anti arpc5l  (Proteintech)

Journal: bioRxiv

Article Title: Branched actin networks in innate immune cells mediate host microbiota homeostasis

doi: 10.1101/2024.07.18.604111

Figure Lengend Snippet: (A) The level of Arpc5 and Arpc5l in C5 fl/fl , C5 HetVav or C5 1′Vav bone marrow extracts. ( B ) Lipocalin-2 (LPCN-2) faecal levels in the indicated mice. ( C ) H&E images of intestines (left) together with quantification of the histopathology score (middle) and disease activity index (DAI) (right). ( D ) Immunohistochemistry reveals the increased prevalence of macrophages (top) and neutrophils (bottom) in ileal sections in mice lacking Arpc5. ( E ) Loss of Arpc5 increases the size and cell counts of Mesenteric lymph nodes (mLNs) but not the inguinal lymph node (iLN). ( F ). Mice lacking Arpc5 have increased levels C-reactive protein (CRP) as well as phagocytes in their blood. ( G ) Quantification of the counts of neutrophils (NØs) and Ly6C hi/lo monocytes (MØs) in peripheral blood of the indicated mice. (H) Representative images together with the quantification of the counts of erythroid lineage and immature neutrophil (NØ) in the bone marrow of the indicated mice. ( I ) Quantification of the % of multipotent progenitors (MPP) among the lineage - Sca1 + c-Kit + (LSK) cells. MPP-Ly: lymphoid-biased MPPs; MPP-G/M: myeloid-biased MPPs; MPP-MkE: megakaryocyte and erythroid-biased MPPs; MPP: unbiased MPPs. ( J ) Representative image and weight of spleen in the indicated mice. ( K ) Bacterial colony forming units (CFU) in C5 HetVav or C5 1′Vav mice spleens. ( L ) Schematic of the bone marrow transplantation protocol: CD45.1_wild type mice were lethally irradiated and reconstituted with either CD45.2_controls (WT-Control) or CD45.2_C5 1′Vav (WT-C5 1′Vav ) bone marrow. H&E image of intestines of animals which received wild-type (WT-control) or C5 1′Vav (WT-C5 1′Vav ) bone marrow transplantation. Animals assessed in this figure were 8-15 week old mice n=7 of two independent experiments. Data shown as means ± SEM of at least three independent experiments, n=8. Statistical analysis was performed using Kruskal-Wallis test in B , Mann-Whitney test in C , E, F , H, J, K, L, one-way ANOVA in G and I, or multiple Mann-Whitney test in D . Significant p values are indicated on the graphs. C5 fl/fl (arpc5 fl/fl ), C5 HetVav (arpc5 HetVav ) and C5 1′Vav (arpc5 1′Vav ). ns = not significant.* = p-value < 0.05. ** = p-value < 0.01. *** = p-value < 0.001 and **** = p-value < 0.0001.

Article Snippet: Antibodies used for immunoblots: Arp3 (Sigma-Aldrich, A5979), ArpC1A (Atlas antibodies, HPA004334), ArpC1B (Bethyl Lab, A302-781A), ArpC2 (Abcam, ab133315), ArpC5 (Synaptic Systems, 305011), ArpC5L (Proteintech, 22025-a-AP), β -actin (Abcam, ab179467), anti-mouse IgG-IRDye® 800CW (LI-COR Biosciences, 926-32210) and anti-rabbit IgG-IRDye® 680RD (LI-COR Biosciences, 926-68071).

Techniques: Histopathology, Activity Assay, Immunohistochemistry, Transplantation Assay, Irradiation, Control, MANN-WHITNEY

Journal: eLife

Article Title: ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells

doi: 10.7554/eLife.82450

Figure Lengend Snippet: ( A ) Expression of all the subunits of the Arp2/3 complex along with the respective isoforms of ARPC1 and ARPC5 across Jurkat cell line and primary human CD4 T cells from two representative healthy donors were verified using western blotting. Representative immunoblots compare the protein levels of each subunit and their isoforms in CD4 T cells. Additional comparisons for expression of these proteins in Resting (R) and Activated (A) CD4 T cells from donors 3 and 4 are shown, respectively. Black arrowheads indicate the specific bands. Note that the ARPC5L antibody also detects ARPC5 (marked by red asterisk). The numbers indicated below each row represents the mean ± SD values from three independent experiments of the densitometric quantification of the bands compared to Jurkat protein levels (which is set to 1). Saturated exposure of the ARPC5 immunoblot is shown for better visualization of the ARPC5 levels in the JNLA cell line. ( B, C ) Single-cell RNA-sequencing analysis of Jurkat CD4 T cells ( B ) and primary CD4 T cells ( C ). Expression of selected genes in UMAP embedded cells with adjacent histograms of their frequency distributions. See for UMAP embedding of ( C ). Dashed line represents threshold for frequency quantification at 1 TPM. Source data is avaialble at https://doi.org/10.11588/data/YVYEO8 .

Article Snippet: Antibody , Anti-human ARPC5L (rabbit polyclonal) , GeneTex , Cat# GTX120725; RRID: AB_11172404 , WB (1:1000).

Techniques: Expressing, Western Blot, RNA Sequencing

Journal: eLife

Article Title: ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells

doi: 10.7554/eLife.82450

Figure Lengend Snippet: ( A ) Representative immunoblots show knockdown of ARPC1 and ARPC5 isoforms in bulk JNLA cells treated with indicated shRNAs. Black arrowheads indicate the specific bands, and black asterisks mark unspecific bands. Note that the ARPC5L antibody also detects ARPC5 (marked by red asterisk). The numbers indicated below the respective blots represent the mean ± SD values from four independent experiments, based on the densitometric quantification of the bands, normalized to GAPDH and compared to the non-targeting control (NTC) protein levels (which is set to 1). ( B ) Representative spinning-disk confocal still images of JNLA cells treated with indicated shRNA show stills post activation with PMA/ionomycin (P/I). Arrows point to the nuclear F-actin (NFA). Relates to . Scale bar, 3 µm. ( C ) Quantification of NFA formation in shRNA-treated cells relative to the scrambled control-treated cells. Mean ± SD of four independent experiments where 30 cells were analyzed per condition per experiment. Each dot represents the mean of each independent experiment. ( D ) Representative immunofluorescence images indicate averaged intensity projections of Phalloidin-647-stained F-actin ring (AR) formation in JNLA cells treated with indicated shRNA upon activation on coverslips coated with anti-CD3+CD28 antibodies. Arrowheads point to the F-actin ring at the PM. ( E ) Quantification of Phalloidin-stained AR formation in shRNA-treated cells relative to the control-treated cells. In ( C , E ), each data point indicates the mean value of an independent experiment consisting of two technical replicates with at least 100 cells analyzed per condition with indicated mean ± SD from four independent experiments. One-way ANOVA with Kruskal–Wallis test was used to determine statistical significances, where * p≤0.0332 , ** p≤0.0021, and ns: not significant. Scale bar, 5 µm. See also related . Source data is avaialble at https://doi.org/10.11588/data/YVYEO8 .

Article Snippet: Antibody , Anti-human ARPC5L (rabbit polyclonal) , GeneTex , Cat# GTX120725; RRID: AB_11172404 , WB (1:1000).

Techniques: Western Blot, Knockdown, Control, shRNA, Activation Assay, Immunofluorescence, Staining

Journal: eLife

Article Title: ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells

doi: 10.7554/eLife.82450

Figure Lengend Snippet: ( A ) shRNA-mediated knockdown of scrambled (control), ARPC5 or ARPC5L in JNLA cells was put on TCR stimulatory GBDs and subjected to live-cell microscopy. Shown are representative still images from the spinning-disk confocal microscope indicate the time of cells forming nuclear F-actin (NFA) after falling and contacting the GBDs. Movies were acquired every 30 s for a total of 6 min. Arrows indicate the NFA. Scale bar, 4 µm. ( B ) Quantification of nuclear actin filaments (NFA) was performed upon contact with TCR stimulatory surface. Data points indicate mean values from two independent experiments where 40–60 cells were analyzed per condition in each experiment. ( C ) Representative bigger field of view of average intensity projections of confocal images showing Phalloidin-647-stained F-actin ring (AR) formation in JNLA cells, treated with indicated shRNA upon activation on coverslips coated with anti-CD3+CD28 antibodies. Scale bar, 15 µm. ( D ) Representative confocal still images (average intensity projection) of JNLA cells upon activation for 5 min on stimulatory coverslip showing formation of distinct morphologies of actin ring formed. Cells were fixed, permeabilized, and stained for F-actin (with Phalloidin-647) and counterstained with DAPI. Scale bar, 10 µm. ( E, F ) Cells with knockdown, exhibiting different morphologies (bars in different colors, stacked) based on classification shown above in ( D ), upon TCR activation, were quantified as % of cells of the total 100 cells quantified per condition is represented in ( E ). ( F ) shows the fold change in the different morphotypes exhibited by the cells upon knockdown relative to the control cells starts forming (top panel) or has fully formed into a classical actin ring structure (bottom).

Article Snippet: Antibody , Anti-human ARPC5L (rabbit polyclonal) , GeneTex , Cat# GTX120725; RRID: AB_11172404 , WB (1:1000).

Techniques: shRNA, Knockdown, Control, Microscopy, Staining, Activation Assay

Journal: eLife

Article Title: ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells

doi: 10.7554/eLife.82450

Figure Lengend Snippet: ( A ) Representative immunoblots show levels of each of the Arp2/3 complex subunits in JNLA cells with the indicated KO of each of the ARPC5 isoforms. Black arrowheads indicate the specific bands, and black asterisks mark unspecific bands. Immunoblots are representative of three independent experiments. The numbers indicated below the respective immunoblots (only the blots where differences were observed are indicated) represent the mean ± SD values from three independent experiments, based on the densitometric quantification of the bands, normalized to GAPDH and compared to the nontargeting control (NTC) protein levels (which is set to 1). ( B, C ) Shown are representative immunoblots confirming the successful overexpression of each of the ARPC5 isoforms in JNLA cells on the indicated KO of each of the ARPC5 isoforms compared to the NTC cells. Note that the ARPC5L antibody also detects ARPC5 (marked by red asterisk). ( D ) Representative immunoblots (from n = 3) show expression of respective subunits of the Arp2/3 complex when immunoblotted against each subunit antibody from mCherry-expressing and mCherry-C5/C5L-expressing JNLA cells. JNLA cells stably expressing either mCherry or mCherry-C5/C5L were lysed and 500 µg of cell lysates were immunoprecipitated with 25 µl RFP-TRAP beads overnight before pulling down on the mCherry-bound fraction from all the samples. All samples were run in denaturing condition on a 4–12% gradient SDS-PAGE with fractions loaded as total cell lysate (TCL), flow through (FT), or unbound fraction and bound or immunoprecipitated fraction. Source data is avaialble at https://doi.org/10.11588/data/YVYEO8 .

Article Snippet: Antibody , Anti-human ARPC5L (rabbit polyclonal) , GeneTex , Cat# GTX120725; RRID: AB_11172404 , WB (1:1000).

Techniques: Western Blot, Control, Over Expression, Expressing, Stable Transfection, Immunoprecipitation, SDS Page

Journal: eLife

Article Title: ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells

doi: 10.7554/eLife.82450

Figure Lengend Snippet: ( A ) Schematic of the experimental timeline for generating knockdown of ARPC5 isoforms and PMA + ionomycin (P/I) stimulation of the transduced cells. ( B ) The bar graph represents the mean of three independent experiments showing the relative mRNA expression (fold change) of ARPC5 and ARPC1 isoforms along with ARP3, between control and ARPC5 isoform KD cells, 72 hr post transduction with their respective shRNAs. Dots represent the mean of each independent experiment run in a technical triplicate for each sample, and error bars represent the mean ± SD of three independent experiments. Two-way ANOVA with Tukey’s multiple-comparison test was performed to calculate statistical significance with * p = 0.032 , ** p = 0.0075 , *** p = 0.0005 , **** p ≤ 0.0001, and ns: not significant . ( C ) Representative histogram of control and respective ARPC5 isoform KD cells, fixed, permeabilized, and stained with ARPC5 or ARPC5L-FITC showing the normalized count (mode), 72 hr post transduction with their respective shRNAs. In gray is the isotype control for each antibody based on which the gating was done, in red is the shRNA control cells, and in orange is the C5/C5L shRNA-treated cells. ( D ) Representative dot plots of intracellular TNFα in A.301 WT (parental/unmodified cells), control shRNA-treated cells, and ARPC5 isoform KD cells show the percentage of TNFα-positive cells in unstimulated and P/I-stimulated conditions. A.301 WT and sh_Control cells were stimulated with P/I in the presence of DMSO or CK869 for 4 hr plus monensin, before being fixed, permeabilized, and stained with TNFα-BV421. Whereas ARPC5 isoform KD cells were stimulated only in the presence or absence of DMSO and monensin, before being fixed, permeabilized, and stained with TNFα-BV421. ( E ) Representative dot plots of intracellular IL-2-stained control shRNA-treated and ARPC5 isoform KD A.301 cells show the percentage of IL2-positive cells in unstimulated and P/I-stimulated conditions. sh_Control cells and ARPC5 isoform KD cells were stimulated with P/I in the presence of DMSO for 16 hr, with monensin added only in the last 4 hr before being fixed, permeabilized, and stained with IL2-APC. Source data is avaialble at https://doi.org/10.11588/data/YVYEO8 .

Article Snippet: Antibody , Anti-human ARPC5L (rabbit polyclonal) , GeneTex , Cat# GTX120725; RRID: AB_11172404 , WB (1:1000).

Techniques: Knockdown, Expressing, Control, Transduction, Comparison, Staining, shRNA

Journal: eLife

Article Title: ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells

doi: 10.7554/eLife.82450

Figure Lengend Snippet: ( A ) Shown are representative spinning-disk confocal images (maximum projection) of ARPC5.mCherry and ARPC5L.mCherry distribution in unstimulated JNLA cells. White arrows point to the respective C5 or C5L punctae seen in the nucleus. Also see related . ( B ) Subcellular distribution of the ARPC5 subunit and its isoform ARPC5L was determined by biochemical fractionation of JNLA cells with respective knockout of either C5 or C5L in the bulk culture. Representative immunoblots reveal levels of ARPC5 isoforms in the whole cell extract (WCE), cytoplasmic (C), and nuclear (N) fractions in the indicated JNLA knockout cells post fractionation. GAPDH and hnRNPL were used as markers for cytoplasmic and nuclear compartments, respectively. ( C ) Representative, deconvoluted, and segmented stimulated emission depletion (STED) single-plane images show endogenous nuclear actin filaments (stained with Phalloidin-647N) and ARPC5.mCherry/ARPC5L.mCherry (signal enhanced with anti-mCherry with secondary antibody in atto-594 channel) in A3.01 T cells, stimulated with PMA/ionomycin (P/I) for 30 s. Arrows (in white) point to the colocalization events. Percent colocalization is mentioned as mean ± SD (in white bar, top right) for each of the isoforms from three independent experiments. Scale bar, 500 nm. ( D ) The dot plot shows the frequency of colocalization of ARPC5 and ARPC5L with nuclear F-actin (from representative STED-deconvolved and segmented super-resolved images shown in ) in A3.01 cells post 30 s of stimulation with PMA + ionomycin. Each dot represents colocalization events per cell that was analyzed. Source data is avaialble at https://doi.org/10.11588/data/YVYEO8 .

Article Snippet: Antibody , Anti-human ARPC5L (rabbit polyclonal) , GeneTex , Cat# GTX120725; RRID: AB_11172404 , WB (1:1000).

Techniques: Fractionation, Knock-Out, Western Blot, Staining

Journal: eLife

Article Title: ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells

doi: 10.7554/eLife.82450

Figure Lengend Snippet: ( A ) Representative maximum intensity projection of confocal images showing respective ARPC5/ARPC5L antibody staining (on the left) of the endogenous ARPC5 isoforms expressed in the JNLA cells in unstimulated condition, where cells were fixed on poly-lysine-coated coverslips. Additional, Phalloidin-stained channel (on the right) is also shown for each image. Scale bar, 3 µm. ( B ) Representative maximum intensity projection of confocal images showing respective ARPC5/ARPC5L antibody staining of the endogenous ARPC5 isoforms, Phalloidin staining as well as merged view of DAPI, phalloidin, and ARPC5/C5L antibody staining are shown in the JNLA cells upon TCR activation on coverslips coated with anti-CD3+CD28 antibodies. The high background observed in C5 antibody-stained channel is unavoidable as the CD3/28 antibodies coated on the coverslips and the ARPC5 antibody are of the same species (mouse). Scale bar, 5 µm. Source data is avaialble at https://doi.org/10.11588/data/YVYEO8 .

Article Snippet: Antibody , Anti-human ARPC5L (rabbit polyclonal) , GeneTex , Cat# GTX120725; RRID: AB_11172404 , WB (1:1000).

Techniques: Staining, Activation Assay

Journal: eLife

Article Title: ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells

doi: 10.7554/eLife.82450

Figure Lengend Snippet: ( A–C ) Representative immunoblots show levels of ARP3, ARPC1, and ARPC5 subunits in JNLA.GFP cells with the indicated KO of respective class I NPFs. Black arrowheads indicate the specific bands, and black asterisks mark unspecific bands. Note that the ARPC5L antibody also detects ARPC5 (marked by red asterisk). Immunoblots are representative of three independent experiments. The numbers indicated below the respective immunoblots represent the mean ± SD values from three independent experiments, based on the densitometric quantification of the bands, normalized to either GAPDH or Tubulin and compared to the non-targeting control (NTC) protein levels (which is set to 1). ( D, E ) Effects of NWASPi on NFA in A3.01 T cells expressing nuclear lifeact.GFP. ( D ) Representative images upon stimulation with PMA/ionomycin (P/I). ( F ) Quantification with statistical ( t -test, * p ≤ 0.05). Dots represent the mean of each independent experiment, and the bar graph represents the mean of three experiments with error bars calculated from mean ± SD of three independent experiments where at least 30 cells were analyzed per condition per experiment. ( F ) Stacked bar graph represents the frequency of JNLA cells (%) with respective class I NPF KO, exhibiting different morphologies based on classification shown in , upon TCR activation, were quantified as % of cells of the total 100 cells quantified per condition.

Article Snippet: Antibody , Anti-human ARPC5L (rabbit polyclonal) , GeneTex , Cat# GTX120725; RRID: AB_11172404 , WB (1:1000).

Techniques: Western Blot, Control, Expressing, Activation Assay

Journal: eLife

Article Title: ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells

doi: 10.7554/eLife.82450

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-human ARPC5L (rabbit polyclonal) , GeneTex , Cat# GTX120725; RRID: AB_11172404 , WB (1:1000).

Techniques: Stable Transfection, Expressing, Recombinant, Construct, Plasmid Preparation, Flow Cytometry, Sequencing, shRNA, Immunoprecipitation, CRISPR, Software, Microscopy, Live Cell Imaging, Super-Resolution Microscopy, Confocal Microscopy