Journal: NPJ Vaccines

Article Title: Deimmunization of flagellin for repeated administration as a vaccine adjuvant

doi: 10.1038/s41541-021-00379-4

Figure Lengend Snippet: a Characterization of dFlaB and FlaB ΔD2D3 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), native-PAGE, and subsequent Western blot analysis with mouse anti-FlaB serum induced by intraperitoneal immunization of FlaB formulated complete Freund’s adjuvant. b Determination of TLR5-dependent NF-κB stimulating activity by FlaB, dFlaB, and FlaB ΔD2D3 . The relative NF-κB activities were analyzed by using HEK-Blue TM hTLR5 cells and HEK-Blue TM detection assay systems. EC50 were calculated using triplicate OD 620 nm values for each protein concentration over a wide range of protein concentrations (0.03719 nM to 76.16 nM at the AAT Bioquest website ( https://www.aatbio.com/tools/ec50-calculator ). The same molar ratio of proteins was used, and PBS was used as a negative control.

Article Snippet: We measured TLR5-dependent NFκB stimulating activity of the recombinant proteins by using HEK-Blue TM hTLR5 cells (InvivoGen, hκb-htlr-5) and HEK-Blue TM Detection (InvivoGen, hb-det2) assay systems following the manufacturer’s instruction.

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Clear Native PAGE, Western Blot, Adjuvant, Activity Assay, Detection Assay, Protein Concentration, Negative Control

Journal: EMBO Reports

Article Title: LRBA deficiency impairs autophagy and contributes to enhanced antigen presentation and T-cell dysregulation

doi: 10.1038/s44319-025-00504-7

Figure Lengend Snippet: ( A ) Schematic representation of PI3K-III complex activity and the initiation of autophagosome formation (omegasome). ( B ) PI(3)P levels were measured in lipid extracts of WT (grey) and LRBA-KO (red) HEK293T cells under basal conditions, or upon treatment with 333 nM of Torin 1 alone, or in combination with 1 nM of VPS34-IN1 (PI3K-III complex inhibitor) for 4 h. Each dot represents technical replicates ( n = 2) from one experiment, while bars represent the mean ± SD of n = 4 independent biological experiments. ( C , D ) Quantification of ( C ) DFCP1 and ( D ) WIPI2 signal in WT (grey) and LRBA-KO (red) HEK293T cells immunostained with either ( C ) anti-DFCP-1 or ( D ) anti-WIPI2 antibody after incubation for 1 h with either control medium, 50 µM Rapamycin, or 333 nM of Torin 1. Each dot represents the mean of one experiment while the bars represent the mean ± SD of n = 3 independent biological experiments. Total cells analyzed in ( C , D ) were: basal conditions, WT = 27/59, KO = 21/35; Rapamycin, WT = 44/70, KO = 49/91 and Torin 1, WT = 52/51, KO = 49/41. ( E ) Representative confocal microscopy images of WT (left) and LRBA-KO (right) HEK293T cells immunostained with anti-DFCP1 (green) or anti-WIPI2 (red) under the indicated conditions. Scale bar = 5 µm. ( F ) Representative confocal microscopy images (Scale bar=5 µm) and ( G ) quantification of DFCP1 signal in WT (grey) and LRBA-KO (red) HEK293T cells immunostained with anti-DFCP1 after treatment with the indicated concentrations of VPS34-IN1 for 1 h followed by 1 h incubation with 333 nM Torin 1. Each dot represents one image analyzed and the mean ± SD was calculated from n = 2 independent biological experiments. Total cells analyzed in WT: 0 μM = 46, 0.01 μM = 42, 0.1 μM = 42 and in KO: 0 μM = 29, 0.01 μM = 110, 0.1 μM = 94. Scale bar = 5 µm. ( H , I ) LRBA reconstitution restores DFCP1 recruitment. ( H ) Representative confocal microscopy images and ( I ) quantification of DFCP-1 expression in WT (grey), LRBA-KO (red) and Myc-LRBA (teal) HEK293T cells after stimulation for 1 h with control medium or 333 nM Torin 1. Each dot represents the mean of one experiment while bars represent the mean ± SD of n = 3 independent biological replicates. Total cells analyzed in WT (basal/Torin): 158/244 cells; LRBA-KO: 269/294 cells and Myc-LRBA: 152/273. Statistical analyses of ( B – D ) was performed using a two-way ANOVA with Bonferroni’s multiple comparisons test and for ( I ) a two-way ANOVA with Tukey’s multiple comparisons test, ** P < 0.01 (( I ): P = 0.0014, Basal LRBA-KO vs Myc-LRBA), (( I ): P = 0.0037, Torin1 WT vs Myc-LRBA), **** P < 0.0001. .

Article Snippet: Rapamycin , InvivoGen , tlrl-rap.

Techniques: Activity Assay, Incubation, Control, Confocal, Microscopy, Expressing, Immunofluorescence

Journal: EMBO Reports

Article Title: LRBA deficiency impairs autophagy and contributes to enhanced antigen presentation and T-cell dysregulation

doi: 10.1038/s44319-025-00504-7

Figure Lengend Snippet: ( A ) Representative immunoblot analyses of p62 degradation in WT, LRBA-KO and Myc-LRBA HEK293T cells at basal conditions or after Torin 1 stimulation. ( B ) Densitometry analysis of ( A ) of WT (grey), LRBA-KO (red) and Myc-LRBA (teal) HEK293T cells. Each dot represents the densitometry analysis of one blot while bars represent the mean ± SD from n = 6 independent biological replicates. ( C , D ) Representative histograms of p62 degradation in WT (grey) and LRBA-KO (red) Ramos B cells in the presence and absence of Rapamycin, with or without Bafilomycin A1 and ( D ) fold change of p62 degradation normalized to basal levels. Each dot represents the fold change of one technical replicate while bars represent the mean ± SD from n = 4–5 independent biological replicates. ( E , F ) Reduced autophagy flux in splenic naive B cells from Lrba −/− mice. ( E ) Representative immunoblot analyses of LC3-I and LC3-II processing and Tubulin in isolated splenocytes from WT and Lrba −/− mice at day 0 or upon stimulation for 3 days with 20 µg/ml LPS alone or in the presence of 100 nM Bafilomycin A1 or protease inhibitors E64D and Pepstatin. ( F ) Densitometry analyses of LC3-II expression relative to Tubulin of splenic murine naive B cells from WT (grey) and Lrba−/− (red) mice. Each dot represents the densitometry analysis of one blot while bars represent the mean ± SD from n = 2 independent biological replicates. ( G ) Representative immunoblot analyses of the processing of endogenous unconjugated LC3-I to lipid-conjugated LC3-II in shControl and shLRBA HeLa cells at resting conditions or after 16 h or 24 h of MG132 alone or in the presence of 100 nM Bafilomycin A1. ( H ) Densitometry analyses of LC3-II expression relative to GAPDH of shControl (grey) and shLRBA (red) HeLa cells. Each dot represents the densitometry analysis of one blot while bars represent the mean ± SD from n = 4 independent biological replicates. ( I ) Representative immunoblot analyses of p62 and GAPDH in shControl and shLRBA HeLa cells at resting conditions or after 16 h treatment with MG132 alone or along with 100 nM Bafilomycin A1. ( J ) Densitometry analyses of p62 relative to GAPDH and normalized to basal conditions of shControl (grey) and shLRBA (red) HeLa cells. Each dot represents the densitometry analysis of one blot while bars represent the mean ± SD from n = 4 independent biological replicates. Statistical analysis for ( B ) was performed using a one-way ANOVA with Tukey’s multiple comparisons test and for ( D , H , I ) a two-way ANOVA with Bonferroni’s multiple comparisons test, * P < 0.05 ( B : P = 0.02 WT vs KO and P = 0.0146 KO vs Myc-LRBA), ( D : P = 0.0237 Rapamycin and P = 0.0132 Rapamycin+Bafilomycin A1), ** P < 0.01 (( H ): P = 0.0027), *** P < 0.001 (( H ): P = 0.0002), **** P < 0.0001.

Article Snippet: Rapamycin , InvivoGen , tlrl-rap.

Techniques: Western Blot, Analysis, Isolation, Expressing, FACS

Journal: EMBO Reports

Article Title: LRBA deficiency impairs autophagy and contributes to enhanced antigen presentation and T-cell dysregulation

doi: 10.1038/s44319-025-00504-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rapamycin , InvivoGen , tlrl-rap.

Techniques: Generated, Virus, Recombinant, Gentle, Cell-Signaling, Sequencing, CRISPR, Cell Isolation, In Situ, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Membrane, Software, Docking Assay, Tissue, Transmission Assay, Microscopy