anti lrba polyclonal antibody  (Millipore)

Journal: bioRxiv

Article Title: Endosomal LRBA regulates the endo-lysosomal pathway

doi: 10.1101/2024.02.07.579084

Figure Lengend Snippet: Distinct point mutations in the LRBA gene of two LRBA deficient patients cause mRNA decay and loss of the protein . (A) Schematic of LRBA protein structure with annotated domains. The genetic mutations carried by the two LRBA deficient patients investigated in this study are shown. Dermal fibroblasts obtained from these patients were used in this study. (B) LRBA mRNA levels in the two patient and two healthy donor (HD) fibroblast cell lines were determined by qRT-PCR. Mean and standard deviation are shown from n=4 biological replicates; one- way ANOVA using Dunnett’s multiple comparison, ****P<0.0001. (C) Immunoblot analysis of LRBA presence in fibroblasts of two HDs and two patient donors using polyclonal LRBA antibody and actin as a loading control. (D) Colocalization of LRBA and TGN46 in fibroblasts of HDs. LRBA is absent in patients’ fibroblasts. Cells were fixed, immunostained and imaged using a confocal microscope. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. H1: HD 1, H2: HD 2, P1: patient 1, P2: patient 2.

Article Snippet: The following antibodies were used in this study: sheep polyclonal anti-TGN46 (#AHP500G, BioRad, 1:2000), rabbit polyclonal LRBA antibody (#HPA023567, Atlas Antibodies, 1:1000), mouse AP1 100/3 hybridoma antibody (home-made), rabbit monoclonal EEA1 (C45B10) antibody (#3288, Cell Signaling, 1:2000), rabbit monoclonal anti LAMP1 (D2D11) XP® antibody (#9091, Cell Signaling, 1:200 for immunofluorescence, 1:1000 for western blot), rabbit monoclonal Rab7 (D95F2) XP® antibody (#9367, Cell Signaling, 1:400 for immunofluorescence, 1:1000 for western blotting), mouse monoclonal anti M6PR (cation dependent) (2G11) antibody (#ab2733, Abcam), rabbit polyclonal anti giantin antibody (#924302, BioLegend, 1:500), goat polyclonal anti Vps35 antibody (#ab10099, Abcam, 1:50), rabbit monoclonal anti EGF receptor (D38B1) XP® antibody (#4267, Cell Signaling, 1:1000), rabbit monoclonal phospho-EGF receptor (Tyr1068) (D7A5) XP® antibody (#3777, Cell Signaling, 1:1000), mouse monoclonal anti p44/42 MAPK (Erk1/2) (L334F10) antibody (#4696, Cell Signaling, 1:2000), rabbit polyclonal anti phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (#9101, Cell Signaling, 1:1000), mouse anti calnexin antibody clone 37 (#610523, BD Biosciences, 1:2000), mouse monoclonal anti Pan Actin (#LCU9001, Linaris, 1:1000), mouse monoclonal anti TCIRG1/ lysosomal ATPase V0 subunit a3 antibody (M01), clone 6H3 (#H00010312-M01, Abnova), rabbit monoclonal recombinant anti Cathepsin D [EPR3057Y] antibody (#ab75852, Abcam, 1:100 for immunofluorescence, 1:2000 for western blot), Hoechst 33342 Fluorescent Nucleic Acid Stain (#639, ImmunoChemistry Technologies, 1:200), goat anti-rabbit IgG(H+L) secondary antibody, AlexaFluor TM 488 (#A-11034, Invitrogen, 1:500), goat anti-Mouse IgG (H+L) secondary antibody, AlexaFluor TM 488 (#A11001, Invitrogen, 1:500), goat anti-mouse IgG (H+L) secondary antibody, AlexaFluor TM 633 (#A-21052, Invitrogen, 1:500), donkey anti-goat IgG (H+L) secondary antibody, AlexaFluor TM 488 (#A11055, Invitrogen, 1:500), donkey anti-mouse IgG (H+L) Secondary antibody, AlexaFluor TM 568 (#A10037, Invitrogen, 1:500), donkey anti-sheep IgG (H+L) secondary antibody, AlexaFluor TM 568 (#A21099, Invitrogen, 1:500), mouse monoclonal alpha-tubulin antibody (#T5168, Sigma-Aldrich, 1:10.000), goat anti-mouse IgG, (H+L), HRP-coupled (#31430, Pierce/Invitrogen, 1:10000), goat anti-rabbit IgG, (H+L), HRP- coupled (#31460, Pierce/Invitrogen, 1:10000), anti-CD107a-PB (#B13978, Beckman Coulter), anti-CD45-KrO (#B36294, Beckman Coulter), anti-CD3-APC (#IM2467, Beckman Coulter), anti-CD8-APC-AF700 (#B49181, Beckman Coulter), anti-CD69-PE (#IM1943U, Beckman Coulter), CD56-PC7 (#A21692, Beckman Coulter).

Techniques: Quantitative RT-PCR, Standard Deviation, Comparison, Western Blot, Control, Microscopy, Labeling

Journal: bioRxiv

Article Title: Endosomal LRBA regulates the endo-lysosomal pathway

doi: 10.1101/2024.02.07.579084

Figure Lengend Snippet: Immunoblot analysis of LRBA presence in HeLa cells of 2 control and 4 LRBA KO clones using polyclonal LRBA antibody and α -tubulin as a loading control. (B) LRBA is localized at the perinuclear region and on vesicular structures in HeLa cells. Immunofluorescence analysis of endogenous LRBA in fixed HeLa cells. Scale bar, 10 μm. (C) LRBA appears juxtaposed to the cis-Golgi in HeLa cells. Immunoblot analysis of endogenous LRBA and the cis- Golgi marker giantin colocalization in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (D) LRBA does not colocalize with M6PR in HeLa cells. Immunoblot analysis of endogenous LRBA and endogenous M6PR colocalization in fixed HeLa cells. Squares show magnification of the perinuclear area. The labeling of the single channels represents the color of the channel on the merged image. Scale bar, 10 μm, inlays 2 μm. (E) LRBA dissociates from membranes upon ArfGEF inhibitors. Live-cell imaging of 3xFlagEGFP-LRBA upon BFA (top panels) and GCA (lower panels) inhibition. Scale bar, 10 μm. (F) The amino acids isoleucine 49 and the aspargine 52 of Arf1 and Arf3 were predicted to interact with LRBA. Both amino acids are conserved across species. The amino acid sequences of the yeast, C.elegans (CAEEL), Drosophila melanogaster (DROME), mouse and human Arf1 and human Arf3 were aligned. Labels: (*) conserved sequence; (:) conservative mutation; (.) semi-conservative mutation; (-) gap.

Article Snippet: The following antibodies were used in this study: sheep polyclonal anti-TGN46 (#AHP500G, BioRad, 1:2000), rabbit polyclonal LRBA antibody (#HPA023567, Atlas Antibodies, 1:1000), mouse AP1 100/3 hybridoma antibody (home-made), rabbit monoclonal EEA1 (C45B10) antibody (#3288, Cell Signaling, 1:2000), rabbit monoclonal anti LAMP1 (D2D11) XP® antibody (#9091, Cell Signaling, 1:200 for immunofluorescence, 1:1000 for western blot), rabbit monoclonal Rab7 (D95F2) XP® antibody (#9367, Cell Signaling, 1:400 for immunofluorescence, 1:1000 for western blotting), mouse monoclonal anti M6PR (cation dependent) (2G11) antibody (#ab2733, Abcam), rabbit polyclonal anti giantin antibody (#924302, BioLegend, 1:500), goat polyclonal anti Vps35 antibody (#ab10099, Abcam, 1:50), rabbit monoclonal anti EGF receptor (D38B1) XP® antibody (#4267, Cell Signaling, 1:1000), rabbit monoclonal phospho-EGF receptor (Tyr1068) (D7A5) XP® antibody (#3777, Cell Signaling, 1:1000), mouse monoclonal anti p44/42 MAPK (Erk1/2) (L334F10) antibody (#4696, Cell Signaling, 1:2000), rabbit polyclonal anti phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (#9101, Cell Signaling, 1:1000), mouse anti calnexin antibody clone 37 (#610523, BD Biosciences, 1:2000), mouse monoclonal anti Pan Actin (#LCU9001, Linaris, 1:1000), mouse monoclonal anti TCIRG1/ lysosomal ATPase V0 subunit a3 antibody (M01), clone 6H3 (#H00010312-M01, Abnova), rabbit monoclonal recombinant anti Cathepsin D [EPR3057Y] antibody (#ab75852, Abcam, 1:100 for immunofluorescence, 1:2000 for western blot), Hoechst 33342 Fluorescent Nucleic Acid Stain (#639, ImmunoChemistry Technologies, 1:200), goat anti-rabbit IgG(H+L) secondary antibody, AlexaFluor TM 488 (#A-11034, Invitrogen, 1:500), goat anti-Mouse IgG (H+L) secondary antibody, AlexaFluor TM 488 (#A11001, Invitrogen, 1:500), goat anti-mouse IgG (H+L) secondary antibody, AlexaFluor TM 633 (#A-21052, Invitrogen, 1:500), donkey anti-goat IgG (H+L) secondary antibody, AlexaFluor TM 488 (#A11055, Invitrogen, 1:500), donkey anti-mouse IgG (H+L) Secondary antibody, AlexaFluor TM 568 (#A10037, Invitrogen, 1:500), donkey anti-sheep IgG (H+L) secondary antibody, AlexaFluor TM 568 (#A21099, Invitrogen, 1:500), mouse monoclonal alpha-tubulin antibody (#T5168, Sigma-Aldrich, 1:10.000), goat anti-mouse IgG, (H+L), HRP-coupled (#31430, Pierce/Invitrogen, 1:10000), goat anti-rabbit IgG, (H+L), HRP- coupled (#31460, Pierce/Invitrogen, 1:10000), anti-CD107a-PB (#B13978, Beckman Coulter), anti-CD45-KrO (#B36294, Beckman Coulter), anti-CD3-APC (#IM2467, Beckman Coulter), anti-CD8-APC-AF700 (#B49181, Beckman Coulter), anti-CD69-PE (#IM1943U, Beckman Coulter), CD56-PC7 (#A21692, Beckman Coulter).

Techniques: Western Blot, Control, Clone Assay, Immunofluorescence, Marker, Labeling, Live Cell Imaging, Inhibition, Sequencing, Mutagenesis

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Standardization of the LRBA staining in human peripheral blood cells. Phytohemaglutinin A (PHA)-stimulated peripheral blood mononuclear cells (PBMC) cells from healthy donors (HD) were obtained and immediately lysed to extract and quantified proteins (40 µg) that were tested in a Western blot with the antibody anti-LRBA (HPA023597, Sigma) using GAPDH as a constitutively expressed protein. Bands were detected at 319 and 37 KDa, respectively (A). Consecutively, ex vivo PB cells and PBMC stimulated w/o PHA (1 µg/mL) were intracellularly stained with the antibody anti-LRBA (HPA019366, Sigma) using anti Rabbit PE- F(ab)`2 Donkey IgG as a secondary antibody. Dotted lines indicate the staining only with the secondary antibody. Continued lines represent the staining with the anti-LRBA antibody together with the secondary antibody. Shown is the LRBA staining gating only the CD3+ cells (B). A Pearson correlation analysis was performed by comparing the intensity of the LRBA detection by western blot (LRBA band intensity) and the LRBA staining by FACS (Mean fluorescence intensity, MFI) (C). Also, a BLAST analysis (https://blast.ncbi.nlm.nih.gov) was performed to evaluate if the peptide recognized by the anti-LRBA antibodies used in this study were cross-reacting with NBEA (Sequence from www.ncbi.nlm.nih.gov/nuccore/NC_000013.11) (NBEA is a paralog of LRBA) (D). Considering that anti-LRBA antibodies obtained commercially are policlonal, the specificity of recognition was also evaluated using the LRBA peptide SVLMVSKYRDILEPQNERHSQSCTETGSENENVSLSEITPAAFSTLTTASVEESESTSSARRRDSGIGEETATGLGSHVEVTPHTAPPGVSAGPDAISEVLSTLSLEVNKSPETKNDRGNDLDTKATPSVSV as a competitive reagent at different concentrations. The percentage of the recognition of the cell LRBA protein was calculated (E).

Article Snippet: Furthermore, to evaluate LRBA staining specificity from the polyclonal antibody by FACS, the immunization LRBA peptide of the anti-LRBA polyclonal antibody (HPA019366, Sigma-Aldrich) was synthesized by ProteoGenix (Schiltigheim, France) and then diluted in dPBS (Sigma-Aldrich) at a final concentration of 4 mg/mL.

Techniques: Staining, Western Blot, Ex Vivo, Fluorescence, Sequencing

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: LRBA expression in PHA-stimulated PBMC from CVID022 by FACS. Ex-vivo periphericalblood cells and PBMC stimulated w/o PHA (1 μg/ml) were intracellularly stained with the antibody anti-LRBA (HPA019366, Sigma) using anti Rabbit PE- F(ab)`2 Donkey IgG as a secondary antibody. Dotted lines indicate the staining only with the secondary antibody. Continued lines represent the staining with the anti-LRBA antibody together with the secondary antibody. Shown is the LRBA staining gating only the CD3+ cells. The red line represents the LRBA mean fluorescence intensity (MFI) from the corresponding experiment in the HD

Article Snippet: Furthermore, to evaluate LRBA staining specificity from the polyclonal antibody by FACS, the immunization LRBA peptide of the anti-LRBA polyclonal antibody (HPA019366, Sigma-Aldrich) was synthesized by ProteoGenix (Schiltigheim, France) and then diluted in dPBS (Sigma-Aldrich) at a final concentration of 4 mg/mL.

Techniques: Expressing, Ex Vivo, Staining, Fluorescence

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Estandarización de la tinción de LRBA en células de sangre periférica humana. Se obtuvieron células mononucleares de sangre periférica (CMSP) estimuladas con fitohemaglutinina A (PHA) de donantes sanos (DS) y se lisaron inmediatamente para extraer y cuantificar proteínas (40 ug) que se analizaron en una transferencia Western Blot (WB) con el anticuerpo anti-LRBA (HPA023597 , Sigma) usando GAPDH como una proteína expresada constitutivamente. Se detectaron bandas a 319 y 37 KDa, respectivamente (A). Consecutivamente, las células de sangre periférica ex vivo y las CMSP estimuladas sin PHA (1 µg / ml) se tiñeron intracelularmente con el anticuerpo anti-LRBA (HPA019366, Sigma) usando anti-conejo PE-F (ab) `2 Donkey IgG como anticuerpo secundario. Las líneas punteadas indican la tinción solo con el anticuerpo secundario. Las líneas continuas representan la tinción con el anticuerpo anti-LRBA junto con el anticuerpo secundario. Se muestra la tinción de LRBA solamente las células CD3+ activadas (B). Se realizó un análisis de correlación de Pearson comparando la intensidad de la detección de LRBA por WB (intensidad de banda de LRBA) y la tinción de LRBA por FACS (intensidad de fluorescencia media, IMF) (C). Además, se realizó un análisis BLAST (https://blast.ncbi.nlm.nih.gov) para evaluar si el péptido reconocido por los anticuerpos anti-LRBA utilizados en este estudio tenían reacción cruzada con NBEA (Secuencia de www.ncbi .nlm.nih.gov/nuccore/NC_000013.11 ) (NBEA es un paralog de LRBA) (D). En función de los anticuerpos anti-LRBA que se comercializan son policlonales, la especificidad de reconocimiento también se evaluó mediante el péptido LRBA SVLMVSKYRDILEPQNERHSQSCTETGSENENVSLSEITPAAFSTLTTASVEESESTSSARRRDSGIGEETATGLGSHVEVTPHTAPPGVSAGPDAISEVLSTLSLEVNKSPETKNDRGNDLDTKATPSVSV como un reactivo competitivo a diferentes concentraciones. Se calculó el porcentaje de reconocimiento de la proteína LRBA celular (E).

Article Snippet: Furthermore, to evaluate LRBA staining specificity from the polyclonal antibody by FACS, the immunization LRBA peptide of the anti-LRBA polyclonal antibody (HPA019366, Sigma-Aldrich) was synthesized by ProteoGenix (Schiltigheim, France) and then diluted in dPBS (Sigma-Aldrich) at a final concentration of 4 mg/mL.

Techniques: Western Blot, Ex Vivo

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Expresión de LRBA en CMSP estimulada por PHA de CVID022 por FACS. Las células de sangre periférica ex-vivo y CMSP estimuladas sin PHA (1 μg / ml) se tiñeron intracelularmente con el anticuerpo anti-LRBA (HPA019366, Sigma) usando IgG de burro anti-conejo PE-F (ab) `2 como anticuerpo secundario. Las líneas punteadas indican la tinción solo con el anticuerpo secundario. Las líneas continuas representan la tinción con el anticuerpo anti-LRBA junto con el anticuerpo secundario. Se muestra la tinción de LRBA que activa solo las células CD3+. La línea roja representa la intensidad media de fluorescencia de LRBA (IMF) del experimento correspondiente en DS

Article Snippet: Furthermore, to evaluate LRBA staining specificity from the polyclonal antibody by FACS, the immunization LRBA peptide of the anti-LRBA polyclonal antibody (HPA019366, Sigma-Aldrich) was synthesized by ProteoGenix (Schiltigheim, France) and then diluted in dPBS (Sigma-Aldrich) at a final concentration of 4 mg/mL.

Techniques: Ex Vivo

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Standardization of the LRBA staining in human peripheral blood cells. Phytohemaglutinin A (PHA)-stimulated peripheral blood mononuclear cells (PBMC) cells from healthy donors (HD) were obtained and immediately lysed to extract and quantified proteins (40 µg) that were tested in a Western blot with the antibody anti-LRBA (HPA023597, Sigma) using GAPDH as a constitutively expressed protein. Bands were detected at 319 and 37 KDa, respectively (A). Consecutively, ex vivo PB cells and PBMC stimulated w/o PHA (1 µg/mL) were intracellularly stained with the antibody anti-LRBA (HPA019366, Sigma) using anti Rabbit PE- F(ab)`2 Donkey IgG as a secondary antibody. Dotted lines indicate the staining only with the secondary antibody. Continued lines represent the staining with the anti-LRBA antibody together with the secondary antibody. Shown is the LRBA staining gating only the CD3+ cells (B). A Pearson correlation analysis was performed by comparing the intensity of the LRBA detection by western blot (LRBA band intensity) and the LRBA staining by FACS (Mean fluorescence intensity, MFI) (C). Also, a BLAST analysis (https://blast.ncbi.nlm.nih.gov) was performed to evaluate if the peptide recognized by the anti-LRBA antibodies used in this study were cross-reacting with NBEA (Sequence from www.ncbi.nlm.nih.gov/nuccore/NC_000013.11) (NBEA is a paralog of LRBA) (D). Considering that anti-LRBA antibodies obtained commercially are policlonal, the specificity of recognition was also evaluated using the LRBA peptide SVLMVSKYRDILEPQNERHSQSCTETGSENENVSLSEITPAAFSTLTTASVEESESTSSARRRDSGIGEETATGLGSHVEVTPHTAPPGVSAGPDAISEVLSTLSLEVNKSPETKNDRGNDLDTKATPSVSV as a competitive reagent at different concentrations. The percentage of the recognition of the cell LRBA protein was calculated (E).

Article Snippet: Ex vivo PB cells and PBMC stimulated w/o PHA (10 µg/mL), were intracellularly stained with the polyclonal anti-LRBA antibody (HPA019366, Sigma-Aldrich) using anti Rabbit PE-F(ab)`2 Donkey IgG (Sigma-Aldrich) as a secondary antibody.

Techniques: Staining, Western Blot, Ex Vivo, Fluorescence, Sequencing

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: LRBA expression in PHA-stimulated PBMC from CVID022 by FACS. Ex-vivo periphericalblood cells and PBMC stimulated w/o PHA (1 μg/ml) were intracellularly stained with the antibody anti-LRBA (HPA019366, Sigma) using anti Rabbit PE- F(ab)`2 Donkey IgG as a secondary antibody. Dotted lines indicate the staining only with the secondary antibody. Continued lines represent the staining with the anti-LRBA antibody together with the secondary antibody. Shown is the LRBA staining gating only the CD3+ cells. The red line represents the LRBA mean fluorescence intensity (MFI) from the corresponding experiment in the HD

Article Snippet: Ex vivo PB cells and PBMC stimulated w/o PHA (10 µg/mL), were intracellularly stained with the polyclonal anti-LRBA antibody (HPA019366, Sigma-Aldrich) using anti Rabbit PE-F(ab)`2 Donkey IgG (Sigma-Aldrich) as a secondary antibody.

Techniques: Expressing, Ex Vivo, Staining, Fluorescence

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Estandarización de la tinción de LRBA en células de sangre periférica humana. Se obtuvieron células mononucleares de sangre periférica (CMSP) estimuladas con fitohemaglutinina A (PHA) de donantes sanos (DS) y se lisaron inmediatamente para extraer y cuantificar proteínas (40 ug) que se analizaron en una transferencia Western Blot (WB) con el anticuerpo anti-LRBA (HPA023597 , Sigma) usando GAPDH como una proteína expresada constitutivamente. Se detectaron bandas a 319 y 37 KDa, respectivamente (A). Consecutivamente, las células de sangre periférica ex vivo y las CMSP estimuladas sin PHA (1 µg / ml) se tiñeron intracelularmente con el anticuerpo anti-LRBA (HPA019366, Sigma) usando anti-conejo PE-F (ab) `2 Donkey IgG como anticuerpo secundario. Las líneas punteadas indican la tinción solo con el anticuerpo secundario. Las líneas continuas representan la tinción con el anticuerpo anti-LRBA junto con el anticuerpo secundario. Se muestra la tinción de LRBA solamente las células CD3+ activadas (B). Se realizó un análisis de correlación de Pearson comparando la intensidad de la detección de LRBA por WB (intensidad de banda de LRBA) y la tinción de LRBA por FACS (intensidad de fluorescencia media, IMF) (C). Además, se realizó un análisis BLAST (https://blast.ncbi.nlm.nih.gov) para evaluar si el péptido reconocido por los anticuerpos anti-LRBA utilizados en este estudio tenían reacción cruzada con NBEA (Secuencia de www.ncbi .nlm.nih.gov/nuccore/NC_000013.11 ) (NBEA es un paralog de LRBA) (D). En función de los anticuerpos anti-LRBA que se comercializan son policlonales, la especificidad de reconocimiento también se evaluó mediante el péptido LRBA SVLMVSKYRDILEPQNERHSQSCTETGSENENVSLSEITPAAFSTLTTASVEESESTSSARRRDSGIGEETATGLGSHVEVTPHTAPPGVSAGPDAISEVLSTLSLEVNKSPETKNDRGNDLDTKATPSVSV como un reactivo competitivo a diferentes concentraciones. Se calculó el porcentaje de reconocimiento de la proteína LRBA celular (E).

Article Snippet: Ex vivo PB cells and PBMC stimulated w/o PHA (10 µg/mL), were intracellularly stained with the polyclonal anti-LRBA antibody (HPA019366, Sigma-Aldrich) using anti Rabbit PE-F(ab)`2 Donkey IgG (Sigma-Aldrich) as a secondary antibody.

Techniques: Western Blot, Ex Vivo

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Expresión de LRBA en CMSP estimulada por PHA de CVID022 por FACS. Las células de sangre periférica ex-vivo y CMSP estimuladas sin PHA (1 μg / ml) se tiñeron intracelularmente con el anticuerpo anti-LRBA (HPA019366, Sigma) usando IgG de burro anti-conejo PE-F (ab) `2 como anticuerpo secundario. Las líneas punteadas indican la tinción solo con el anticuerpo secundario. Las líneas continuas representan la tinción con el anticuerpo anti-LRBA junto con el anticuerpo secundario. Se muestra la tinción de LRBA que activa solo las células CD3+. La línea roja representa la intensidad media de fluorescencia de LRBA (IMF) del experimento correspondiente en DS

Article Snippet: Ex vivo PB cells and PBMC stimulated w/o PHA (10 µg/mL), were intracellularly stained with the polyclonal anti-LRBA antibody (HPA019366, Sigma-Aldrich) using anti Rabbit PE-F(ab)`2 Donkey IgG (Sigma-Aldrich) as a secondary antibody.

Techniques: Ex Vivo

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Standardization of the LRBA staining in human peripheral blood cells. Phytohemaglutinin A (PHA)-stimulated peripheral blood mononuclear cells (PBMC) cells from healthy donors (HD) were obtained and immediately lysed to extract and quantified proteins (40 µg) that were tested in a Western blot with the antibody anti-LRBA (HPA023597, Sigma) using GAPDH as a constitutively expressed protein. Bands were detected at 319 and 37 KDa, respectively (A). Consecutively, ex vivo PB cells and PBMC stimulated w/o PHA (1 µg/mL) were intracellularly stained with the antibody anti-LRBA (HPA019366, Sigma) using anti Rabbit PE- F(ab)`2 Donkey IgG as a secondary antibody. Dotted lines indicate the staining only with the secondary antibody. Continued lines represent the staining with the anti-LRBA antibody together with the secondary antibody. Shown is the LRBA staining gating only the CD3+ cells (B). A Pearson correlation analysis was performed by comparing the intensity of the LRBA detection by western blot (LRBA band intensity) and the LRBA staining by FACS (Mean fluorescence intensity, MFI) (C). Also, a BLAST analysis (https://blast.ncbi.nlm.nih.gov) was performed to evaluate if the peptide recognized by the anti-LRBA antibodies used in this study were cross-reacting with NBEA (Sequence from www.ncbi.nlm.nih.gov/nuccore/NC_000013.11) (NBEA is a paralog of LRBA) (D). Considering that anti-LRBA antibodies obtained commercially are policlonal, the specificity of recognition was also evaluated using the LRBA peptide SVLMVSKYRDILEPQNERHSQSCTETGSENENVSLSEITPAAFSTLTTASVEESESTSSARRRDSGIGEETATGLGSHVEVTPHTAPPGVSAGPDAISEVLSTLSLEVNKSPETKNDRGNDLDTKATPSVSV as a competitive reagent at different concentrations. The percentage of the recognition of the cell LRBA protein was calculated (E).

Article Snippet: The peptides of the anti-LRBA polyclonal antibodies (HPA019366 and HPA023597, Sigma-Aldrich) were subjected to a blast analysis against all reference proteins using the following parameters: Database: refseq protein; Organism: Homo sapiens (taxid:9606); Algorithm: blatstp (protein-protein BLAST).

Techniques: Staining, Western Blot, Ex Vivo, Fluorescence, Sequencing

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: LRBA expression in patients. One hundred and twelve patients were recruited to evaluate the LRBA expression by westerm blot (WB). PHA-stimulated PBMC from 25 healthy donors and patients were obtain and lysed to extract and quantify proteins (40 μg) that were tested in a WB with the anti-LRBA antibody Ab HPA023597 (Sigma) using either GAPDH or TUBA as a constitutively expressed protein. Bands were detected at 319 kDa for LRBA, 37 KDa for GAPDH and 49 KDa for TUBA. Normalization of the LRBA band intensity against the constitutive protein (GAPDH or TUBA) is shown in (A). WB band intensities of LRBA in PHA-stimulated cells from patients with LRBA decreased expression is shown in (B). CVID: Common Variable Immunodeficiency, XLA: X-linked agammaglobulinemia, IHG: Hypogammaglobulinemia, SIgAD: Selective IgA Deficiency, ITP: Idiopathic thrombocytopenic purpura, SLE: Systemic Lupus Erythematosus. The gray bars represent the repeated samples. The red arrows indicate the patients with decrease in LRBA who continued in the study

Article Snippet: The peptides of the anti-LRBA polyclonal antibodies (HPA019366 and HPA023597, Sigma-Aldrich) were subjected to a blast analysis against all reference proteins using the following parameters: Database: refseq protein; Organism: Homo sapiens (taxid:9606); Algorithm: blatstp (protein-protein BLAST).

Techniques: Expressing

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: LRBA expression in PHA-stimulated PBMC from CVID022 by FACS. Ex-vivo periphericalblood cells and PBMC stimulated w/o PHA (1 μg/ml) were intracellularly stained with the antibody anti-LRBA (HPA019366, Sigma) using anti Rabbit PE- F(ab)`2 Donkey IgG as a secondary antibody. Dotted lines indicate the staining only with the secondary antibody. Continued lines represent the staining with the anti-LRBA antibody together with the secondary antibody. Shown is the LRBA staining gating only the CD3+ cells. The red line represents the LRBA mean fluorescence intensity (MFI) from the corresponding experiment in the HD

Article Snippet: The peptides of the anti-LRBA polyclonal antibodies (HPA019366 and HPA023597, Sigma-Aldrich) were subjected to a blast analysis against all reference proteins using the following parameters: Database: refseq protein; Organism: Homo sapiens (taxid:9606); Algorithm: blatstp (protein-protein BLAST).

Techniques: Expressing, Ex Vivo, Staining, Fluorescence

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Estandarización de la tinción de LRBA en células de sangre periférica humana. Se obtuvieron células mononucleares de sangre periférica (CMSP) estimuladas con fitohemaglutinina A (PHA) de donantes sanos (DS) y se lisaron inmediatamente para extraer y cuantificar proteínas (40 ug) que se analizaron en una transferencia Western Blot (WB) con el anticuerpo anti-LRBA (HPA023597 , Sigma) usando GAPDH como una proteína expresada constitutivamente. Se detectaron bandas a 319 y 37 KDa, respectivamente (A). Consecutivamente, las células de sangre periférica ex vivo y las CMSP estimuladas sin PHA (1 µg / ml) se tiñeron intracelularmente con el anticuerpo anti-LRBA (HPA019366, Sigma) usando anti-conejo PE-F (ab) `2 Donkey IgG como anticuerpo secundario. Las líneas punteadas indican la tinción solo con el anticuerpo secundario. Las líneas continuas representan la tinción con el anticuerpo anti-LRBA junto con el anticuerpo secundario. Se muestra la tinción de LRBA solamente las células CD3+ activadas (B). Se realizó un análisis de correlación de Pearson comparando la intensidad de la detección de LRBA por WB (intensidad de banda de LRBA) y la tinción de LRBA por FACS (intensidad de fluorescencia media, IMF) (C). Además, se realizó un análisis BLAST (https://blast.ncbi.nlm.nih.gov) para evaluar si el péptido reconocido por los anticuerpos anti-LRBA utilizados en este estudio tenían reacción cruzada con NBEA (Secuencia de www.ncbi .nlm.nih.gov/nuccore/NC_000013.11 ) (NBEA es un paralog de LRBA) (D). En función de los anticuerpos anti-LRBA que se comercializan son policlonales, la especificidad de reconocimiento también se evaluó mediante el péptido LRBA SVLMVSKYRDILEPQNERHSQSCTETGSENENVSLSEITPAAFSTLTTASVEESESTSSARRRDSGIGEETATGLGSHVEVTPHTAPPGVSAGPDAISEVLSTLSLEVNKSPETKNDRGNDLDTKATPSVSV como un reactivo competitivo a diferentes concentraciones. Se calculó el porcentaje de reconocimiento de la proteína LRBA celular (E).

Article Snippet: The peptides of the anti-LRBA polyclonal antibodies (HPA019366 and HPA023597, Sigma-Aldrich) were subjected to a blast analysis against all reference proteins using the following parameters: Database: refseq protein; Organism: Homo sapiens (taxid:9606); Algorithm: blatstp (protein-protein BLAST).

Techniques: Western Blot, Ex Vivo

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Expresión de LRBA en pacientes . Ciento doce pacientes fueron reclutados para evaluar la expresión de LRBA por westerm blot (WB). Se obtuvieron CMSP estimuladas con PHA de 25 donantes sanos para extraer y cuantificar proteínas (40 μg) que se analizaron en un WB con el anticuerpo anti-LRBA Ab HPA023597 (Sigma) usando GAPDH o TUBA como proteína expresada constitutivamente. Se detectaron bandas a 319 kDa para LRBA, 37 KDa para GAPDH y 49 KDa para TUBA. La normalización de la intensidad de la banda LRBA contra la proteína constitutiva (GAPDH o TUBA) se muestra en (A). Las intensidades de la banda WB de LRBA en células estimuladas con PHA de pacientes con expresión disminuida de LRBA se muestran en (B). ICV o CVID: inmunodeficiencia común variable, XLA: agammaglobulinemia ligada a X, IHG: hipogammaglobulinemia, SIgAD: deficiencia selectiva de IgA, PTI: púrpura trombocitopénica idiopática, LES: lupus eritematoso sistémico. Las barras grises representan las muestras repetidas. Las flechas rojas indican los pacientes con disminución de LRBA que continuaron en el estudio.

Article Snippet: The peptides of the anti-LRBA polyclonal antibodies (HPA019366 and HPA023597, Sigma-Aldrich) were subjected to a blast analysis against all reference proteins using the following parameters: Database: refseq protein; Organism: Homo sapiens (taxid:9606); Algorithm: blatstp (protein-protein BLAST).

Techniques:

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Expresión de LRBA en CMSP estimulada por PHA de CVID022 por FACS. Las células de sangre periférica ex-vivo y CMSP estimuladas sin PHA (1 μg / ml) se tiñeron intracelularmente con el anticuerpo anti-LRBA (HPA019366, Sigma) usando IgG de burro anti-conejo PE-F (ab) `2 como anticuerpo secundario. Las líneas punteadas indican la tinción solo con el anticuerpo secundario. Las líneas continuas representan la tinción con el anticuerpo anti-LRBA junto con el anticuerpo secundario. Se muestra la tinción de LRBA que activa solo las células CD3+. La línea roja representa la intensidad media de fluorescencia de LRBA (IMF) del experimento correspondiente en DS

Article Snippet: The peptides of the anti-LRBA polyclonal antibodies (HPA019366 and HPA023597, Sigma-Aldrich) were subjected to a blast analysis against all reference proteins using the following parameters: Database: refseq protein; Organism: Homo sapiens (taxid:9606); Algorithm: blatstp (protein-protein BLAST).

Techniques: Ex Vivo

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Standardization of the LRBA staining in human peripheral blood cells. Phytohemaglutinin A (PHA)-stimulated peripheral blood mononuclear cells (PBMC) cells from healthy donors (HD) were obtained and immediately lysed to extract and quantified proteins (40 µg) that were tested in a Western blot with the antibody anti-LRBA (HPA023597, Sigma) using GAPDH as a constitutively expressed protein. Bands were detected at 319 and 37 KDa, respectively (A). Consecutively, ex vivo PB cells and PBMC stimulated w/o PHA (1 µg/mL) were intracellularly stained with the antibody anti-LRBA (HPA019366, Sigma) using anti Rabbit PE- F(ab)`2 Donkey IgG as a secondary antibody. Dotted lines indicate the staining only with the secondary antibody. Continued lines represent the staining with the anti-LRBA antibody together with the secondary antibody. Shown is the LRBA staining gating only the CD3+ cells (B). A Pearson correlation analysis was performed by comparing the intensity of the LRBA detection by western blot (LRBA band intensity) and the LRBA staining by FACS (Mean fluorescence intensity, MFI) (C). Also, a BLAST analysis (https://blast.ncbi.nlm.nih.gov) was performed to evaluate if the peptide recognized by the anti-LRBA antibodies used in this study were cross-reacting with NBEA (Sequence from www.ncbi.nlm.nih.gov/nuccore/NC_000013.11) (NBEA is a paralog of LRBA) (D). Considering that anti-LRBA antibodies obtained commercially are policlonal, the specificity of recognition was also evaluated using the LRBA peptide SVLMVSKYRDILEPQNERHSQSCTETGSENENVSLSEITPAAFSTLTTASVEESESTSSARRRDSGIGEETATGLGSHVEVTPHTAPPGVSAGPDAISEVLSTLSLEVNKSPETKNDRGNDLDTKATPSVSV as a competitive reagent at different concentrations. The percentage of the recognition of the cell LRBA protein was calculated (E).

Article Snippet: Proteins were transferred onto polyvinylidenedifluoride (PVDF) membranes (Bio-Rad) and incubated with polyclonal rabbit anti-human LRBA antibody (HPA023597, Sigma-Aldrich), anti-GAPDH (Sigma-Aldrich) and anti-rabbit IgG-peroxidase antibody as secondary antibody (Sigma-Aldrich), were used for the protein detection.

Techniques: Staining, Western Blot, Ex Vivo, Fluorescence, Sequencing

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: LRBA expression in patients. One hundred and twelve patients were recruited to evaluate the LRBA expression by westerm blot (WB). PHA-stimulated PBMC from 25 healthy donors and patients were obtain and lysed to extract and quantify proteins (40 μg) that were tested in a WB with the anti-LRBA antibody Ab HPA023597 (Sigma) using either GAPDH or TUBA as a constitutively expressed protein. Bands were detected at 319 kDa for LRBA, 37 KDa for GAPDH and 49 KDa for TUBA. Normalization of the LRBA band intensity against the constitutive protein (GAPDH or TUBA) is shown in (A). WB band intensities of LRBA in PHA-stimulated cells from patients with LRBA decreased expression is shown in (B). CVID: Common Variable Immunodeficiency, XLA: X-linked agammaglobulinemia, IHG: Hypogammaglobulinemia, SIgAD: Selective IgA Deficiency, ITP: Idiopathic thrombocytopenic purpura, SLE: Systemic Lupus Erythematosus. The gray bars represent the repeated samples. The red arrows indicate the patients with decrease in LRBA who continued in the study

Article Snippet: Proteins were transferred onto polyvinylidenedifluoride (PVDF) membranes (Bio-Rad) and incubated with polyclonal rabbit anti-human LRBA antibody (HPA023597, Sigma-Aldrich), anti-GAPDH (Sigma-Aldrich) and anti-rabbit IgG-peroxidase antibody as secondary antibody (Sigma-Aldrich), were used for the protein detection.

Techniques: Expressing

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Estandarización de la tinción de LRBA en células de sangre periférica humana. Se obtuvieron células mononucleares de sangre periférica (CMSP) estimuladas con fitohemaglutinina A (PHA) de donantes sanos (DS) y se lisaron inmediatamente para extraer y cuantificar proteínas (40 ug) que se analizaron en una transferencia Western Blot (WB) con el anticuerpo anti-LRBA (HPA023597 , Sigma) usando GAPDH como una proteína expresada constitutivamente. Se detectaron bandas a 319 y 37 KDa, respectivamente (A). Consecutivamente, las células de sangre periférica ex vivo y las CMSP estimuladas sin PHA (1 µg / ml) se tiñeron intracelularmente con el anticuerpo anti-LRBA (HPA019366, Sigma) usando anti-conejo PE-F (ab) `2 Donkey IgG como anticuerpo secundario. Las líneas punteadas indican la tinción solo con el anticuerpo secundario. Las líneas continuas representan la tinción con el anticuerpo anti-LRBA junto con el anticuerpo secundario. Se muestra la tinción de LRBA solamente las células CD3+ activadas (B). Se realizó un análisis de correlación de Pearson comparando la intensidad de la detección de LRBA por WB (intensidad de banda de LRBA) y la tinción de LRBA por FACS (intensidad de fluorescencia media, IMF) (C). Además, se realizó un análisis BLAST (https://blast.ncbi.nlm.nih.gov) para evaluar si el péptido reconocido por los anticuerpos anti-LRBA utilizados en este estudio tenían reacción cruzada con NBEA (Secuencia de www.ncbi .nlm.nih.gov/nuccore/NC_000013.11 ) (NBEA es un paralog de LRBA) (D). En función de los anticuerpos anti-LRBA que se comercializan son policlonales, la especificidad de reconocimiento también se evaluó mediante el péptido LRBA SVLMVSKYRDILEPQNERHSQSCTETGSENENVSLSEITPAAFSTLTTASVEESESTSSARRRDSGIGEETATGLGSHVEVTPHTAPPGVSAGPDAISEVLSTLSLEVNKSPETKNDRGNDLDTKATPSVSV como un reactivo competitivo a diferentes concentraciones. Se calculó el porcentaje de reconocimiento de la proteína LRBA celular (E).

Article Snippet: Proteins were transferred onto polyvinylidenedifluoride (PVDF) membranes (Bio-Rad) and incubated with polyclonal rabbit anti-human LRBA antibody (HPA023597, Sigma-Aldrich), anti-GAPDH (Sigma-Aldrich) and anti-rabbit IgG-peroxidase antibody as secondary antibody (Sigma-Aldrich), were used for the protein detection.

Techniques: Western Blot, Ex Vivo

Journal: Colombia Médica : CM

Article Title: Clinical, immunological and genetic characteristic of patients with clinical phenotype associated to LRBA-deficiency in Colombia

doi: 10.25100/cm.v50i3.3969

Figure Lengend Snippet: Expresión de LRBA en pacientes . Ciento doce pacientes fueron reclutados para evaluar la expresión de LRBA por westerm blot (WB). Se obtuvieron CMSP estimuladas con PHA de 25 donantes sanos para extraer y cuantificar proteínas (40 μg) que se analizaron en un WB con el anticuerpo anti-LRBA Ab HPA023597 (Sigma) usando GAPDH o TUBA como proteína expresada constitutivamente. Se detectaron bandas a 319 kDa para LRBA, 37 KDa para GAPDH y 49 KDa para TUBA. La normalización de la intensidad de la banda LRBA contra la proteína constitutiva (GAPDH o TUBA) se muestra en (A). Las intensidades de la banda WB de LRBA en células estimuladas con PHA de pacientes con expresión disminuida de LRBA se muestran en (B). ICV o CVID: inmunodeficiencia común variable, XLA: agammaglobulinemia ligada a X, IHG: hipogammaglobulinemia, SIgAD: deficiencia selectiva de IgA, PTI: púrpura trombocitopénica idiopática, LES: lupus eritematoso sistémico. Las barras grises representan las muestras repetidas. Las flechas rojas indican los pacientes con disminución de LRBA que continuaron en el estudio.

Article Snippet: Proteins were transferred onto polyvinylidenedifluoride (PVDF) membranes (Bio-Rad) and incubated with polyclonal rabbit anti-human LRBA antibody (HPA023597, Sigma-Aldrich), anti-GAPDH (Sigma-Aldrich) and anti-rabbit IgG-peroxidase antibody as secondary antibody (Sigma-Aldrich), were used for the protein detection.

Techniques: