCR4. (A) Immunofluorescence microscopy of HEK293-US27 or HEK293-US28 cells stained with anti-FLAG (green) and anti-CXCR4 (red) followed by goat anti-mouse FITC and donkey anti-goat TRITC secondary antibodies, respectively. Nuclei were visualized with DAPI (blue). Bar, 10 μm. (B) HEK293-US27 or HEK293-US28 cells were stained with anti-FLAG and anti-CXCR4 followed by oligonucleotide-conjugated secondary antibodies and amplification using the DuoLink kit for the proximity ligation assay (PLA). Red PLA spots indicate receptors in close proximity. (C) NuFF cells were infected with AD169-GFP (MOI = 1, 72 hpi) and stained with antibodies against CXCR4 and US27 (left) or CXCR4 and US28 (right) followed by DuoLink PLA. Infected cells are shown in green, and red indicates PLA spots. Images were captured using a Zeiss LSM700 laser scanning confocal microscope, and representative images are shown. For PLA (B, C), images are maximum-intensity projections from z-stacks.

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus UL111A and US27 Gene Products Enhance the CXCL12/CXCR4 Signaling Axis via Distinct Mechanisms

    doi: 10.1128/JVI.01981-17

    Figure Lengend Snippet: US27 is found in close proximity to CXCR4. (A) Immunofluorescence microscopy of HEK293-US27 or HEK293-US28 cells stained with anti-FLAG (green) and anti-CXCR4 (red) followed by goat anti-mouse FITC and donkey anti-goat TRITC secondary antibodies, respectively. Nuclei were visualized with DAPI (blue). Bar, 10 μm. (B) HEK293-US27 or HEK293-US28 cells were stained with anti-FLAG and anti-CXCR4 followed by oligonucleotide-conjugated secondary antibodies and amplification using the DuoLink kit for the proximity ligation assay (PLA). Red PLA spots indicate receptors in close proximity. (C) NuFF cells were infected with AD169-GFP (MOI = 1, 72 hpi) and stained with antibodies against CXCR4 and US27 (left) or CXCR4 and US28 (right) followed by DuoLink PLA. Infected cells are shown in green, and red indicates PLA spots. Images were captured using a Zeiss LSM700 laser scanning confocal microscope, and representative images are shown. For PLA (B, C), images are maximum-intensity projections from z-stacks.

    Article Snippet: For standard immunofluorescence (IF), cells were costained with anti-CXCR4 goat polyclonal (1:100) and anti-FLAG mouse monoclonal antibody (1:500) followed by goat anti-mouse FITC and donkey anti-goat tetramethylrhodamine (TRTIC; 1:250), and then Prolong Gold mounting medium with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen) was added.

    Techniques: Immunofluorescence, Microscopy, Staining, Amplification, Proximity Ligation Assay, Infection

    cmvIL-10 enhances CXCR4 signaling in HCMV-infected cells. (A) NuFF cells were infected with TB40/E- mCherry (MOI = 1). Total RNA was harvested from mock- or HCMV-infected NuFFs at 72 h postinfection (hpi) and reverse transcribed, and UL123 (IE1) or β-actin genes were amplified. The resulting bands were visualized via agarose gel electrophoresis. (B) Mock- or HCMV-infected NuFF cells were stained with IL-10R-FITC or isotype control antibody and analyzed by flow cytometry. (C) Flow cytometric comparison of mock- or HCMV-infected NuFF cells for mCherry fluorescence as a measure of infection (left) or IL-10R stained as described above (right). (D) Calcium response was evaluated in Fluo-4 AM-loaded mock- or HCMV-infected cells stimulated with 100 ng/ml CXCL12 ± 100 ng/ml cmvIL-10 at 72 hpi. Relative fluorescence units (RFU) were measured by flow cytometry; the arrow indicates stimulus addition. (E) Transwell migration of mock-infected (gray bars) or HCMV-infected (black bars) NuFF cells toward CXCL12 in the lower chamber ± 100 ng/ml cmvIL-10 (striped bars, mock infected; white bars, HCMV infected) was measured at 72 hpi. Error bars, standard error for 3 replicates; *, P

    Journal: Journal of Virology

    Article Title: Human Cytomegalovirus UL111A and US27 Gene Products Enhance the CXCL12/CXCR4 Signaling Axis via Distinct Mechanisms

    doi: 10.1128/JVI.01981-17

    Figure Lengend Snippet: cmvIL-10 enhances CXCR4 signaling in HCMV-infected cells. (A) NuFF cells were infected with TB40/E- mCherry (MOI = 1). Total RNA was harvested from mock- or HCMV-infected NuFFs at 72 h postinfection (hpi) and reverse transcribed, and UL123 (IE1) or β-actin genes were amplified. The resulting bands were visualized via agarose gel electrophoresis. (B) Mock- or HCMV-infected NuFF cells were stained with IL-10R-FITC or isotype control antibody and analyzed by flow cytometry. (C) Flow cytometric comparison of mock- or HCMV-infected NuFF cells for mCherry fluorescence as a measure of infection (left) or IL-10R stained as described above (right). (D) Calcium response was evaluated in Fluo-4 AM-loaded mock- or HCMV-infected cells stimulated with 100 ng/ml CXCL12 ± 100 ng/ml cmvIL-10 at 72 hpi. Relative fluorescence units (RFU) were measured by flow cytometry; the arrow indicates stimulus addition. (E) Transwell migration of mock-infected (gray bars) or HCMV-infected (black bars) NuFF cells toward CXCL12 in the lower chamber ± 100 ng/ml cmvIL-10 (striped bars, mock infected; white bars, HCMV infected) was measured at 72 hpi. Error bars, standard error for 3 replicates; *, P

    Article Snippet: For standard immunofluorescence (IF), cells were costained with anti-CXCR4 goat polyclonal (1:100) and anti-FLAG mouse monoclonal antibody (1:500) followed by goat anti-mouse FITC and donkey anti-goat tetramethylrhodamine (TRTIC; 1:250), and then Prolong Gold mounting medium with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen) was added.

    Techniques: Infection, Amplification, Agarose Gel Electrophoresis, Staining, Flow Cytometry, Cytometry, Fluorescence, Migration

    Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Journal: Infection and Immunity

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    doi: 10.1128/IAI.00864-13

    Figure Lengend Snippet: Confocal microscopy and adherence-invasion assays with pharmacologic inhibitors. Lipid raft-independent invasion of bronchial epithelial cells by NTHI. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI does not colocalize with vesicles positive for the following markers of lipid rafts: caveolin-1 (shown at 24 h), flotillin-1 (shown at 24 h), and cholesterol (shown at 4 h). NTHI was labeled with anti-11P6H antibody conjugated to Alexa Fluor 488 (green). Caveolin-1 was labeled with anti-caveolin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Flotillin-1 was labeled with anti-flotillin-1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). Cholesterol was labeled with filipin (blue), and those samples were also labeled with anti-human secretory component antibody and donkey anti-goat antibody conjugated to Alexa Fluor 568 (red) to provide additional visualization of the plasma membrane. (B) Confocal microscopy. Filipin (FLP) and nystatin (NST) inhibit the internalization of fluorescently conjugated cholera toxin B subunit (white arrows), a known cargo of lipid raft-mediated endocytosis. H292 cells were pretreated with sRPMI containing inhibitor or inhibitor diluent, followed by addition of the cargo conjugate, incubation for 20 min on ice, and incubation for 2 h at 37°C. (C) Adherence-invasion assays. Filipin and nystatin do not inhibit invasion by NTHI. H292 cells were pretreated with inhibitor or inhibitor diluent for 2 h, followed by a 4-h infection conducted according to the adherence-invasion assay protocol. Data are normalized to samples in the absence of inhibitor. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Article Snippet: Host cell components were visualized using filipin III fluorescent dye (final concentration of 333 μg/ml; Cayman Chemical), primary monoclonal antibodies including mouse anti-caveolin-1 antibody (1:50; BD), mouse anti-early endosomal antigen 1 (EEA-1) antibody (1:200; BD), mouse anti-flotillin-1 antibody (1:20; BD), mouse anti-human CD107a antibody (LAMP1) (1:200; BD), and goat anti-human secretory component antibody (1:200; Sigma), and secondary antibodies including highly cross-adsorbed goat anti-mouse IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200) and donkey anti-goat IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200).

    Techniques: Confocal Microscopy, Labeling, Incubation, Infection, Invasion Assay

    Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Journal: Infection and Immunity

    Article Title: Internalization and Trafficking of Nontypeable Haemophilus influenzae in Human Respiratory Epithelial Cells and Roles of IgA1 Proteases for Optimal Invasion and Persistence

    doi: 10.1128/IAI.00864-13

    Figure Lengend Snippet: Confocal microscopy and survival assay with pharmacologic inhibitor of lysosome acidification. NTHI traffics to early endosomes and lysosomes and is killed in lysosomes. IgA1 proteases are required for optimal survival in lysosomes. (A) Confocal microscopy. At 4 and 24 h postinoculation of H292 cells, NTHI is found within vesicles positive for EEA1 (shown at 4 h) and within vesicles positive for LAMP1 (shown at 24 h). NTHI was labeled using anti-11P6H antibody conjugated to Alexa Fluor 488 (green). EEA1 was labeled using anti-EEA1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). LAMP1 was labeled using anti-human LAMP1 antibody and goat anti-mouse antibody conjugated to Alexa Fluor 568 (red). (B) Survival assay in the presence and absence of concanamycin A (CMA). H292 cells were infected for 16 h, treated with gentamicin for 1 h, and treated with concanamycin or diluent for 3 h. Survival data are normalized to samples treated with diluent. Error bars represent standard errors of the means of three independent experiments. A paired Student t test was used to calculate statistical significance. *, P ≤ 0.05; **, P ≤ 0.005.

    Article Snippet: Host cell components were visualized using filipin III fluorescent dye (final concentration of 333 μg/ml; Cayman Chemical), primary monoclonal antibodies including mouse anti-caveolin-1 antibody (1:50; BD), mouse anti-early endosomal antigen 1 (EEA-1) antibody (1:200; BD), mouse anti-flotillin-1 antibody (1:20; BD), mouse anti-human CD107a antibody (LAMP1) (1:200; BD), and goat anti-human secretory component antibody (1:200; Sigma), and secondary antibodies including highly cross-adsorbed goat anti-mouse IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200) and donkey anti-goat IgG conjugated to Alexa Fluor 568 (Life Technologies, 1:200).

    Techniques: Confocal Microscopy, Clonogenic Cell Survival Assay, Labeling, Infection