mouse anti insulin  (HyTest)

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: INSR, CAV1, and CLTC were detected by the following Cell Signaling primary antibodies: INSRβ (1:1000, Mouse mAb, Cat. #3020S, RRID: AB_2249166), CLTC (1:1000, Rabbit mAb, Cat. #4796, RRID: AB_10828486), Caveolin-1 (1:1000, Rabbit mAb, Cat. #3267, RRID: AB_2275453), and HRP-conjugated VeriBlot secondary antibodies (1:200, anti-mouse Cat. #ab131368, RRID: AB_2895114; anti-rabbit ab131366, RRID: AB_2892718; Abcam) that do not bind to IgG heavy and light chains to reduce background noise.

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: 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: INSR, CAV1, and CLTC were detected by the following Cell Signaling primary antibodies: INSRβ (1:1000, Mouse mAb, Cat. #3020S, RRID: AB_2249166), CLTC (1:1000, Rabbit mAb, Cat. #4796, RRID: AB_10828486), Caveolin-1 (1:1000, Rabbit mAb, Cat. #3267, RRID: AB_2275453), and HRP-conjugated VeriBlot secondary antibodies (1:200, anti-mouse Cat. #ab131368, RRID: AB_2895114; anti-rabbit ab131366, RRID: AB_2892718; Abcam) that do not bind to IgG heavy and light chains to reduce background noise.

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: INSR, CAV1, and CLTC were detected by the following Cell Signaling primary antibodies: INSRβ (1:1000, Mouse mAb, Cat. #3020S, RRID: AB_2249166), CLTC (1:1000, Rabbit mAb, Cat. #4796, RRID: AB_10828486), Caveolin-1 (1:1000, Rabbit mAb, Cat. #3267, RRID: AB_2275453), and HRP-conjugated VeriBlot secondary antibodies (1:200, anti-mouse Cat. #ab131368, RRID: AB_2895114; anti-rabbit ab131366, RRID: AB_2892718; Abcam) that do not bind to IgG heavy and light chains to reduce background noise.

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: INSR, CAV1, and CLTC were detected by the following Cell Signaling primary antibodies: INSRβ (1:1000, Mouse mAb, Cat. #3020S, RRID: AB_2249166), CLTC (1:1000, Rabbit mAb, Cat. #4796, RRID: AB_10828486), Caveolin-1 (1:1000, Rabbit mAb, Cat. #3267, RRID: AB_2275453), and HRP-conjugated VeriBlot secondary antibodies (1:200, anti-mouse Cat. #ab131368, RRID: AB_2895114; anti-rabbit ab131366, RRID: AB_2892718; Abcam) that do not bind to IgG heavy and light chains to reduce background noise.

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: 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