syntaxin 16 (sx16 Search Results


syntaxin 16 (sx16; #110 162, rrid:ab_887799)  (Synaptic Systems)
90
Synaptic Systems syntaxin 16 (sx16; #110 162, rrid:ab_887799)
EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 (Sx4), SNAP23, VAMP2, and Syntaxin16 <t>(Sx16),</t> as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.
Syntaxin 16 (Sx16; #110 162, Rrid:Ab 887799), supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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syntaxin 16 (sx16; #110 162, rrid:ab_887799) - by Bioz Stars, 2026-05
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syntaxin 4 (sx4; #110 042, rrid:ab_887853)  (Synaptic Systems)
90
Synaptic Systems syntaxin 4 (sx4; #110 042, rrid:ab_887853)
EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 <t>(Sx4),</t> SNAP23, VAMP2, and Syntaxin16 (Sx16), as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.
Syntaxin 4 (Sx4; #110 042, Rrid:Ab 887853), supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/syntaxin 4 (sx4; #110 042, rrid:ab_887853)/product/Synaptic Systems
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syntaxin 4 (sx4; #110 042, rrid:ab_887853) - by Bioz Stars, 2026-05
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mammalian uncoordinated-18c (munc18c)  (Synaptic Systems)
90
Synaptic Systems mammalian uncoordinated-18c (munc18c)
EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 <t>(Sx4),</t> SNAP23, VAMP2, and Syntaxin16 (Sx16), as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.
Mammalian Uncoordinated 18c (Munc18c), supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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synaptosome associated protein 23 (snap23; #111 213, rrid:ab_10805651)  (Synaptic Systems)
90
Synaptic Systems synaptosome associated protein 23 (snap23; #111 213, rrid:ab_10805651)
EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 (Sx4), <t>SNAP23,</t> VAMP2, and Syntaxin16 (Sx16), as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.
Synaptosome Associated Protein 23 (Snap23; #111 213, Rrid:Ab 10805651), supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/synaptosome associated protein 23 (snap23; #111 213, rrid:ab_10805651)/product/Synaptic Systems
Average 90 stars, based on 1 article reviews
synaptosome associated protein 23 (snap23; #111 213, rrid:ab_10805651) - by Bioz Stars, 2026-05
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Image Search Results


EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 (Sx4), SNAP23, VAMP2, and Syntaxin16 (Sx16), as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.

Journal: Molecular Biology of the Cell

Article Title: EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events

doi: 10.1091/mbc.E23-03-0078

Figure Lengend Snippet: EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 (Sx4), SNAP23, VAMP2, and Syntaxin16 (Sx16), as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.

Article Snippet: Mammalian uncoordinated-18c (Munc18c; #116 202, RRID:AB_2619785), Syntaxin 4 (Sx4; #110 042, RRID:AB_887853), vesicle-associated membrane protein 2 (VAMP2; #104 403, RRID:AB_2864782), synaptosome associated protein 23 (SNAP23; #111 213, RRID:AB_10805651), and Syntaxin 16 (Sx16; #110 162, RRID:AB_887799) were all from Synaptic Systems (Coventry, UK).

Techniques: Western Blot, Expressing, Labeling

Summary of mass spectrometry data of Sx16 coimmunoprecipitation analysis.

Journal: Molecular Biology of the Cell

Article Title: EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events

doi: 10.1091/mbc.E23-03-0078

Figure Lengend Snippet: Summary of mass spectrometry data of Sx16 coimmunoprecipitation analysis.

Article Snippet: Mammalian uncoordinated-18c (Munc18c; #116 202, RRID:AB_2619785), Syntaxin 4 (Sx4; #110 042, RRID:AB_887853), vesicle-associated membrane protein 2 (VAMP2; #104 403, RRID:AB_2864782), synaptosome associated protein 23 (SNAP23; #111 213, RRID:AB_10805651), and Syntaxin 16 (Sx16; #110 162, RRID:AB_887799) were all from Synaptic Systems (Coventry, UK).

Techniques: Mass Spectrometry

EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 (Sx4), SNAP23, VAMP2, and Syntaxin16 (Sx16), as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.

Journal: Molecular Biology of the Cell

Article Title: EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events

doi: 10.1091/mbc.E23-03-0078

Figure Lengend Snippet: EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 (Sx4), SNAP23, VAMP2, and Syntaxin16 (Sx16), as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.

Article Snippet: Mammalian uncoordinated-18c (Munc18c; #116 202, RRID:AB_2619785), Syntaxin 4 (Sx4; #110 042, RRID:AB_887853), vesicle-associated membrane protein 2 (VAMP2; #104 403, RRID:AB_2864782), synaptosome associated protein 23 (SNAP23; #111 213, RRID:AB_10805651), and Syntaxin 16 (Sx16; #110 162, RRID:AB_887799) were all from Synaptic Systems (Coventry, UK).

Techniques: Western Blot, Expressing, Labeling

Reduced cholesterol and altered PM lipid content in EHD2 KO adipocytes. (A) Whole-cell and (B) PM samples were subjected to immunoblotting for Munc18c, Sx4, and SNAP23. All data are presented as percentage of WT levels (WT = 100%, dashed line), n = 3 biological replicates (A) and n = 3–6 (B). Data are displayed as mean ± SD, and unpaired two-sample t test was used for statistical analysis. Significance was determined according to ** p ≤ 0.01. (C) Cholesterol levels in FC, PM, and serum (S) from WT and EHD2 KO mice. Differences assessed by two-way ANOVA, involving an interaction (x) between genotype (g) and sample (t) with a posthoc Student’s t test. (D) Score plots from OPLS-DA calculated on membrane lipidomic data acquired in positive (top) and negative (below) electrospray ionization mode. t1, first predictive component; to[1], first orthogonal component. WT in white, EHD2 in gray. (E) Lipids showing significant differences between WT and EHD2 KO PMs (variable importance of projection (VIP >1). PE, PE_ep, PC, and SM. Enrichment was accessed using χ 2 statistics; PC_ep, q = 0.0013, PE, q = 0.011, PE_ep, q = 0.00081, SM, q = 0.00022. Significance was determined according to * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001, n = 3 biological replicates. NKA = Na + /K + -ATPase. All displayed results were obtained from inguinal adipocytes 2 wk of HFD.

Journal: Molecular Biology of the Cell

Article Title: EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events

doi: 10.1091/mbc.E23-03-0078

Figure Lengend Snippet: Reduced cholesterol and altered PM lipid content in EHD2 KO adipocytes. (A) Whole-cell and (B) PM samples were subjected to immunoblotting for Munc18c, Sx4, and SNAP23. All data are presented as percentage of WT levels (WT = 100%, dashed line), n = 3 biological replicates (A) and n = 3–6 (B). Data are displayed as mean ± SD, and unpaired two-sample t test was used for statistical analysis. Significance was determined according to ** p ≤ 0.01. (C) Cholesterol levels in FC, PM, and serum (S) from WT and EHD2 KO mice. Differences assessed by two-way ANOVA, involving an interaction (x) between genotype (g) and sample (t) with a posthoc Student’s t test. (D) Score plots from OPLS-DA calculated on membrane lipidomic data acquired in positive (top) and negative (below) electrospray ionization mode. t1, first predictive component; to[1], first orthogonal component. WT in white, EHD2 in gray. (E) Lipids showing significant differences between WT and EHD2 KO PMs (variable importance of projection (VIP >1). PE, PE_ep, PC, and SM. Enrichment was accessed using χ 2 statistics; PC_ep, q = 0.0013, PE, q = 0.011, PE_ep, q = 0.00081, SM, q = 0.00022. Significance was determined according to * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001, n = 3 biological replicates. NKA = Na + /K + -ATPase. All displayed results were obtained from inguinal adipocytes 2 wk of HFD.

Article Snippet: Mammalian uncoordinated-18c (Munc18c; #116 202, RRID:AB_2619785), Syntaxin 4 (Sx4; #110 042, RRID:AB_887853), vesicle-associated membrane protein 2 (VAMP2; #104 403, RRID:AB_2864782), synaptosome associated protein 23 (SNAP23; #111 213, RRID:AB_10805651), and Syntaxin 16 (Sx16; #110 162, RRID:AB_887799) were all from Synaptic Systems (Coventry, UK).

Techniques: Western Blot, Membrane

EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 (Sx4), SNAP23, VAMP2, and Syntaxin16 (Sx16), as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.

Journal: Molecular Biology of the Cell

Article Title: EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events

doi: 10.1091/mbc.E23-03-0078

Figure Lengend Snippet: EHD2 KD in 3T3-L1 adipocytes is associated with impaired insulin signaling. (A) Representative Western blots of total phospho-tyrosine in 3T3-L1 adipocyte lysates collected 96 h after gene silencing with control siRNA or EHD2 siRNA, either untreated or following 20-min stimulation with 100 nM insulin ( n = 2) and of protein levels of EHD2, SNARE proteins Syntaxin4 (Sx4), SNAP23, VAMP2, and Syntaxin16 (Sx16), as well as regulatory protein Munc18c, and GLUT4 ( n = 3). GAPDH was used as a loading control and the calculations displayed in C were performed exclusively on basal samples. (B) Glucose uptake (2-deoxy-D-glucose) of 3T3-L1 adipocytes, 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), with (INS) or without (Basal) 20-min stimulation with 100 nM insulin. Data were corrected for nonspecific cellular isotope uptake by performing parallel assays in the presence of 10 μM cytochalasin B and normalized to those obtained in the insulin-stimulated control adipocytes for each data set. Mean ± SD of n = 4 independent experiments are shown. Statistical analysis was done using two-way ANOVA Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05. (C) Corresponding quantification of protein expression in EHD2 siRNA KD adipocytes in the absence of an acute insulin challenge (lanes labeled “-” in A) is normalized to GAPDH and expressed as a percentage of protein expression in control siRNA adipocytes. Mean and SD of n = 3 independent experiments are shown. Statistical analysis was conducted using unpaired two-sample t test, * p < 0.05, ** p < 0.01.

Article Snippet: Mammalian uncoordinated-18c (Munc18c; #116 202, RRID:AB_2619785), Syntaxin 4 (Sx4; #110 042, RRID:AB_887853), vesicle-associated membrane protein 2 (VAMP2; #104 403, RRID:AB_2864782), synaptosome associated protein 23 (SNAP23; #111 213, RRID:AB_10805651), and Syntaxin 16 (Sx16; #110 162, RRID:AB_887799) were all from Synaptic Systems (Coventry, UK).

Techniques: Western Blot, Expressing, Labeling

EHD2 KD in 3T3-L1 adipocytes causes impaired SNARE protein assembly. PLA was used to assess pairwise interactions of proteins in 3T3-L1 adipocytes, fixed 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), and following 0 (Basal), 5- or 20-min stimulation with 100 nM insulin. Representative images with PLA signal in green and nuclei staining (DAPI) in blue are shown alongside corresponding quantification of PLA signal per cell normalized to basal control for the following protein pairs: (A) SNAP23 and VAMP2; (B) SNAP23 and Munc18c; (C) VAMP2 and Munc18c; and (D) Syntaxin4 and Munc18c. Mean ± SD of n = 3 independent experiments are shown. Quantification was performed in ImageJ, and statistical analysis was done using two-way ANOVA, Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar = 10 µm.

Journal: Molecular Biology of the Cell

Article Title: EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events

doi: 10.1091/mbc.E23-03-0078

Figure Lengend Snippet: EHD2 KD in 3T3-L1 adipocytes causes impaired SNARE protein assembly. PLA was used to assess pairwise interactions of proteins in 3T3-L1 adipocytes, fixed 96 h after gene silencing with control siRNA (Control) or EHD2 siRNA (EHD2 KD), and following 0 (Basal), 5- or 20-min stimulation with 100 nM insulin. Representative images with PLA signal in green and nuclei staining (DAPI) in blue are shown alongside corresponding quantification of PLA signal per cell normalized to basal control for the following protein pairs: (A) SNAP23 and VAMP2; (B) SNAP23 and Munc18c; (C) VAMP2 and Munc18c; and (D) Syntaxin4 and Munc18c. Mean ± SD of n = 3 independent experiments are shown. Quantification was performed in ImageJ, and statistical analysis was done using two-way ANOVA, Tukey’s Honest Significant Difference (TukeyHSD), * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar = 10 µm.

Article Snippet: Mammalian uncoordinated-18c (Munc18c; #116 202, RRID:AB_2619785), Syntaxin 4 (Sx4; #110 042, RRID:AB_887853), vesicle-associated membrane protein 2 (VAMP2; #104 403, RRID:AB_2864782), synaptosome associated protein 23 (SNAP23; #111 213, RRID:AB_10805651), and Syntaxin 16 (Sx16; #110 162, RRID:AB_887799) were all from Synaptic Systems (Coventry, UK).

Techniques: Staining

Reduced cholesterol and altered PM lipid content in EHD2 KO adipocytes. (A) Whole-cell and (B) PM samples were subjected to immunoblotting for Munc18c, Sx4, and SNAP23. All data are presented as percentage of WT levels (WT = 100%, dashed line), n = 3 biological replicates (A) and n = 3–6 (B). Data are displayed as mean ± SD, and unpaired two-sample t test was used for statistical analysis. Significance was determined according to ** p ≤ 0.01. (C) Cholesterol levels in FC, PM, and serum (S) from WT and EHD2 KO mice. Differences assessed by two-way ANOVA, involving an interaction (x) between genotype (g) and sample (t) with a posthoc Student’s t test. (D) Score plots from OPLS-DA calculated on membrane lipidomic data acquired in positive (top) and negative (below) electrospray ionization mode. t1, first predictive component; to[1], first orthogonal component. WT in white, EHD2 in gray. (E) Lipids showing significant differences between WT and EHD2 KO PMs (variable importance of projection (VIP >1). PE, PE_ep, PC, and SM. Enrichment was accessed using χ 2 statistics; PC_ep, q = 0.0013, PE, q = 0.011, PE_ep, q = 0.00081, SM, q = 0.00022. Significance was determined according to * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001, n = 3 biological replicates. NKA = Na + /K + -ATPase. All displayed results were obtained from inguinal adipocytes 2 wk of HFD.

Journal: Molecular Biology of the Cell

Article Title: EHD2 regulates plasma membrane integrity and downstream insulin receptor signaling events

doi: 10.1091/mbc.E23-03-0078

Figure Lengend Snippet: Reduced cholesterol and altered PM lipid content in EHD2 KO adipocytes. (A) Whole-cell and (B) PM samples were subjected to immunoblotting for Munc18c, Sx4, and SNAP23. All data are presented as percentage of WT levels (WT = 100%, dashed line), n = 3 biological replicates (A) and n = 3–6 (B). Data are displayed as mean ± SD, and unpaired two-sample t test was used for statistical analysis. Significance was determined according to ** p ≤ 0.01. (C) Cholesterol levels in FC, PM, and serum (S) from WT and EHD2 KO mice. Differences assessed by two-way ANOVA, involving an interaction (x) between genotype (g) and sample (t) with a posthoc Student’s t test. (D) Score plots from OPLS-DA calculated on membrane lipidomic data acquired in positive (top) and negative (below) electrospray ionization mode. t1, first predictive component; to[1], first orthogonal component. WT in white, EHD2 in gray. (E) Lipids showing significant differences between WT and EHD2 KO PMs (variable importance of projection (VIP >1). PE, PE_ep, PC, and SM. Enrichment was accessed using χ 2 statistics; PC_ep, q = 0.0013, PE, q = 0.011, PE_ep, q = 0.00081, SM, q = 0.00022. Significance was determined according to * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001, n = 3 biological replicates. NKA = Na + /K + -ATPase. All displayed results were obtained from inguinal adipocytes 2 wk of HFD.

Article Snippet: Mammalian uncoordinated-18c (Munc18c; #116 202, RRID:AB_2619785), Syntaxin 4 (Sx4; #110 042, RRID:AB_887853), vesicle-associated membrane protein 2 (VAMP2; #104 403, RRID:AB_2864782), synaptosome associated protein 23 (SNAP23; #111 213, RRID:AB_10805651), and Syntaxin 16 (Sx16; #110 162, RRID:AB_887799) were all from Synaptic Systems (Coventry, UK).

Techniques: Western Blot, Membrane