ampr richard murray lab cidar moclo extension unpublished 120975 c91m superfolder cyan fluorescent protein sfcfp  (Addgene inc)

Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: CFP fluorescence was examined in whole mount fourth-instar larvae and pupae from PPO6 ( a ) or SPARC ( b ) transgenic lines. Arrowheads highlight presumed hemocytes. Scale bars: 1 mm. Potential differences in CFP expression between larvae and adult mosquitoes were examined by qPCR for both PPO6 ( c ) or SPARC ( d ) transgenic lines. For ( c and d ), expression data are displayed as the mean ± SE from three independent replicates ( N = 3, dots) of pooled ( n = ~10) larvae or adult female mosquitoes, with values relative to rpS7 expression. Significance was determined using a two-tailed unpaired Student’s t -test. Exact P values are displayed in the figure where significant. ns not significant. Additional ex vivo analysis of perfused hemocytes from PPO6 ( e ) or SPARC ( f ) transgenic lines using microscopy was used to examine hemocyte fluorescence. The injection of fluorescent beads (red) prior to perfusion indicates that both PPO6-CFP + and SPARC-CFP + immune cell populations are comprised of phagocytic and non-phagocytic cells. Scale bars: 10 μm. Subpanels demonstrate different representative cell phenotypes. The specificity of CFP expression was examined in hemocytes and carcass samples via qPCR for the PPO6 ( g ) and SPARC ( h ) constructs across individual transgenic lines. The hemocyte-specific expression of NimB2 was used as a positive control for gene expression analysis. Expression data are displayed as the mean ± SE from three independent replicates ( N = 3, dots) of pooled hemolymph perfusions ( n = >30 adult female mosquitoes) or adult female mosquitoes ( n = ~10) following perfusion (carcass), with values relative to rpS7 expression. Significance was determined using multiple two-tailed unpaired t -tests and a Holm–Šídák correction. Adjusted P values are displayed in the figure where significant. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Fluorescence, Transgenic Assay, Expressing, Two Tailed Test, Ex Vivo, Microscopy, Injection, Construct, Positive Control, Gene Expression

Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: Flow cytometry analysis was performed on immune cell populations resulting from LRIM15 + , SPARC + , and PPO6 + individual transgenic lines ( a – f ). Representative UMAPs resulting from spectral imaging flow cytometry for LRIM15-GFP ( a ), SPARC-CFP ( c ), and PPO6-CFP transgenics ( e ) display the overall cell populations and the composition of fluorescent cells as an overlay using the established gating for each of the 12 hemocyte subpopulations identified in wild-type mosquitoes. The total number of events for each cell classification are displayed on the right of each figure subpanel. These distributions are summarized in pie charts to display fluorescent cell subtypes for LRIM15-GFP ( b ), SPARC-CFP ( d ), and PPO6-CFP transgenics ( f ). Data were averaged from three independent biological replicates. To determine potential overlap between transgenic markers, crosses were performed to establish either LRIM15 + /SPARC + ( g ) or LRIM15 + /PPO6 + ( h ) genetic backgrounds. For each genetic background, the abundance of GFP + , CFP + , and GFP + /CFP + cells were examined by overlaying fluorescent cell populations on the UMAP and summarized in bar graphs to denote the hemocyte clusters represented for each fluorescent cell subtype. Data display the average of three independent biological replicates. UMAP analysis was performed using the Euclidean distance metric using FlowJo V10.10.0, including the following parameters: DRAQ5 signal, Maximum Intensity Forward Scatter (FSC), Maximum Intensity Side Scatter (SSC), and Maximum Intensity Light Loss. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Flow Cytometry, Transgenic Assay, Imaging

Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: CFP fluorescence was examined in whole mount fourth-instar larvae and pupae from PPO6 ( a ) or SPARC ( b ) transgenic lines. Arrowheads highlight presumed hemocytes. Scale bars: 1 mm. Potential differences in CFP expression between larvae and adult mosquitoes were examined by qPCR for both PPO6 ( c ) or SPARC ( d ) transgenic lines. For ( c and d ), expression data are displayed as the mean ± SE from three independent replicates ( N = 3, dots) of pooled ( n = ~10) larvae or adult female mosquitoes, with values relative to rpS7 expression. Significance was determined using a two-tailed unpaired Student’s t -test. Exact P values are displayed in the figure where significant. ns not significant. Additional ex vivo analysis of perfused hemocytes from PPO6 ( e ) or SPARC ( f ) transgenic lines using microscopy was used to examine hemocyte fluorescence. The injection of fluorescent beads (red) prior to perfusion indicates that both PPO6-CFP + and SPARC-CFP + immune cell populations are comprised of phagocytic and non-phagocytic cells. Scale bars: 10 μm. Subpanels demonstrate different representative cell phenotypes. The specificity of CFP expression was examined in hemocytes and carcass samples via qPCR for the PPO6 ( g ) and SPARC ( h ) constructs across individual transgenic lines. The hemocyte-specific expression of NimB2 was used as a positive control for gene expression analysis. Expression data are displayed as the mean ± SE from three independent replicates ( N = 3, dots) of pooled hemolymph perfusions ( n = >30 adult female mosquitoes) or adult female mosquitoes ( n = ~10) following perfusion (carcass), with values relative to rpS7 expression. Significance was determined using multiple two-tailed unpaired t -tests and a Holm–Šídák correction. Adjusted P values are displayed in the figure where significant. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Fluorescence, Transgenic Assay, Expressing, Two Tailed Test, Ex Vivo, Microscopy, Injection, Construct, Positive Control, Gene Expression

Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: The percentage of PPO6 + ( a ), SPARC + ( b ), and LRIM15 + ( c ) hemocytes were evaluated under sugar-fed (SF), at 24 h post-feeding (24 h PBF), and at 48 h post-feeding (48 PBF). For each experimental condition in ( a – c ), data display the percentage of CFP+ ( a , b ) or GFP+ cells ( c ) of the total hemocytes collected from individual mosquitoes (dots). n number of individual mosquitoes examined. Data from three independent biological replicates were pooled and displayed as the mean ± SE. Differences in the shading of the bar graphs highlight differences between sugar-fed (light gray) and blood-fed (dark gray) conditions. Statistical analysis was performed using a one-way ANOVA with a Holm–Šídák comparison test. Adjusted P values are displayed in the figure where significant. ns not significant. For each transgenic construct, representative images are displayed at right for each experimental condition. Scale bars represent 10 μm. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Comparison, Transgenic Assay, Construct

Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: Flow cytometry analysis was performed on immune cell populations resulting from LRIM15 + , SPARC + , and PPO6 + individual transgenic lines ( a – f ). Representative UMAPs resulting from spectral imaging flow cytometry for LRIM15-GFP ( a ), SPARC-CFP ( c ), and PPO6-CFP transgenics ( e ) display the overall cell populations and the composition of fluorescent cells as an overlay using the established gating for each of the 12 hemocyte subpopulations identified in wild-type mosquitoes. The total number of events for each cell classification are displayed on the right of each figure subpanel. These distributions are summarized in pie charts to display fluorescent cell subtypes for LRIM15-GFP ( b ), SPARC-CFP ( d ), and PPO6-CFP transgenics ( f ). Data were averaged from three independent biological replicates. To determine potential overlap between transgenic markers, crosses were performed to establish either LRIM15 + /SPARC + ( g ) or LRIM15 + /PPO6 + ( h ) genetic backgrounds. For each genetic background, the abundance of GFP + , CFP + , and GFP + /CFP + cells were examined by overlaying fluorescent cell populations on the UMAP and summarized in bar graphs to denote the hemocyte clusters represented for each fluorescent cell subtype. Data display the average of three independent biological replicates. UMAP analysis was performed using the Euclidean distance metric using FlowJo V10.10.0, including the following parameters: DRAQ5 signal, Maximum Intensity Forward Scatter (FSC), Maximum Intensity Side Scatter (SSC), and Maximum Intensity Light Loss. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Flow Cytometry, Transgenic Assay, Imaging

Journal: Nature Communications

Article Title: Characterization of Anopheles gambiae immune cells through genetic and functional immunophenotyping

doi: 10.1038/s41467-025-65895-6

Figure Lengend Snippet: The injection of red fluorescent beads prior to perfusion enabled flow cytometry analysis of phagocytosis in mosquito immune cells ( a ) and the identification of multiple phagocytic immune cell phenotypes based on the intensity of bead signal ( b ). This resulted in the identification of non-phagocytic and phagocytic immune cell subtypes ( c ) that were confirmed by imaging ( d ). When phagocytic cells were further distinguished by bead signal intensity, we identified immune cell subtypes with low, medium, or high phagocytic capacity ( e ). Similar experiments with our LRIM15, SPARC, and PPO6 transgenic lines revealed the phagocytic ability of fluorescent immune cells ( f ) and the overall proportion of phagocytic immune cells ( g ), which are displayed as the mean ± SE of three biological replicates. h The phagocytic capacity of each transgenic line is visualized as the percentage of fluorescent cells displaying low, medium, or high bead-positive cells and displayed as the average of three independent biological replicates. Dot plots represent single replicates of three independent biological experiments. Source data are provided as a Source Data file.

Article Snippet: For immunostaining, samples were co-incubated overnight at 4 °C with a mouse monoclonal anti-cyan fluorescent protein (CFP) antibody (Biosensis, #M-1300-100) and a rabbit anti-PPO6 antibody , diluted at 1:250 and 1:500, respectively, in blocking buffer.

Techniques: Injection, Flow Cytometry, Imaging, Transgenic Assay

Journal: Science Advances

Article Title: Mechanistic insights into TTLL11 polyglutamylase–mediated primary tubulin chain elongation

doi: 10.1126/sciadv.adw1561

Figure Lengend Snippet: ( A ) TTLL11 binding to pMTs analyzed using TIRF microscopy. The TTLL11 representation shows the catalytic domain (purple), MT-BHB (pink), and site-specific mutations (described in fig. S6). MTs were attached to coverslips and incubated with 100 nM Janelia Fluor 549–labeled TTLL11 variants (red). Scale bars, 2 μm. ( B ) Quantification of TIRF images for full-length (FL) TTLL11, the N-terminally truncated variant (122 to 710), the catalytically inactive E441G, and the KKKR-EEEE mutant show binding with similar intensity. Deleting the MT-BHB or catalytic domains abolishes TTLL11 binding. Mutations in TTLL11 helix α 11 (R601E and I594W) reduced binding by 60 and 80%, respectively. Norm., normalized. ( C to E ) Tubulin C-tails are critical for TTLL11 interactions. (C) CBB-stained gel of truncated MTs. AspN truncates the β-tubulin, and subtilisin truncates both α- and β-tubulin C-tails. TIRF microscopy images (D) and quantification (E) show that AspN-treated MTs exhibit ~50% reduction in TTLL11 binding, while the complete loss of binding is observed for subtilisin-treated MTs. Data are shown as mean fluorescent intensity from n = 2 replicates with 114, 93, and 58 MTs quantified in each sample. Statistical significance: Unpaired t test with Welch correction; **** P < 0.0001. The black bar: Median value with 95% confidence interval. Scale bars, 5 μm. Ctrl, control. Created in BioRender. Barinka, C. (2025) https://BioRender.com/sr4q7do . ( F ) In vitro polyglutamylation of MTs from HEK293T cells by TTLL11 variants, analyzed by WB, confirms activity for FL and 122 to 710. Variants lacking the MT-BHB domain show no activity. I594W and R601E mutants show reduced activity. ( G ) Assay in HEK293T cells transfected with TTLL11 variants shows polyglutamylation consistent with in vitro findings. TTLL6 and TTLL7 show specificity for α- and β-tubulin, respectively. An unidentified TTLL11 substrate, represented by a polyE-stained band in between α- and β-tubulin (between red lines), is glutamylated regardless of the MT-BHB mutations. Only TTLL7 shows a signal of the GT335 antibody specific for branching.

Article Snippet: Membranes were incubated with primary antibodies, with secondary fluorescent antibodies (antimouse conjugated to Alexa Fluor 488 and anti–guinea pig, antirabbit, and antirat conjugated to cyanine-3, all used at 1:2000) and last revealed with ChemiDoc camera (Bio-Rad Laboratories).

Techniques: Binding Assay, Microscopy, Incubation, Labeling, Variant Assay, Mutagenesis, Staining, Control, In Vitro, Activity Assay, Transfection