anti p2x7r  (Alomone Labs)

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: ( A ) Schematic representation of the P2X7 labeling strategy using ligand-directed N -cyanomethyl NASA chemistry. A P2X7 ligand (Lg) binds to allosteric sites on the receptor, bringing the N -cyanomethyl NASA warhead into close proximity with endogenous lysine (K) residues. This spatial arrangement enables the covalent transfer of a biotin moiety via amide bond formation, resulting in highly specific biotinylation of P2X7. The biotin tag allows super-resolution imaging of nanoscale P2X7 localization using a Streptavidin-Alexa 647 probe (Strept-A 647). Notably, following biotin transfer, the ligand can dissociate from P2X7, leaving the allosteric sites unoccupied. ( B ) Crystal structure of panda P2X7 (pdP2X7) shown in ribbon representation, bound to AZ10606120 depicted as spheres (PDB:5U1W; ). One of the three ATP-binding sites and the approximate location of the membrane are also indicated. Inset, enlarged view of the AZ10606120-binding pocket, rotated 180°. Distances (in Å) between the α-carbon of selected lysines and the hydroxyl group of AZ10606120 are displayed. Note that K300 is not visible in this view. ( C ) Chemical structure of X7-uP .

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: Labeling, Imaging, Binding Assay, Membrane

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: ( A ) Sequence alignment of selected regions from P2X receptors: panda P2X7 (pdP2X7), rat P2X7 (rP2X7), mouse P2X7 (mP2X7), human P2X7 (hP2X7), and rat and human P2X1–6 (rP2X1–6, hP2X1–6). Lysine residues are highlighted in red. Secondary structure elements are depicted in cartoon representation and are labeled as in the crystal structure of pdP2X7 . ( B ) Chemical structures of compound 1 and AZ10606120. Red star indicates electrophilic carbon of the N -cyanomethyl NASA reacting group. ( C ) Right, crystal structure of AZ10606120 (stick representation) bound to pdP2X7 (PDB:5 U1W) . Left, same view with 1 docked to pdP2X7 (left). Distances (in Å) separating the reactive carbonyl of 1 and selected α-carbons of nearby residues (red stick) are shown, along with residues (blue stick) important for AZ10606120 binding . Residues shown in parentheses are the equivalent rat P2X7 residues.

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: Sequencing, Labeling, Binding Assay

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: ( A ) Whole-cell currents evoked by 10 µM BzATP are reversibly inhibited by co-applying 1 µM X7-uP (upper trace) or 1 µM AZ10606120 (middle trace) in cells transiently transfected with rP2X7. Inhibitors were pre-applied alone for 10 s before 2 s of co-application. In the control (absence of inhibitors), BzATP-induced currents further increased, demonstrating current facilitation, which is an expected feature of P2X7 activation . ( B ) Summary of whole-cell inhibition at the indicated concentrations (n=4–8 cells for each condition). Bars represent mean ± s.e.m. ( C ) Western blot analysis of P2X7 labeling by X7-uP . Cells transiently transfected with P2X7c-myc were treated with 1 µM X7-uP for 0–60 minutes, in the absence or presence of 10 µM AZ10606120 or 10 µM A740003 (as indicated), followed by extensive washing. After cell lysis, biotinylated proteins were pulled down, separated on SDS-PAGE, and Western blotting was revealed using an anti-c-myc antibody (@c-myc). Molecular mass markers are shown on the right. Control of P2X7c-myc expression is presented in the corresponding input. β-Actin was used as a loading control. ( D ) Time course plot of P2X7 labeling with 1 µM X7-uP . Data (mean ± s.e.m., n=3 independent transfections) were fitted with to determine the pseudo-first-order reaction rate k app (mean ± s.e.m.). Figure 2—source data 1. PDF file containing the original western blots used for , indicating the relevant bands. Figure 2—source data 2. Original image files of western blots used for . Figure 2—source data 3. Numerical values used to generate .

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: Transfection, Control, Activation Assay, Inhibition, Western Blot, Labeling, Lysis, SDS Page, Expressing

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: ( A ) Western blot analysis showing the specificity of P2X7 labeling by X7-uP in untransfected (-) or P2X7c-myc-transfected (+) HEK293T cells (refer to for details). Molecular mass markers are shown on the right. ( B ) Western blot analysis of P2X7 labeling using different concentrations of X7-uP at various time points in HEK293T cells expressing P2X7c-myc. β-Actin was used as a loading control. Molecular mass markers are shown on the right. ( C ) Time course of P2X7 labeling using 0.5 µM and 2.5 µM X7-uP . Data (mean ± s.e.m., n=3 independent transfections) were fitted using , providing pseudo-first-order reaction rate constants ( k app ) (mean ± s.e.m.). ( D ) Concentration-dependence of k app values for X7-uP . Data were fitted with , yielding the labeling rate constant ( k L = 0.011 ± 0.003 s –1 , mean of triplicate ± standard deviation (s.d.)) and the dissociation constant ( K d = 7.3 ± 2.7 µM, mean ± s.d.). Figure 2—figure supplement 1—source data 1. PDF file containing the original western blots used for , indicating the relevant bands. Figure 2—figure supplement 1—source data 2. Original image files of western blots used for . Figure 2—figure supplement 1—source data 3. Numerical values used to generate .

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: Western Blot, Labeling, Transfection, Expressing, Control, Concentration Assay, Standard Deviation

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: ( A ) Western blot analysis of P2X7 in the supernatants of cell lysates from P2X7c-myc-transfected HEK293T cells (left) or BV2 cells (right), either untreated (-) or treated with X7-uP (+) for 60 minutes (HEK293T cells) or 10 minutes (BV2 cells). P2X7 was detected using either an anti-c-myc antibody (for HEK293T cells) or a mouse anti-P2X7 antibody (for BV2 cells). β-Actin was used as a loading control. Molecular mass markers are shown on the right. ( B ) Quantification of P2X7 labeling yield, calculated using the following equation: ( 1 − a / b ) × 100 \begin{document}$\left (1- a/b\right)\times 100$\end{document} , where a and b represent the amounts of P2X7 in lanes 2 and 1 of the supernatants, respectively. Figure 2—figure supplement 2—source data 1. PDF file containing the original western blots used for , indicating the relevant bands. Figure 2—figure supplement 2—source data 2. Original image files of western blots used for . Figure 2—figure supplement 2—source data 3. Numerical values used to generate .

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: Western Blot, Transfection, Control, Labeling

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: ( A–B ) Confocal images of HEK293T cells transiently transfected with either P2X7-mScarlet ( A ) or various P2X subunits tagged with GFP (P2X1-GFP, P2X2-GFP, P2X3-GFP, P2X4-GFP, P2X5-GFP, or P2X6-GFP) ( B ) were labeled with X7-uP and revealed using Strept-A 647 (red) in FBS-free DMEM. Labeling was performed in the presence of 10 µM AZ10606120 or 10 µM A740003 ( A ). Nuclei were stained with Hoechst (blue). For clarity, mScarlet and GFP signals are displayed in orange and green, respectively. Scale bars, 10 µm. ( C ) Quantification of Alexa 647 fluorescence. Bars represent mean ± standard deviation (s.d.) (n=75–129 cells, t -test comparisons to P2X7-mScarlet, ****p<0.0001). Figure 3—source code 1. RStudio code to generate the histogram of . Figure 3—source data 1. Data set used to generate .

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: Transfection, Labeling, Staining, Fluorescence, Standard Deviation

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: ( A ) Molecular docking of 1 (same pose as shown in ) in pdP2X7, showing distances (in Å) between the reactive carbonyl of 1 (stick representation) and selected α-carbons of nearby residues (blue). Residues shown in parentheses correspond to equivalent rP2X7 residues. ( B ) Confocal images of HEK293T cells transiently transfected with different P2X7 constructs: P2X7-mScarlet, K82A, K117A, and K82A/K117A. Refer to the legend of for additional details. Scale bars, 10 µm. ( C ) Quantification of Alexa 647 fluorescence. Bars represent mean ± s.d. (n=90–190 cells, t -test comparisons to indicated conditions, ****p<0.0001). ( D ) Whole-cell currents evoked by 10 µM BzATP are reversibly inhibited by co-application of 0.5 µM X7-uP (upper trace) to BzATP in a cell transiently transfected with the double mutant K82A/K117A. The control (absence of X7-uP ) is shown in the bottom trace. ( E ) Summary of whole-cell inhibition for K82A/K117A (n=7 cells for X7-uP and 5 cells for control). Bars represent mean ± s.e.m.; Mann-Whitney test (**p<0.005). Figure 4—source data 1. Data set and numerical values used to generate .

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: Transfection, Construct, Fluorescence, Mutagenesis, Control, Inhibition, MANN-WHITNEY

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: . Confocal images of HEK293T cells transiently transfected with different P2X7 constructs (P2X7-mScarlet, K110A, and K300A). Refer to the legend of for additional details. Scale bars, 10 µm.

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: Transfection, Construct

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: ( A ) Cartoon and experimental timeline of BV2 cell treatments. IL-1β release was assessed in the supernatant (sup), and the same cells were labeled with 1 µM X7-uP after extensive washout. ( B ) Quantification of IL-1β release by ELISA following the indicated treatments: LPS (1 μg/mL for 24 hr), ATP (1 mM for 30 min), BzATP (300 μM for 30 min), and MβCD (15 mM for 15 min). Bars represent mean ± s.e.m. (n=12 samples from three independent experiments). Data were compared using Kruskal-Wallis followed by Dunn’s multiple comparisons (*p=0.0208, #p=0.0362, **p=0.0014, ****p<0.0001). ( C ) Normalized quantification of IL-1β release induced by LPS +ATP or LPS +BzATP in the presence of P2X7 inhibitors AZ10606120 or A740003. Bars represent mean ± s.e.m. (n=6 samples from six independent experiments). One-way ANOVA with Dunnett’s multiple comparisons to control condition for ATP data (****p<0.0001). Kruskal-Wallis followed by Dunn’s multiple comparisons to control condition for BzATP data (*p=0.0414, *** p=0.0006). ( D ) Bright-field and dSTORM images of X7-uP -labeled BV2 cells revealed with Strept-A 647 corresponding to experiments shown in panel B. Scale bars, 10 μm. Insets: Magnified dSTORM images. Scale bars, 1 μm. ( E ) Quantification of single P2X7 localization density. Bars represent mean ± s.e.m. (each data point represents a cell, n=3 independent experiments). One-way ANOVA with Tukey’s multiple comparisons (*p<0.019, #p<0.0194, @p<0.0477, ***p<0.0002, ###p<0.0008, ****p<0.0001). ( F ) Relative frequency of cluster size. Inset: percentage of clusters larger than 0.025 mm 2 . ( G ) Number of detections per cluster. Bars represent mean ± s.e.m. One-way ANOVA with Tukey’s multiple comparisons (*p<0.0197, #p<0.0277, @p<0.0389, ****p<0.0001). ( H ) Images showing tessellation analysis of cells treated either with LPS +ATP or left untreated. Inset: magnification. Scale bars, 200 nm. ( I ) Number of fluorophores per cluster. Bars represent mean ± s.e.m. One-way ANOVA with Tukey’s multiple comparisons (****p<0.0001). Figure 5—source data 1. Numerical values used to generate .

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Control

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: ( A ) Western blot analysis of P2X7 labeling in BV2 cells treated with 1 µM X7-uP for 10 min in the absence or presence of 10 µM AZ10606120 or 10 µM A740003. After extensive washing, cells were lysed, and biotinylated proteins were pulled down and separated on SDS-PAGE. Western blot was revealed using a mouse anti-P2X7 antibody (@mP2X7). Molecular mass markers are shown on the right. ( B ) Corresponding input control for the blot shown in panel A. β-Actin was used as a loading control. ( C ) Confocal images of BV2 cells labeled with X7-uP and visualized with Strept-A 647 in the absence or presence of AZ10606120 or A740003, or in the absence of X7-uP (control, Strept-A 647 alone). Alexa 647 signals are shown in red, and nuclei are stained with Hoechst (blue). Scale bars, 10 µm. ( D ) Quantification of Alexa 647 fluorescence. Bars represent mean ± s.d. (n=46–220 cells, t -test comparisons to control, ****p<0.0001). Figure 5—figure supplement 1—source data 1. PDF file containing the original western blots used for , indicating the relevant bands. Figure 5—figure supplement 1—source data 2. Original image files of western blots used for . Figure 5—figure supplement 1—source data 3. Data set used to generate .

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: Western Blot, Labeling, SDS Page, Control, Staining, Fluorescence

Journal: eLife

Article Title: Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

doi: 10.7554/eLife.106096

Figure Lengend Snippet: The cartoon illustrates two distinct clusters of P2X7 receptors (blue), each adorned with one, two, or three fluorescently tagged tetrameric biotin-bound streptavidin (red). In untreated cells, each cluster contains an average of 1.5 fluorophores per P2X7 receptor. Treatment with LPS and ATP promotes P2X7 clustering by increasing the average number of fluorophores per cluster to between 4 and 5, resulting in an increased number of P2X7 receptors per cluster, from one to three. This redistribution synergistically triggers IL-1β release.

Article Snippet: The membrane was then incubated overnight at 4 °C under gentle agitation with primary antibodies: 1:2000 anti-GFP antibody (MA5-15256, Thermo Fisher), 1:500 anti-c-Myc mouse antibody (13–2500, Invitrogen, Thermo Fisher), 1:500 rabbit anti-P2X7 antibody (APR-008, Alomone Labs, Israel), or mouse 1:5000 anti-β-actin (A5441, Sigma Aldrich).

Techniques: