phosphorylated cd79a (pcd79a, y182)  (Cell Signaling Technology Inc)

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 KD does not reduce the expression of Hem1 protein and does not affect BCR signaling in response to immobilized anti-Ig. ( A ) A20 cells were transfected with control siRNA or WAVE2 siRNA, cultured for 48 h, and then analyzed via immunoblotting with Abs to WAVE2, Hem1, or actin (loading control). Normalized levels of WAVE2 or Hem1 relative to control siRNA-transfected cells are shown. ( B ) A20 cells that had been transfected with control siRNA or WAVE2 siRNA were allowed to spread on anti-IgG-coated tissue culture wells for the indicated times before analyzing BCR signaling by immunoblotting for the phosphorylation of the CD79a ITAM (pCD79a) or the phosphorylated (activated) forms of ERK (pERK) or Akt (pAkt). The same cell lysates were probed for total CD79a, ERK, or Akt. The dashed red line was overlaid on the images of the blots to visually separate the time courses for the control siRNA and WAVE2 siRNA-transfected cells. For both panels, one of three independent experiments with similar results is shown. Full uncropped blots are shown in .

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Expressing, Transfection, Control, Cell Culture, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: N-WASP-dependent branched actin polymerization attenuates B-cell receptor signaling by increasing the molecular density of receptor clusters

doi: 10.1101/2023.03.14.532631

Figure Lengend Snippet: ( A-G ) Flox control and cNKO B-cells incubated with AF546-Fab’-PLB were fixed at 1, 3, 5, and 7 min, permeabilized, stained for phosphorylated CD79a (pCD79a, Tyr182), and imaged using TIRF and IRM. ( A ) Representative IRM and TIRF images of a flox control versus a cNKO B-cell at 7 min. Scale bars, 2 µm. ( B-D ) Ratios of pCD79a MFI relative to AF546-Fab’ MFI were plotted against AF546-Fab’ peak FI in individual AF546-Fab’ clusters in the contact zone of flox control ( B ), cNKO B-cells ( C ), or flox control and cNKO B-cells overlay ( D ). AF546-Fab’ clusters were identified as described in and Figure 6-figure supplement 2, from an equal number of cells at the 4 time points. Data points represent individual AF546-Fab’ clusters with an equal number of clusters from each time point. The black diamond symbols represent the average ratios of pCD79a MFI to Fab’ MFI in individual BCR-Fab’ clusters within the indicated Fab’ peak FI range. The brown square symbols represent the fraction of the AF546-Fab’ clusters out of the total, within the indicated Fab’ peak FI range. Data were generated from 3 independent experiments with ∼20 cells and ≥125 clusters per condition per experiment. * p <0.05, *** p <0.001, by non-parametric student’s t -test. ( E-G ) The MFI (±SEM) of AF546-Fab’ ( E ) and pCD79a ( F ) and the MFI ratio (±SEM) of pCD79a relative to AF546-Fab’ ( G ) in individual AF546-Fab’ clusters at indicated times were compared between flox control and cNKO B-cells and between different times. ( H-K ) WT B-cells treated with CK-689 or CK-666 after 2 min-incubation with AF546-Fab’-PLB. ( H ) Representative IRM and TIRF images of a CK-689-versus a CK-666-treated B-cell at 7 min. Scale bars, 2 µm. ( I-K ) The MFI (±SEM) of AF546-Fab’ ( I ) and pCD79a ( J ) and the MFI ratio (±SEM) of pCD79a relative to AF546-Fab’ ( K ) in individual AF546-Fab’ clusters were compared between CK-689- and CK-666-treated B-cells after 7 min stimulation. Data points represent individual clusters. Horizontal solid lines in the violin plots represent the mean, while the dotted lines represent the quartiles of the distribution. Data were generated from 3 independent experiments with ∼20 cells per condition per experiment. * p <0.05, *** p <0.001, by non-parametric student’s t -test. The p -values in D were corrected using the Benjamini-Hochberg/Yekutieli method for false discovery rate control.

Article Snippet: Splenic B-cells were incubated with AF546-Fab’-PLB at 37°C and fixed at 1, 3, 5, 7, and 9 min. After fixation, cells were stained for phosphorylated CD79a (pCD79a, Y182) (Cell Signaling Technology), Syk (pSyk, Y519/520) (Cell Signaling Technology), SHIP-1 (pSHIP-1, Y1020) (Cell Signaling Technology), or total Syk or SHIP-1 proteins (Thermo Fisher Scientific).

Techniques: Incubation, Staining, Generated

Journal: International immunology

Article Title: Multiple tolerance checkpoints restrain affinity maturation of B cells expressing the germline precursor of a lupus patient-derived anti-dsDNA antibody in knock-in mice.

doi: 10.1093/intimm/dxab111

Figure Lengend Snippet: Fig. 5. CpG-containing ssDNA enhances proliferation and CSR, but inhibits plasma cell differentiation. (A and B) Fo B cells from B6, B1-8hi, or G9gl HL homozygous mice (A) or Tlr9-decificent G9gl homozygous mice (B) were stimulated in vitro with anti-CD40 antibody, IL-4, and IL-5 for 4 d with or without endotoxin-free bacterial ssDNA (1 μg/ml). To antagonize TLR9, 1 μM of ODN2088 (TLR9 antagonist) was added to the cul- ture 1 h before ssDNA stimulation. n = 2–5. Two-tailed Mann–Whitney test. *P < 0.05. (C) Phosphorylated CD79a of Fo B cells from B6 or G9gl HL homozygous mice before or after BCR ligation was detected by intracellular FACS. FACS sorted Fo B cells were stimulated by 10 μg/ml of anti-IgM F(ab’)2 for 5 and 10 min. For the histogram, cells were gated for lymphocyte 7AAD− B220+. ΔMFI is shown in the graph. n = 2 each. The representative results from two experiments are shown.

Article Snippet: For intracellular staining of AKT, phosphorylated CD79a, and SHIP1, cells were stained by antibody against surface markers and then fixed and permeabilized with Cytofix/ Cytoperm (BD) to be reacted with and rabbit anti-AKT (Cell Signaling Technology), anti-phosphorylated CD79a (Tyr 182) (Cell Signaling Technology), or anti-SHIP1 (Abcam) followed by Alexa647-conjugated anti-rabbit IgG (Thermo Fisher).

Techniques: Clinical Proteomics, Cell Differentiation, In Vitro, Two Tailed Test, MANN-WHITNEY, Ligation

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

doi: 10.3389/fcell.2021.647063

Figure Lengend Snippet: Depleting GMFγ reduces cSMAC formation and proximal BCR signaling at the immune synapse. Raji D1.3 B cells were transfected with either control siRNA or GMFγ siRNA and added to COS-7 APCs expressing the mHEL-HaloTag Ag (magenta). The cells were fixed at the indicated times and stained with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79, cyan). The B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images from one of five independent experiments. Scale bars: 5 μm. (B) The total fluorescence intensity of the mHEL-HaloTag Ag that had been gathered into clusters at the B cell-APC contact site was quantified for each B cell and the median values were calculated for each time point. Each symbol on the graph represents the median value for the GMFγ knockdown cells, expressed as a percent of the median value for the control siRNA-transfected cells for the same time point in the same experiment. The differently shaped symbols represent five independent experiments. Paired t -tests were used to calculate p -values. (C) The percent of cells that had formed a cSMAC, defined as > 90% of the total Ag fluorescence intensity being contained in one or two clusters, is graphed. The different symbols represent independent experiments. Paired t -tests were used to calculate p -values. (D) The total fluorescence intensity of pCD79 that was present in clusters at the B cell-APC contact site was quantified for each B cell. The left panel shows representative data from one experiment. Each dot is one cell. n > 31 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. The Mann-Whitney U -test was used to calculate p -values. The right panel shows the results from five independent experiments, presented as in (B) , with n > 30 cells per condition in each experiment. Each symbol represents a single experiment in which the median pCD79 fluorescence intensity for GMFγ-depleted cells is expressed as a percent of the corresponding median value for the control cells. Paired t -tests were used to calculate p -values. (E) For each B cell represented in (D) , the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of the clustered mHEL-HaloTag Ag. The median (blue line) and interquartile ranges (black box) are shown. The data are presented as in (B , D) . **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p ≤ 0.05; ns, not significant ( p > 0.05).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:200 in PBS + 2% BSA), washed, and then incubated for 30 min at room temperature with PBS + 2% BSA containing Alexa Fluor-647-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, #A21244, 1:400) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific, #A12379, 1:400).

Techniques: Transfection, Control, Expressing, Staining, Microscopy, Fluorescence, Knockdown, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

doi: 10.3389/fcell.2021.647063

Figure Lengend Snippet: Depleting GMFγ does not impair CD79 phosphorylation induced by soluble anti-Ig antibodies. Cells were transfected with either control siRNA or GMFγ siRNA. (A) Raji D1.3 B cells were stimulated with 20 μg/mL goat anti-mouse IgM to initiate signaling through the D1.3 BCR. (B) Ramos B cells were stimulated with donkey anti-human IgM. Cell lysates were analyzed by immunoblotting with antibodies that recognize the phosphorylated CD79a ITAM (pCD79a), actin (loading control), or GMFγ. The left panels show a representative experiment. The right panels show results from 4 independent experiments. For each sample, the pCD79a band intensity was divided by the corresponding actin band intensity (loading control). The resulting ratios were normalized to that for the 0 min control siRNA cell sample (defined as 1.0) in the same experiment. On the graph, each of the four experiments is indicated by a different symbol. The bars show the mean ± SEM. Paired t -tests were used to calculate p -values. ns, not significant ( p > 0.05).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:200 in PBS + 2% BSA), washed, and then incubated for 30 min at room temperature with PBS + 2% BSA containing Alexa Fluor-647-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, #A21244, 1:400) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific, #A12379, 1:400).

Techniques: Phospho-proteomics, Transfection, Control, Western Blot

Journal: Cellular and Molecular Immunology

Article Title: The metabolic regulator Lamtor5 suppresses inflammatory signaling via regulating mTOR-mediated TLR4 degradation

doi: 10.1038/s41423-019-0281-6

Figure Lengend Snippet: Lamtor5 negatively regulates TLR4-driven signaling. a, b Immunoblotting of total and phosphorylated IKK, IκBα, and p65 in control, Lamtor5-expressing and Lamtor5-silenced RAW264.7 cells stimulated with LPS (100 ng/ml) for the indicated time periods. c Relative NF-κB promoter activity induced by LPS in control, Lamtor5-expressing and Lamtor5-silenced RAW264.7 cells. d Confocal microscopy analysis of p65 nuclear translocation upon stimulation with p65. p65 immunoreactivity is shown in red, and nuclei are stained with DAPI (blue). e Immunoblotting of total and phosphorylated IKK, IκBα, and p65 in WT and Lamtor5 +/− cells stimulated with LPS (100 ng/ml) for the indicated time periods. f, g Immunoblotting of total and phosphorylated JNK, p38, and ERK in control, Lamtor5-expressing or Lamtor5-silenced RAW264.7 cells stimulated with LPS (100 ng/ml) for the indicated time periods. h, i Immunoblotting of total and phosphorylated TBK1 and IRF3 and j, k quantitative PCR to assess IFNβ production in control, Lamtor5-expressing, or Lamtor5-silenced RAW264.7 cells stimulated with LPS (100 ng/ml) for the indicated time periods. Representative images are shown, and the data from three independent experiments are expressed as the means ± SDs. * P < 0.05, ** P < 0.01 by Student’s t- test

Article Snippet: Antibodies (Abs) specific for total and phosphorylated forms of p38 (Thr180/Tyr182), ERK1/2 (Thr202/Tyr204), JNK (Thr183/Tyr185), p65 (Ser536), IKKα/β (Ser176), TAK1 (Thr187), AKT (Ser473), mTOR (Ser2448), p70 S6 Kinase (Thr421/Ser424), 4E-binding protein 1 (4E-BP1) (Thr37/46), ULK1 (Ser757), IRF3 (Ser396), and IκBα (Ser32) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot, Control, Expressing, Activity Assay, Confocal Microscopy, Translocation Assay, Staining, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Immunology

Article Title: The metabolic regulator Lamtor5 suppresses inflammatory signaling via regulating mTOR-mediated TLR4 degradation

doi: 10.1038/s41423-019-0281-6

Figure Lengend Snippet: Lamtor5 downregulates TLR4 levels by promoting autophagy. Immunoblotting for TLR4 in control, Lamtor5-expressing, and Lamtor5-silenced RAW264.7 cells ( a ) and Lamtor5 +/− and WT macrophages ( b ) stimulated with LPS (100 ng/ml) for the indicated time periods. c FACS to assess surface TLR4 levels in control, Lamtor5-expressing, and Lamtor5-silenced RAW264.7 cells 2 h after LPS stimulation. Representative histograms and mean fluorescence intensities (MFIs) are shown. d Representative immunofluorescence images (left) and fluorescence quantification (right) of DQ-BSA activity in control, Lamtor5-expressing, and Lamtor5-silenced RAW264.7 cells. e Immunoblotting for LC3 and p62 in control, Lamtor5-expressing and Lamtor5-silenced RAW264.7 cells stimulated with LPS for the indicated time periods. f Representative confocal images showing LC3 puncta in control, Lamtor5-expressing and Lamtor5-silenced RAW264.7 cells stimulated with LPS for 2 h. Quantification of LC3 puncta per cell is also shown ( n = 100 cells). g Immunoblotting for LC3 in control and Lamtor5-expressing RAW264.7 cells pretreated with DMSO or Baf-A1 and subjected to LPS stimulation for the indicated time periods. h, i Immunoblotting for the indicated proteins in control, Lamtor5-expressing, and Lamtor5-silenced RAW264.7 cells stimulated with LPS for the indicated time periods. j Immunoblotting for TLR4 and total and phosphorylated p65 in control and Lamtor5-expressing RAW264.7 cells pretreated with DMSO or Baf1 and then stimulated with LPS for the indicated time periods. k Immunoblotting for TLR4 and total and phosphorylated p65 in control and Lamtor5-silenced RAW264.7 cells pretreated with DMSO or torin 1 and then stimulated with LPS for the indicated time periods. l Immunoblotting for TLR4 in control, Lamtor5-expressing, and Lamtor5-silenced RAW264.7 cells transfected with nonspecific control (NC) or Atg5-specific siRNA, followed by LPS stimulation for 2 h. Representative images are shown, and data from three independent experiments are expressed as the means ± SDs. * P < 0.05, ** P < 0.01 by Student’s t- test

Article Snippet: Antibodies (Abs) specific for total and phosphorylated forms of p38 (Thr180/Tyr182), ERK1/2 (Thr202/Tyr204), JNK (Thr183/Tyr185), p65 (Ser536), IKKα/β (Ser176), TAK1 (Thr187), AKT (Ser473), mTOR (Ser2448), p70 S6 Kinase (Thr421/Ser424), 4E-binding protein 1 (4E-BP1) (Thr37/46), ULK1 (Ser757), IRF3 (Ser396), and IκBα (Ser32) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot, Control, Expressing, Fluorescence, Immunofluorescence, Activity Assay, Transfection

Journal: Cellular and Molecular Immunology

Article Title: The metabolic regulator Lamtor5 suppresses inflammatory signaling via regulating mTOR-mediated TLR4 degradation

doi: 10.1038/s41423-019-0281-6

Figure Lengend Snippet: Lamtor5 enhances the autolysosomal gene program via regulating the mTOR/TFEB axis. a Colocalization of mTOR and LAMP1 in control, Lamtor5- or mutant Lamtor5 (ΔLZ)-expressing RAW264.7 cells treated with or without LPS for 1 h. b Colocalization of mTOR and LAMP1 in RAW264.7 cells transfected with intact TLR4- or truncated TLR4 (LRR)-expressing plasmids with or without LPS stimulation for 1 h. c Immunoblotting for total and phosphorylated mTOR in RAW264.7 cells transfected with Lamtor5- or Lamtor5 (ΔLZ)-expressing plasmids (upper) or TLR4-/- macrophages transfected with intact TLR4 or truncated TLR4 (LRR) (lower) upon stimulation with LPS for the indicated time periods. d Immunoblotting for phosphorylated and total TFEB in control, Lamtor5-expressing, and Lamtor5-silenced RAW264.7 cells stimulated with LPS for the indicated time periods. e Immunostaining for TFEB in control and Lamtor5-expressing RAW264.7 cells stimulated with LPS for 1 h. f Heat map showing the levels of autophagy-lysosome genes in control, Lamtor5-expressing or Lamtor5-silenced RAW264.7 cells stimulated with LPS for 3 h. g Transmission electron microscopy (TEM) of RAW264.7 cells transfected with control and Lamtor5-expressing plasmids with or without LPS stimulation. Black box, lysosomes. h Control, Lamtor5-expressing or Lamtor5-silenced RAW264.7 cells were stained with LysoSensor Green and analyzed by FACS. Representative histograms (lower) and relative fluorescence quantification (upper) are shown. i Immunoblotting for TLR4 in control and Lamtor5-expressing RAW264.7 cells transfected with nonspecific control (NC) or TFEB-targeted siRNA followed by LPS stimulation for the indicated time periods. j Confocal microscopy showing LC3 puncta in control and Lamtor5-expressing RAW264.7 cells transfected with nonspecific control (NC) or TFEB-targeted siRNA followed by LPS stimulation for 1 h. Representative images (left) and the quantification of LC3 puncta (100 cells) are shown (right). Data are from three independent experiments and expressed as the means ± SDs. * P < 0.05, ** P < 0.01 by Student’s t- test

Article Snippet: Antibodies (Abs) specific for total and phosphorylated forms of p38 (Thr180/Tyr182), ERK1/2 (Thr202/Tyr204), JNK (Thr183/Tyr185), p65 (Ser536), IKKα/β (Ser176), TAK1 (Thr187), AKT (Ser473), mTOR (Ser2448), p70 S6 Kinase (Thr421/Ser424), 4E-binding protein 1 (4E-BP1) (Thr37/46), ULK1 (Ser757), IRF3 (Ser396), and IκBα (Ser32) were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Control, Mutagenesis, Expressing, Transfection, Western Blot, Immunostaining, Transmission Assay, Electron Microscopy, Staining, Fluorescence, Confocal Microscopy