nav beta subunits nav beta 1 alomone asc 041 rrid ab 2341071 x datasheet nav beta 4 neuromab  (Alomone Labs)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: An Integrative Proteotranscriptomics Approach Reveals New ADAM9 Substrates and Downstream Pathways

doi: 10.1016/j.mcpro.2026.101538

Figure Lengend Snippet: Identification of IGSF8 and IL10RB as new ADAM9 substrates. HCT116 cells were transfected with the indicated siRNA or expression constructs, and Western blotting was performed for conditioned media (CM) or cell lysates (CLs) using antibodies recognizing the ectodomain of IGSF8 ( A , B ) or IL10RB ( C , D ). Three biological replicates are shown in ( A , C ); unpaired two-tailed t test was performed. ADAM, A Disintegrin And Metalloproteinase; IGSF8, immunoglobulin superfamily member 8; IL10RB, interleukin 10 receptor subunit beta.

Article Snippet: Antibodies used in this study include rabbit anti-ADAM9 (CST, 4151, 1:1000 dilution), mouse anti-ALCAM (Santa Cruz, 74558, 1:200 dilution), mouse anti–interleukin 10 receptor subunit beta (IL10RB) (R&D Systems, MAB874, 5 μg/ml), rabbit anti–Forkhead Box O3 (FOXO3) (CST, 2497, 1:1000 dilution for Western blotting, and 1:100 dilution for immunocytochemistry [ICC]), rabbit anti–immunoglobulin superfamily member 8 (IGSF8) (Sigma, 011917, 1:500 dilution), horseradish peroxidase (HRP)–conjugated rabbit anti-mouse (CST, 7076, 1:2000 dilution), HRP-conjugated goat anti-rabbit (CST, 7074; 1:2000 dilution), HRP-conjugated mouse anti-β-actin antibody (CST, 12262, 1:2000 dilution), and Alexa Fluor 594–conjugated goat anti-rabbit antibody (Thermo Fisher, A-11012, 1:1000 dilution for ICC).

Techniques: Transfection, Expressing, Construct, Western Blot, Two Tailed Test

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Biochemical and live-cell imaging characterization of the effects of insulin on INSR. (A) Internalized to total INSR ratio was quantified using surface biotinylation in undifferentiated C2C12 myoblasts. Cells were incubated in PBS (no insulin) or serum-free DMEM containing 0, 0.2, 2, or 20 nM insulin for 15 minutes. The ratios are normalized to the 0 nM group of each gel ( P > .05 when not specified). (B) Western blot showing SNAP-tagged INSR expressed from lentiviral vector in comparison to wild-type INSR in C2C12 myoblasts. (C, D) Representative images of INSR-A-SNAP-labeled using an Alexa Fluor 488 cell nonpermeable dye in 0, 0.2, or 20 nM insulin conditions in live undifferentiated C2C12 myoblasts from (C) 0 to 15 minutes or (D) after 20 minutes using a spinning disk confocal microscope. Time-lapse images of INSR vesicle interactions starting from the selected subregions (white squares) of snapshots are shown in the insets ( n = 3 cells).

Article Snippet: Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA) and probed with antibodies against p-ERK1/2 (Thr202/Tyr204) (1:1000, Cat. #4370, RRID: AB_2315112), ERK1/2 (1:1000, Cat. #4695, RRID: AB_390779), p-AKT (Ser473) (1:1000, Cat. #9271, RRID: AB_329825), p-AKT (Thr308) (1:1000, Cat. #9275, RRID: AB_329828), AKT (1:1000, Cat. #9272, RRID: AB_329827), INSR-β subunit (1:1000, Cat. #3020S, RRID: AB_2249166), p-INSRβ (Tyr1150/1151) (1:1000, Cat. #3024, RRID: AB_331253), FOXO1 (1:1000, Cat. #2880, RRID: AB_2106495), p-FOXO1 (Thr24) (1:1000, Cat. #9464, RRID: AB_329842), all from Cell Signaling (CST), and β-tubulin (1:2000, Cat. #T0198, Sigma, RRID: AB_477556).

Techniques: Live Cell Imaging, Incubation, Western Blot, Plasmid Preparation, Comparison, Labeling, Microscopy

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Live-cell TIRF imaging of cell-surface INSR. (A) Experimental design of TIRF microscopy of the same cells at 3 time points with 2 nM insulin or no insulin (control). (B) Representative TIRF image of INSR-A-EGFP puncta, binary image of detected INSR spots, and INSR tracks. (C) Diffusion coefficient of INSR-A-EGFP tracks in control or insulin group. Data are plotted as SuperPlot with larger dots showing mean values of the cells and smaller dots showing individual INSR tracks of the cells. (D) The cumulative probability shows the distribution of diffusion coefficients in (C). (E) Track radius of INSR-A-EGFP tracks in control or insulin group. (F) The cumulative probability shows the distribution of diffusion coefficient in (E) ( n = 3 cells in control group, n = 9 cells in insulin group).

Article Snippet: Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA) and probed with antibodies against p-ERK1/2 (Thr202/Tyr204) (1:1000, Cat. #4370, RRID: AB_2315112), ERK1/2 (1:1000, Cat. #4695, RRID: AB_390779), p-AKT (Ser473) (1:1000, Cat. #9271, RRID: AB_329825), p-AKT (Thr308) (1:1000, Cat. #9275, RRID: AB_329828), AKT (1:1000, Cat. #9272, RRID: AB_329827), INSR-β subunit (1:1000, Cat. #3020S, RRID: AB_2249166), p-INSRβ (Tyr1150/1151) (1:1000, Cat. #3024, RRID: AB_331253), FOXO1 (1:1000, Cat. #2880, RRID: AB_2106495), p-FOXO1 (Thr24) (1:1000, Cat. #9464, RRID: AB_329842), all from Cell Signaling (CST), and β-tubulin (1:2000, Cat. #T0198, Sigma, RRID: AB_477556).

Techniques: Imaging, Microscopy, Control, Diffusion-based Assay

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Identification of INSR interactors. (A) Proteins that are detected in INSR immunoprecipitation mass spectrometry (IP-MS), and significantly different between wild-type and INSR knockout groups. Proteins highlighted in the square were specific interactors that had little detection in knockout cells. The other 3 proteins are also significantly different but not lower or absent in INSR knockout cells ( n = 3). (B) PPI network of INSR interactors and endocytosis proteins. (C) Modeling the interactions between the cytoplasmic region of INSR (residues 968-1372) and full-length interactor (target) proteins using AlphaFold multimer. pTM scores measure the accuracy of the overall structure of the protein complex and is relatively insensitive to localized inaccuracies. ipTM measures the accuracy of the interacting subunits of the complex, and ipTM > 0.42 (dash line) predicts direct interaction. The target proteins are classified as IP-MS identified novel interactors (unknown ●), the known or expected interactors (positive + ), and an expected noninteractor protein as a negative control (negative × ). The cytoplasmic region of IGF1R (residues 961-1369) is used ( n = 5 predictions). (D) Potential interaction sites based on per-residue scores along the INSR cytoplasmic region (residues 968-1372; top panel) and interactor (target) proteins Ap2m1 (bottom). minD (more accurate) and min-iPAE scores detect interaction sites. pLDDT shows whether the region is structured or unstructured. Shades show confidence errors of 99%. (E) ipTM scores for fragment–protein interactions. Thirty residues around the minD peaks (potential interacting sites) in (D) and Fig. S2 are analyzed. Each box relates to the interaction of the fragments of first protein vs the full-lenght second protein. For example, title “Insr vs Mtco2” means we have cut Insr into fragments and we have run AlphaFold multimer on each of those fragments vs full-length Mtco2. Each dot is the top ipTM score for 1 fragment (we do 5 predictions for each fragment vs protein). The protein residue positions of the interacting fragments (ipTM > 0.42) are labeled.

Article Snippet: Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA) and probed with antibodies against p-ERK1/2 (Thr202/Tyr204) (1:1000, Cat. #4370, RRID: AB_2315112), ERK1/2 (1:1000, Cat. #4695, RRID: AB_390779), p-AKT (Ser473) (1:1000, Cat. #9271, RRID: AB_329825), p-AKT (Thr308) (1:1000, Cat. #9275, RRID: AB_329828), AKT (1:1000, Cat. #9272, RRID: AB_329827), INSR-β subunit (1:1000, Cat. #3020S, RRID: AB_2249166), p-INSRβ (Tyr1150/1151) (1:1000, Cat. #3024, RRID: AB_331253), FOXO1 (1:1000, Cat. #2880, RRID: AB_2106495), p-FOXO1 (Thr24) (1:1000, Cat. #9464, RRID: AB_329842), all from Cell Signaling (CST), and β-tubulin (1:2000, Cat. #T0198, Sigma, RRID: AB_477556).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Knock-Out, Negative Control, Residue, Labeling

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Interaction and colocalization between INSR, CAV3, CAV1, and CLTC under different insulin stimulations. (A) Western blot of mice skeletal muscle lysates after insulin injection. Phospho-AKT (Ser473) to total AKT ratio or phospho-ERK1/2 to total ERK ratio was quantified ( n = 6). (B) Co-immunoprecipitation of skeletal muscle INSR after PBS or insulin injection. CLTC to INSR ratio or CAV3 to INSR ratio were quantified ( n = 7-10). (C) Representative STED microscopy images of C2C12 myoblasts expressing INSR-A-SNAP (surface labeled) that were fixed after stimulation with 0, 0.2, or 20 nM insulin for 30 minutes and stained for CAV1 and CLTC (scale bar = 5 µm). (D, E) Colocalizations between INSR-A-SNAP, CAV1, and CLTC were quantified by Object Pearson Coefficient. Data are plotted to show differences between insulin concentrations (D) or between protein pairs (E) ( n = 7-9 images, 1-4 cells per image. * P < .05, Tukey's multiple comparison after 2-ANOVA. Box represents median and 25th to 75th percentiles).

Article Snippet: Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA) and probed with antibodies against p-ERK1/2 (Thr202/Tyr204) (1:1000, Cat. #4370, RRID: AB_2315112), ERK1/2 (1:1000, Cat. #4695, RRID: AB_390779), p-AKT (Ser473) (1:1000, Cat. #9271, RRID: AB_329825), p-AKT (Thr308) (1:1000, Cat. #9275, RRID: AB_329828), AKT (1:1000, Cat. #9272, RRID: AB_329827), INSR-β subunit (1:1000, Cat. #3020S, RRID: AB_2249166), p-INSRβ (Tyr1150/1151) (1:1000, Cat. #3024, RRID: AB_331253), FOXO1 (1:1000, Cat. #2880, RRID: AB_2106495), p-FOXO1 (Thr24) (1:1000, Cat. #9464, RRID: AB_329842), all from Cell Signaling (CST), and β-tubulin (1:2000, Cat. #T0198, Sigma, RRID: AB_477556).

Techniques: Western Blot, Injection, Immunoprecipitation, Microscopy, Expressing, Labeling, Staining, Comparison

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Colocalization and interaction between INSR and ANXA2 under different insulin concentrations. (A, B) Representative STED microscopy images of C2C12 myoblasts expressing INSR-A-SNAP (surface labeled) that were fixed after stimulation with 0, 0.2, or 20 nM insulin for (A) 15 or (B) 30 minutes and stained for ANXA2 (scale bar = 5 µm). (C, D) Colocalization between INSR-A-SNAP and ANXA2 at (C) 15 or (D) 30 minutes of insulin-stimulated were quantified by Object Pearson Coefficient ( n = 4-13 images, 1-3 cells per image. # P < .05, 1-ANOVA of all groups. Box represents median and 25th to 75th percentiles.).

Article Snippet: Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA) and probed with antibodies against p-ERK1/2 (Thr202/Tyr204) (1:1000, Cat. #4370, RRID: AB_2315112), ERK1/2 (1:1000, Cat. #4695, RRID: AB_390779), p-AKT (Ser473) (1:1000, Cat. #9271, RRID: AB_329825), p-AKT (Thr308) (1:1000, Cat. #9275, RRID: AB_329828), AKT (1:1000, Cat. #9272, RRID: AB_329827), INSR-β subunit (1:1000, Cat. #3020S, RRID: AB_2249166), p-INSRβ (Tyr1150/1151) (1:1000, Cat. #3024, RRID: AB_331253), FOXO1 (1:1000, Cat. #2880, RRID: AB_2106495), p-FOXO1 (Thr24) (1:1000, Cat. #9464, RRID: AB_329842), all from Cell Signaling (CST), and β-tubulin (1:2000, Cat. #T0198, Sigma, RRID: AB_477556).

Techniques: Microscopy, Expressing, Labeling, Staining

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Live-cell TIRF imaging of cell-surface INSR, CAV1, and CLTC. (A, B) TIRF microscopy (2 seconds/frame) showed colocalization between INSR-B-TagBFP and (A) CAV1-mRFP or (B) CLTC-mRFP. (C, D) The associated fluorescent intensity of representative INSR-B-TagBFP and colocalized (C) CAV1-mRFP or (D) CLTC-mRFP. (E-G) The (E) diffusion coefficient (μm 2 /s), (F) track radius, and (G) lifetime of INSR-B-TagBFP tracks in cells expressing CAV1-mRFP under 0 nM ( n = 2 cells, 360 tracks) or 2 nM insulin ( n = 2 cells, 324 tracks) or expressing CLTC-mRFP under 0 nM ( n = 4 cells, 917 tracks) or 2 nM insulin ( n = 2 cells, 146 tracks). Data are plotted as SuperPlot, with bigger dots showing mean values of the cells and smaller dots showing individual INSR tracks of the cells. The cumulative probability shows the distribution of the diffusion coefficient, with the dots and numbers showing the median values. (2-ANOVA: # effect of co-expression; post hoc Tukey test: * P < .05; Kolmogorov–Smirnov (KS) test: * P < .05, ** P < .005, *** P < .0005.) Experiments shown were conducted in Ringer's buffer.

Article Snippet: Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA) and probed with antibodies against p-ERK1/2 (Thr202/Tyr204) (1:1000, Cat. #4370, RRID: AB_2315112), ERK1/2 (1:1000, Cat. #4695, RRID: AB_390779), p-AKT (Ser473) (1:1000, Cat. #9271, RRID: AB_329825), p-AKT (Thr308) (1:1000, Cat. #9275, RRID: AB_329828), AKT (1:1000, Cat. #9272, RRID: AB_329827), INSR-β subunit (1:1000, Cat. #3020S, RRID: AB_2249166), p-INSRβ (Tyr1150/1151) (1:1000, Cat. #3024, RRID: AB_331253), FOXO1 (1:1000, Cat. #2880, RRID: AB_2106495), p-FOXO1 (Thr24) (1:1000, Cat. #9464, RRID: AB_329842), all from Cell Signaling (CST), and β-tubulin (1:2000, Cat. #T0198, Sigma, RRID: AB_477556).

Techniques: Imaging, Microscopy, Diffusion-based Assay, Expressing

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Analysis of INSR tracks colocalized or not colocalized with CAV1 or CLTC. (A) INSR tracks that colocalized with CAV1 under 0 or 2 nM insulin (referred to as CAV1.Y.0nM or CAV1.Y.2nM below). (B) INSR tracks that colocalized with CLTC under 0 or 2 nM insulin (referred to as CLTC.Y.0nM or CLTC.Y.2nM below). (C) Diffusion coefficient (μm 2 /s) of INSR-B-TagBFP tracks that did not colocalize with CAV1-mRFP tracks under 0 nM insulin (CAV1.N.0nM) or 2 nM insulin (CAV1.N.2nM) or colocalized with CAV1-mRFP tracks under 0 nM insulin (CAV1.Y.0nM) or 2 nM insulin (CAV1.Y.2nM). The cumulative probability shows the distribution of the measurements, with the dots and numbers showing the median values (same for other cumulative probability plots). (D) Diffusion coefficient (μm 2 /s) of INSR-B-TagBFP tracks that did not colocalize with CLTC-mRFP tracks under 0 nM insulin (CLTC.N.0nM) or 2 nM insulin (CLTC.N.2nM) or colocalized with CLTC-mRFP tracks under 0 nM insulin (CLTC.Y.0nM) or 2 nM insulin (CLTC.Y.2nM). (E) Track radius of INSR-B-TagBFP tracks that did not colocalize with CAV1-mRFP tracks under 0 nM insulin (CAV1.N.0nM) or 2 nM insulin (CAV1.N.2nM) or colocalized with CAV1-mRFP tracks under 0 nM insulin (CAV1.Y.0nM) or 2 nM insulin (CAV1.Y.2nM). (F) Track radius of INSR-B-TagBFP tracks that did not colocalize with CLTC-mRFP tracks under 0 nM insulin (CLTC.N.0nM) or 2 nM insulin (CLTC.N.2nM) or colocalized with CLTC-mRFP tracks under 0 nM insulin (CLTC.Y.0nM) or 2 nM insulin (CLTC.Y.2nM). (G) Lifetime of INSR-B-TagBFP tracks that did not colocalize with CAV1-mRFP tracks under 0 nM insulin (CAV1.N.0nM) or 2 nM insulin (CAV1.N.2nM) or colocalized with CAV1-mRFP tracks under 0 nM insulin (CAV1.Y.0nM) or 2 nM insulin (CAV1.Y.2nM). (H) Lifetime of INSR-B-TagBFP tracks that did not colocalize with CLTC-mRFP tracks under 0 nM insulin (CLTC.N.0nM) or 2 nM insulin (CLTC.N.2nM) or colocalized with CLTC-mRFP tracks under 0 nM insulin (CLTC.Y.0nM) or 2 nM insulin (CLTC.Y.2nM). (CAV1.N.0nM, n = 2 cells, 125 tracks; CAV1.N.2 nM, n = 2 cells, 96 tracks; CAV1.Y.0nM, n = 2 cells, 235 tracks; CAV1.Y.2nM, n = 2 cells, 228 tracks; CLTC.N.0nM, n = 4 cells, 597 tracks; CLTC.N.2nM, n = 2 cells, 91 tracks; CLTC.Y.0nM, n = 4 cells, 320 tracks; CLTC.Y.2nM, n = 2 cells, 55 tracks.) (2-ANOVA: # effect of colocalization, $ effect of insulin, % interaction of the 2 variables; Kolmogorov–Smirnov [KS] test: * P < .05, *** P < .0005.) Experiments shown were conducted in Ringer's buffer.

Article Snippet: Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA) and probed with antibodies against p-ERK1/2 (Thr202/Tyr204) (1:1000, Cat. #4370, RRID: AB_2315112), ERK1/2 (1:1000, Cat. #4695, RRID: AB_390779), p-AKT (Ser473) (1:1000, Cat. #9271, RRID: AB_329825), p-AKT (Thr308) (1:1000, Cat. #9275, RRID: AB_329828), AKT (1:1000, Cat. #9272, RRID: AB_329827), INSR-β subunit (1:1000, Cat. #3020S, RRID: AB_2249166), p-INSRβ (Tyr1150/1151) (1:1000, Cat. #3024, RRID: AB_331253), FOXO1 (1:1000, Cat. #2880, RRID: AB_2106495), p-FOXO1 (Thr24) (1:1000, Cat. #9464, RRID: AB_329842), all from Cell Signaling (CST), and β-tubulin (1:2000, Cat. #T0198, Sigma, RRID: AB_477556).

Techniques: Diffusion-based Assay

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Summary of the effects of CAV1 and CLTC on INSR tracks. Higher insulin (20 nM) promoted INSR and CAV1 colocalization, while lower insulin (0.2 nM) promoted INSR and CLTC colocalization. INSR tracks had lower diffusion coefficient and track radius in CAV1-overexpressing cells than CLTC-overexpressing cells. Within the same cells, INSR tracks colocalized with CAV1 had longer lifetimes and larger track radius than noncolocalized tracks. INSR tracks colocalized with CLTC had longer lifetimes than noncolocalized tracks, which is further increased by insulin.

Article Snippet: Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA) and probed with antibodies against p-ERK1/2 (Thr202/Tyr204) (1:1000, Cat. #4370, RRID: AB_2315112), ERK1/2 (1:1000, Cat. #4695, RRID: AB_390779), p-AKT (Ser473) (1:1000, Cat. #9271, RRID: AB_329825), p-AKT (Thr308) (1:1000, Cat. #9275, RRID: AB_329828), AKT (1:1000, Cat. #9272, RRID: AB_329827), INSR-β subunit (1:1000, Cat. #3020S, RRID: AB_2249166), p-INSRβ (Tyr1150/1151) (1:1000, Cat. #3024, RRID: AB_331253), FOXO1 (1:1000, Cat. #2880, RRID: AB_2106495), p-FOXO1 (Thr24) (1:1000, Cat. #9464, RRID: AB_329842), all from Cell Signaling (CST), and β-tubulin (1:2000, Cat. #T0198, Sigma, RRID: AB_477556).

Techniques: Diffusion-based Assay

Journal: The Journal of Clinical Investigation

Article Title: Activin A secretion by muscle-repairing macrophages induces heterotopic ossification in mice

doi: 10.1172/JCI193797

Figure Lengend Snippet: ( A ) Representative flow cytometry plots showing the gating strategy for isolating MuSCs (TER119 – CD45 – CD31 – Sca-1 – CD106 + ) from muscles of WT mice at 1 dpi. ( B ) Representative immunofluorescence images showing MyHC + myotubes (green) and nuclei (Hoechst; blue) in myoblast cultures with or without 100 ng/mL activin A treatment on day 7. MuSCs were expanded for 4 days to over 90% confluency and differentiated for 3 days. The boxed areas are shown at higher magnification (×4) in the adjacent lower panels. Scale bars: 100 μm. ( C and D ) Quantification of myotube fusion in B , showing the number of nuclei per myotube ( C ) and the percentage of myotubes with more than 4 nuclei ( D ). ( E ) Relative count of myoblasts on the indicated days. MuSCs were isolated on day 0 and then cultured for 1 to 3 days with or without 100 ng/mL activin A. ( F ) RT-qPCR analysis showing relative Myog mRNA expression in myoblasts treated with or without 100 ng/mL activin A for 2 days under differentiation conditions. The P values were calculated using unpaired 2-tailed t test ( C , D , and F ) or 2-way ANOVA with Bonferroni correction for multiple comparisons ( E ). A P value < 0.05 was considered significant. Data are shown as the mean ± SEM.

Article Snippet: To analyze activin A expression in muscle tissue, sections were stained with APC anti-F4/80 antibody (clone BM8; BioLegend), anti-mouse activin A antibody (catalog AF338; R&D Systems), and corresponding Alexa Fluor 488 secondary antibody (catalog A-11029; Thermo Fisher Scientific).

Techniques: Flow Cytometry, Muscles, Immunofluorescence, Isolation, Cell Culture, Quantitative RT-PCR, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Activin A secretion by muscle-repairing macrophages induces heterotopic ossification in mice

doi: 10.1172/JCI193797

Figure Lengend Snippet: ( A ) UMAP visualization of cells isolated from muscle at 1 dpi. Phylogenetic tree showing cluster hierarchy and cell numbers (in parentheses). ( B ) Heatmap showing the top 10 DEGs by log 2 FC (adjusted P < 0.001) across clusters. Typical cell surface molecules were selected for naming. Expression of Ly6C is shown in . ( C ) UMAP showing the expression of Inhba in each cluster. ( D ) Dot plot showing the relative Inhba expression across 3 subclusters of MDMs. ( E and F ) RT-qPCR results showing relative Inhba mRNA expression in muscles at 1 dpi from mice treated with 150 μL vehicle ( n = 7) or clodronate ( n = 9) ( E ) and from mice treated with 0.25 mg isotype IgG ( n = 8) or 0.25 mg anti-Ly6G antibodies ( n = 7) ( F ). ( G ) Gating strategy for sorting 4 subpopulations of Ly6C hi CX3CR1 lo MDMs based on the expression of PDPN and CD9. ( H ) Flow cytometric analysis confirming the purity of sorted cells. ( I ) RT-qPCR results showing relative Inhba expression in indicated subpopulations sorted from muscles on 1 dpi ( n = 4 for each). The expression in the PDPN – CD9 – fraction was set to 1. ( J ) ELISA results showing the concentration of activin A in the culture supernatants of indicated MDMs sorted from muscles on 1 dpi. Supernatants were collected 48 hours after seeding. ( K ) Flow cytometric histogram showing IL-7R expression in indicated MDMs at 1 dpi. The P values were calculated using unpaired 2-tailed t test ( D , E , and J ) or 1-way ANOVA with Tukey’s multiple-comparison test ( I ). A P value < 0.05 was considered significant. Data are shown as the mean ± SEM, and symbols represent individual mice ( D , E , I , and J ).

Article Snippet: To analyze activin A expression in muscle tissue, sections were stained with APC anti-F4/80 antibody (clone BM8; BioLegend), anti-mouse activin A antibody (catalog AF338; R&D Systems), and corresponding Alexa Fluor 488 secondary antibody (catalog A-11029; Thermo Fisher Scientific).

Techniques: Isolation, Expressing, Quantitative RT-PCR, Muscles, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Activin A secretion by muscle-repairing macrophages induces heterotopic ossification in mice

doi: 10.1172/JCI193797

Figure Lengend Snippet: ( A ) Representative microCT images of the hind limbs of uninjured ( n = 3) and injured gHO mice treated with vehicle ( n = 4) or ACVR1 kinase activity inhibitor ( n = 5) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. ( B and C ) Quantification showing HO volume ( B ) and bone mineral content ( C ) in the gHO mice treated with vehicle or ACVR1 inhibitor. ( D ) Experimental timeline for the coculture of FAPs with Mrep. FAPs, non-FAPs, Mrep, and other cells (the remaining CD45 + cells) were sorted from muscles of gHO mice at 1 dpi. Anti–activin A antibody (1 mg/mL) was used to neutralize activin A. ( E ) Alizarin red S staining of the coculture experiment in D . Representative data from 3 independent experiments are shown. ( F and G ) Representative microCT images ( F ) and quantification ( G ) of HO in Acvr1 Q207D -induced HO of Inhba fl/fl mice ( n = 26) and Inhba fl/fl LysM-Cre mice ( n = 17) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. ( H ) RT-qPCR results showing the relative Inhba expression in the muscles at 1 dpi from the mice treated with DMSO ( n = 5) or TAK-242 ( n = 5). ( I – K ) Representative microCT images ( I and J ) and quantification ( K ) of HO in gHO mice treated with vehicle ( n = 10), TAK-242 ( n = 7), and clodronate ( n = 7) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. The P values were calculated using unpaired 2-tailed t test ( B , C , G , and H ) and 1-way ANOVA with Tukey’s multiple-comparison test ( K ). A P value < 0.05 was considered significant. Data are shown as the mean ± SEM, and symbols represent individual mice.

Article Snippet: To analyze activin A expression in muscle tissue, sections were stained with APC anti-F4/80 antibody (clone BM8; BioLegend), anti-mouse activin A antibody (catalog AF338; R&D Systems), and corresponding Alexa Fluor 488 secondary antibody (catalog A-11029; Thermo Fisher Scientific).

Techniques: Activity Assay, Muscles, Staining, Quantitative RT-PCR, Expressing, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Activin A secretion by muscle-repairing macrophages induces heterotopic ossification in mice

doi: 10.1172/JCI193797

Figure Lengend Snippet: ( A ) Representative microCT images of the hind limbs of uninjured ( n = 3) and injured gHO mice treated with vehicle ( n = 4) or ACVR1 kinase activity inhibitor ( n = 5) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. ( B and C ) Quantification showing HO volume ( B ) and bone mineral content ( C ) in the gHO mice treated with vehicle or ACVR1 inhibitor. ( D ) Experimental timeline for the coculture of FAPs with Mrep. FAPs, non-FAPs, Mrep, and other cells (the remaining CD45 + cells) were sorted from muscles of gHO mice at 1 dpi. Anti–activin A antibody (1 mg/mL) was used to neutralize activin A. ( E ) Alizarin red S staining of the coculture experiment in D . Representative data from 3 independent experiments are shown. ( F and G ) Representative microCT images ( F ) and quantification ( G ) of HO in Acvr1 Q207D -induced HO of Inhba fl/fl mice ( n = 26) and Inhba fl/fl LysM-Cre mice ( n = 17) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. ( H ) RT-qPCR results showing the relative Inhba expression in the muscles at 1 dpi from the mice treated with DMSO ( n = 5) or TAK-242 ( n = 5). ( I – K ) Representative microCT images ( I and J ) and quantification ( K ) of HO in gHO mice treated with vehicle ( n = 10), TAK-242 ( n = 7), and clodronate ( n = 7) at 28 dpi. White arrows indicate HO. Scale bars: 1 mm. The P values were calculated using unpaired 2-tailed t test ( B , C , G , and H ) and 1-way ANOVA with Tukey’s multiple-comparison test ( K ). A P value < 0.05 was considered significant. Data are shown as the mean ± SEM, and symbols represent individual mice.

Article Snippet: For the inhibition of activin A activity, 1 mg/mL anti–activin A antibody (catalog AF338; R&D Systems) was added to the osteogenic differentiation medium.

Techniques: Activity Assay, Muscles, Staining, Quantitative RT-PCR, Expressing, Comparison