Review



insrβ  (Cell Signaling Technology Inc)


Bioz Verified Symbol Cell Signaling Technology Inc is a verified supplier
Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 95
      Buy from Supplier

    Structured Review

    Cell Signaling Technology Inc insrβ
    Interaction and colocalization between INSR, <t>CAV3,</t> <t>CAV1,</t> 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 <t>INSR</t> 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).
    Insrβ, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+insulin+receptor+%CE%B2/Insulin+Receptor+beta+Mouse+mAb/pmc12963976-233-13-9
    Average 95 stars, based on 117 article reviews
    insrβ - by Bioz Stars, 2026-09
    95/100 stars

    Images

    1) Product Images from "Insulin receptor trafficking and interactions in muscle cells"

    Article Title: Insulin receptor trafficking and interactions in muscle cells

    Journal: Journal of the Endocrine Society

    doi: 10.1210/jendso/bvag020

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Diffusion-based Assay

    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.
    Figure Legend 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.

    Techniques Used: Diffusion-based Assay

    Related Articles

    Western Blot:

    Article Title: Inositol hexakisphosphate kinase 1 is essential for cell junction integrity in the mouse seminiferous epithelium.
    Article Snippet: .. Primary antibodies used for immunoblotting (IB) and immunofluorescence (IF) along with their dilutions were: anti-IP6K1 (GeneTex, GTX103949; 1:3000 IB; Santa Cruz Biotechnology, sc-376290; 1:1000 IB; Sigma, HPA040825; 1:400 IF); anti-β1 integrin (Santa Cruz Biotechnology, sc-6622; 1:1000 IB); anti-α-actinin (Santa Cruz Biotechnology, sc-17829; 1:50 IF, 1:1000 IB); anti-vimentin (Abcam, ab92547; 1:200 IF); anti-occludin (Abcam, ab31721; 1:2500 IB); anticlaudin 11 (Abcam, ab53041; 1:400 IF); anti-claudin 3 (Abcam, ab15102; 1:100 IF, 1:1000 IB); anti-connexin 43 (Abcam, ab11370; 1:1000 IF, 1:5000 IB); anti-nectin-3 (Abcam, ab16913; 1:2500 IB); antiespin (BD Biosciences, 611656; 1:400 IF, 1:2500 IB); anti-phosphoERK1/2 (CST, 9106S; 1:700 IB); anti-ERK1/2 (CST, 9102; 1:1000 IB); anti-β-catenin (Invitrogen, 13–8400; 1:2500 IB); anti-MVH (Abcam, ab13840; 1:300 IF); anti-insulin receptor β (CST, 8338 T; 1:2500 IB); anti-IGF-I receptor β (CST, 8338 T; 1:2500 IB); anti-phospho-AKT S473 (CST, 4060P; 1:1500 IB); anti-Phospho-AKT T308 (CST, 13038S; 1:1000 IB), anti-AKT (CST, 9272S; 1:1500 IB); anti-phospho-cofilin-S3 (St Johns Laboratory, STJ90230; 1:2000 IB); anti-cofilin (CST, 5175 T; 1:2000 IB); anti-phospho-GSK3β S9 (CST, 5558S; 1:3000 IB); anti-GSKβ (CST, 12456S; 1:3000 IB). ..

    Article Title: Multi-layered proteomics identifies insulin-induced upregulation of the EphA2 receptor via the ERK pathway which is dependent on low IGF1R level.
    Article Snippet: Human insulin (Novo Nordisk A/S) and S59726 (Novo Nordisk A/S) was utilized for the experiments. .. The following antibodies were applied for Western blots (WBs): rabbit anti–phospho–IGF-1Rβ (Tyr1131)/insulin receptor β (Tyr1146), rabbit anti–insulin receptor β (4B8), rabbit IGF-I Receptor β (111A9), rabbit anti–phosphoAkt (Thr308) and anti-Akt, mouse anti–phospho-ERK1/2 (Thr202/Tyr204), rabbit anti-ERK1/2 (Cell Signaling Technology); rabbit anti-Eph receptor A2 antibody (ab273118 and ab185156), mouse anti-beta actin antibody (ab8226) (Abcam); insulin receptor β antibody (CT-3) (Santa Cruz); goat anti-rabbit immunoglobulin (IgG) secondary antibody, horseradish peroxidase (HRP)–conjugated, and goat anti-mouse IgG secondary antibody, HRP-conjugated (Bio-Rad). .. For phospho (p)Tyr enrichment in the SILAC experiment, pTyr1000 (8803) and pTyr100 (5636) (Cell Signaling Technology) were used.

    Article Title: Inositol hexakisphosphate kinase 1 is essential for cell junction integrity in the mouse seminiferous epithelium
    Article Snippet: .. Primary antibodies used for immunoblotting (IB) and immunofluorescence (IF) along with their dilutions were: anti-IP6K1 (GeneTex, GTX103949; 1:3000 IB, 1μg IP); anti-IP6K1 (Sigma, HPA040825; 1:400 IF); anti-β1 integrin (Santa Cruz Biotechnology, sc-6622; 1:1000 IB); anti-α-actinin (Santa Cruz Biotechnology, sc-17829; 1:50 IF, 1:1000 IB); anti-vimentin (Abcam, ab92547; 1:200 IF); anti-occludin (Abcam, ab31721; 1:2500 IB); anti-claudin 11 (Abcam, ab53041; 1:400 IF); anti-claudin 3 (Abcam, ab15102; 1:100 IF, 1:1000 IB); anti-connexin 43 (Abcam, ab11370; 1:1000 IF, 1:5000 IB); anti-nectin-3 (Abcam, ab16913; 1:2500 IB); anti-espin (BD Biosciences, 611656; 1:400 IF, 1:2500 IB); anti-phospho-ERK1/2 (CST, 9106S; 1:700 IB); anti-ERK1/2 (CST, 9102; 1:1000 IB); anti-β-catenin (Invitrogen, 13-8400; 1:2500 IB); anti-MVH (Abcam, ab13840; 1:300 IF); anti-insulin receptor β (CST, 8338T; 1:2500 IB); anti-IGF-I receptor β (CST, 8338T; 1:2500 IB); anti-phospho-AKT S473 (CST, 4060P; 1:1500 IB); anti-AKT (CST, 9272S; 1:1500 IB); anti-phospho-cofilin-S3 (St Johns Laboratory, STJ90230; 1:2000 IB); anti-cofilin (CST, 5175T; 1:2000 IB). ..

    Immunofluorescence:

    Article Title: Inositol hexakisphosphate kinase 1 is essential for cell junction integrity in the mouse seminiferous epithelium.
    Article Snippet: .. Primary antibodies used for immunoblotting (IB) and immunofluorescence (IF) along with their dilutions were: anti-IP6K1 (GeneTex, GTX103949; 1:3000 IB; Santa Cruz Biotechnology, sc-376290; 1:1000 IB; Sigma, HPA040825; 1:400 IF); anti-β1 integrin (Santa Cruz Biotechnology, sc-6622; 1:1000 IB); anti-α-actinin (Santa Cruz Biotechnology, sc-17829; 1:50 IF, 1:1000 IB); anti-vimentin (Abcam, ab92547; 1:200 IF); anti-occludin (Abcam, ab31721; 1:2500 IB); anticlaudin 11 (Abcam, ab53041; 1:400 IF); anti-claudin 3 (Abcam, ab15102; 1:100 IF, 1:1000 IB); anti-connexin 43 (Abcam, ab11370; 1:1000 IF, 1:5000 IB); anti-nectin-3 (Abcam, ab16913; 1:2500 IB); antiespin (BD Biosciences, 611656; 1:400 IF, 1:2500 IB); anti-phosphoERK1/2 (CST, 9106S; 1:700 IB); anti-ERK1/2 (CST, 9102; 1:1000 IB); anti-β-catenin (Invitrogen, 13–8400; 1:2500 IB); anti-MVH (Abcam, ab13840; 1:300 IF); anti-insulin receptor β (CST, 8338 T; 1:2500 IB); anti-IGF-I receptor β (CST, 8338 T; 1:2500 IB); anti-phospho-AKT S473 (CST, 4060P; 1:1500 IB); anti-Phospho-AKT T308 (CST, 13038S; 1:1000 IB), anti-AKT (CST, 9272S; 1:1500 IB); anti-phospho-cofilin-S3 (St Johns Laboratory, STJ90230; 1:2000 IB); anti-cofilin (CST, 5175 T; 1:2000 IB); anti-phospho-GSK3β S9 (CST, 5558S; 1:3000 IB); anti-GSKβ (CST, 12456S; 1:3000 IB). ..

    Article Title: Inositol hexakisphosphate kinase 1 is essential for cell junction integrity in the mouse seminiferous epithelium
    Article Snippet: .. Primary antibodies used for immunoblotting (IB) and immunofluorescence (IF) along with their dilutions were: anti-IP6K1 (GeneTex, GTX103949; 1:3000 IB, 1μg IP); anti-IP6K1 (Sigma, HPA040825; 1:400 IF); anti-β1 integrin (Santa Cruz Biotechnology, sc-6622; 1:1000 IB); anti-α-actinin (Santa Cruz Biotechnology, sc-17829; 1:50 IF, 1:1000 IB); anti-vimentin (Abcam, ab92547; 1:200 IF); anti-occludin (Abcam, ab31721; 1:2500 IB); anti-claudin 11 (Abcam, ab53041; 1:400 IF); anti-claudin 3 (Abcam, ab15102; 1:100 IF, 1:1000 IB); anti-connexin 43 (Abcam, ab11370; 1:1000 IF, 1:5000 IB); anti-nectin-3 (Abcam, ab16913; 1:2500 IB); anti-espin (BD Biosciences, 611656; 1:400 IF, 1:2500 IB); anti-phospho-ERK1/2 (CST, 9106S; 1:700 IB); anti-ERK1/2 (CST, 9102; 1:1000 IB); anti-β-catenin (Invitrogen, 13-8400; 1:2500 IB); anti-MVH (Abcam, ab13840; 1:300 IF); anti-insulin receptor β (CST, 8338T; 1:2500 IB); anti-IGF-I receptor β (CST, 8338T; 1:2500 IB); anti-phospho-AKT S473 (CST, 4060P; 1:1500 IB); anti-AKT (CST, 9272S; 1:1500 IB); anti-phospho-cofilin-S3 (St Johns Laboratory, STJ90230; 1:2000 IB); anti-cofilin (CST, 5175T; 1:2000 IB). ..

    Incubation:

    Article Title: Targeting Soluble VCAM1 and GSK3β Improves Cerebrovascular Function and Reduces Stroke Pathology in Diabetic Mice
    Article Snippet: .. Membranes were blocked with 5% nonfat milk and incubated with one of the following primary antibodies overnight at 4 °C: anti-insulin receptor β (#3025), phospho-GSK3β (Ser9; #5558), and histamine H1 Receptor (#55550) were from Cell Signaling, Inc., anti-histidine decarboxylase (#EPR26392-79) was from Abcam Inc. (Waltham, MA, USA), histamine H2 receptor (#NB600-812) was from Novus Biologicals (Centennial, CO, USA), and anti-actin (#MAB1501) was from MilliporeSigma (St. Louis, MO, USA). ..



    Similar Products

    phospho ir t1150  (Novus Biologicals)
    94
    Novus Biologicals phospho ir t1150
    Phospho Ir T1150, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+insulin+receptor+%CE%B2/Insulin+Receptor+beta+%5Bp+Thr1160%5D+Antibody/pmc13078873-293-27-30
    Average 94 stars, based on 1 article reviews
    phospho ir t1150 - by Bioz Stars, 2026-09
    94/100 stars
    		  Buy from Supplier
    insrβ  (Cell Signaling Technology Inc)
    95
    Cell Signaling Technology Inc insrβ
    Interaction and colocalization between INSR, <t>CAV3,</t> <t>CAV1,</t> 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 <t>INSR</t> 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).
    Insrβ, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+insulin+receptor+%CE%B2/Insulin+Receptor+beta+Mouse+mAb/pmc12963976-233-13-9
    Average 95 stars, based on 1 article reviews
    insrβ - by Bioz Stars, 2026-09
    95/100 stars
    		  Buy from Supplier
    anti phosphorylated igf 1r  (Cell Signaling Technology Inc)
    95
    Cell Signaling Technology Inc anti phosphorylated igf 1r
    Interaction and colocalization between INSR, <t>CAV3,</t> <t>CAV1,</t> 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 <t>INSR</t> 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).
    Anti Phosphorylated Igf 1r, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+insulin+receptor+%CE%B2/Phospho-IGF-I+Receptor+beta+(Tyr1131)%2FInsulin+Receptor+beta+(Tyr1146)+Antibody/pm41917778-79-14-16
    Average 95 stars, based on 1 article reviews
    anti phosphorylated igf 1r - by Bioz Stars, 2026-09
    95/100 stars
    		  Buy from Supplier
    insr β subunit  (Cell Signaling Technology Inc)
    95
    Cell Signaling Technology Inc insr β subunit
    Biochemical and live-cell imaging characterization of the effects of insulin on <t>INSR.</t> (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).
    Insr β Subunit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+insulin+receptor+%CE%B2/Insulin+Receptor+beta+Mouse+mAb/pmc12963976-174-49-78
    Average 95 stars, based on 1 article reviews
    insr β subunit - by Bioz Stars, 2026-09
    95/100 stars
    		  Buy from Supplier
    anti p ir tyr1150 1151  (Cell Signaling Technology Inc)
    96
    Cell Signaling Technology Inc anti p ir tyr1150 1151
    Biochemical and live-cell imaging characterization of the effects of insulin on <t>INSR.</t> (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).
    Anti P Ir Tyr1150 1151, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+insulin+receptor+%CE%B2/Phospho-IGF-I+Receptor+beta+(Tyr1135%2F1136)%2FInsulin+Receptor+beta+(Tyr1150%2F1151)+Rabbit+mAb/pm41893475-97-7-39
    Average 96 stars, based on 1 article reviews
    anti p ir tyr1150 1151 - by Bioz Stars, 2026-09
    96/100 stars
    		  Buy from Supplier
    anti ir  (Cell Signaling Technology Inc)
    96
    Cell Signaling Technology Inc anti ir
    Biochemical and live-cell imaging characterization of the effects of insulin on <t>INSR.</t> (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).
    Anti Ir, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+insulin+receptor+%CE%B2/Insulin+Receptor+beta+Rabbit+mAb/pm41893475-97-10-39
    Average 96 stars, based on 1 article reviews
    anti ir - by Bioz Stars, 2026-09
    96/100 stars
    		  Buy from Supplier
    anti phospho igf1r  (Cell Signaling Technology Inc)
    96
    Cell Signaling Technology Inc anti phospho igf1r
    Biochemical and live-cell imaging characterization of the effects of insulin on <t>INSR.</t> (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).
    Anti Phospho Igf1r, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+insulin+receptor+%CE%B2/Phospho-IGF-I+Receptor+beta+(Tyr1135%2F1136)%2FInsulin+Receptor+beta+(Tyr1150%2F1151)+Rabbit+mAb/bio_rxiv__64898__2026__03__25__714159-324-30-34
    Average 96 stars, based on 1 article reviews
    anti phospho igf1r - by Bioz Stars, 2026-09
    96/100 stars
    		  Buy from Supplier
    phospho insr  (Cell Signaling Technology Inc)
    96
    Cell Signaling Technology Inc phospho insr
    Biochemical and live-cell imaging characterization of the effects of insulin on <t>INSR.</t> (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).
    Phospho Insr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+insulin+receptor+%CE%B2/Phospho-IGF-I+Receptor+beta+(Tyr1135%2F1136)%2FInsulin+Receptor+beta+(Tyr1150%2F1151)+Rabbit+mAb/pm41845069-118-3-4
    Average 96 stars, based on 1 article reviews
    phospho insr - by Bioz Stars, 2026-09
    96/100 stars
    		  Buy from Supplier
    insr  (Cell Signaling Technology Inc)
    96
    Cell Signaling Technology Inc insr
    Biochemical and live-cell imaging characterization of the effects of insulin on <t>INSR.</t> (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).
    Insr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+insulin+receptor+%CE%B2/Phospho-IGF-I+Receptor+beta+(Tyr1135%2F1136)%2FInsulin+Receptor+beta+(Tyr1150%2F1151)+Rabbit+mAb/pm41845069-118-9-10
    Average 96 stars, based on 1 article reviews
    insr - by Bioz Stars, 2026-09
    96/100 stars
    		  Buy from Supplier

    Image Search Results


    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).

    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

    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.

    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

    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.

    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

    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.

    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

    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).

    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

    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).

    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

    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.

    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

    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).

    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

    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.).

    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

    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.

    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

    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.

    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

    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.

    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