mig6 cdna  (Addgene inc)

Journal: Journal of Cell Communication and Signaling

Article Title: Critical role of mitogen-inducible gene 6 in restraining endothelial cell permeability to maintain vascular homeostasis

doi: 10.1007/s12079-022-00704-z

Figure Lengend Snippet: Vascular permeability is increased in Mig6 knockout mice and endothelial barrier is compromised by MIG6 knockdown. ( a ) Representative images of WT (top) and Mig6 −/− (bottom) mouse showing Evans blue leakage, after treatment with 50 ng/ml of VEGFA (left) or BSA (right) for 1 h. ( b ) Images of Evans blue leakage in excised ears of WT and Mig6 −/− mouse. ( c ) Quantification of Evans blue leakage in ear tissues. Evans blue dye was extracted from ear skin of WT and Mig6 −/− mice to read an absorbance ( n = 3 – 5). ( d ) Histamine (100 μM) or VEGFA (50 ng/ml)-induced EC permeability in siControl or siMIG6-treated HUVECs ( n = 4). ( e ) Normalized resistance of siControl or siMIG6-treated ECs after stimulation with VEGFA. HUVECs were treated with siControl or siMIG6, and grown to confluence on gelatin-coated electrode arrays. After low serum starvation for 3 h, EC monolayer was stimulated with VEGFA (50 ng/ml) and TEER was measured by ECIS at a frequency of 4000 Hz. One-way ANOVA with Sidak multiple comparison test ( c , d) and unpaired Student’s t-test ( e ) were performed. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, NS: not statistically significant

Article Snippet: The cDNAs encoding human MIG6 and VEGFR2 in pCMV3 vector were obtained from SinoBiological (Beijing, China) and subcloned into pGEX-4 T-1 vector (GE Healthcare Life Sciences).

Techniques: Permeability, Knock-Out

Journal: Journal of Cell Communication and Signaling

Article Title: Critical role of mitogen-inducible gene 6 in restraining endothelial cell permeability to maintain vascular homeostasis

doi: 10.1007/s12079-022-00704-z

Figure Lengend Snippet: MIG6 expression is induced by VEGFA and suppressed by VEGFR2 inhibition in ECs. ( a ) VEGFA-induced MIG6 expression. Serum-starved HUVECs were stimulated with a different amount of VEGFA for 30 min. VEGFR2 activation was monitored by phosphorylation of Tyr1175. ( b ) MIG6 induction was analyzed by densitometry and normalized by tubulin. ( c ) Time kinetic assay of MIG6 induction in HUVECs. Serum-starved ECs were stimulated with VEGFA (20 ng/ml) for different time points. ( d ) MIG6 induction at different time points was measured by densitometry and normalized by tubulin. ( e ) HUVECs were treated with a VEGFR2 tyrosine kinase inhibitor Axitinib (10 nM) for 3 h prior to VEGFA treatment (20 ng/ml) for 30 min. The diminished VEGFA-induced MIG6 expression is shown by western blot. ( f ) MIG6 induction was quantified by densitometry and normalized by tubulin. Fold induction relative to the control is shown as the mean ± SEM ( n = 3 for b , d , and f ). Statistical significance was determined by one-way ANOVA with Sidak multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, NS: not statistically significant

Article Snippet: The cDNAs encoding human MIG6 and VEGFR2 in pCMV3 vector were obtained from SinoBiological (Beijing, China) and subcloned into pGEX-4 T-1 vector (GE Healthcare Life Sciences).

Techniques: Expressing, Inhibition, Activation Assay, Kinetic Assay, Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: Critical role of mitogen-inducible gene 6 in restraining endothelial cell permeability to maintain vascular homeostasis

doi: 10.1007/s12079-022-00704-z

Figure Lengend Snippet: MIG6 binds to VEGFR2. ( a ) Association of MIG6 with VEGFR2 was assessed by GST pull-down assay, followed by western blot. ( b ) Schematic representation of VEGFR2 protein domains and the truncated proteins of GST-fusion VEGFR2 domains. Y951, Y1054, Y1059, Y1175, and Y1214 are major tyrosine phosphorylation sites located in the indicated domains of VEGFR2. TM, transmembrane; JX, juxtamembrane domain; KD1, kinase domain 1; KI, kinase insert domain; KD2, kinase domain 2; Cyto, whole cytoplasmic domain of VEGFR2; C-ter, C-terminus domain of VEGFR2 excluding JX, KD1, KI, and KD2. ( c ) Association of Flag-tagged MIG6 with truncated VEGFR2 proteins was assessed by GST pull-down assay, displaying that MIG6 binds to the cytoplasmic domain and kinase domain 2 of VEGFR2. Inputs are shown on the right

Article Snippet: The cDNAs encoding human MIG6 and VEGFR2 in pCMV3 vector were obtained from SinoBiological (Beijing, China) and subcloned into pGEX-4 T-1 vector (GE Healthcare Life Sciences).

Techniques: Pull Down Assay, Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: Critical role of mitogen-inducible gene 6 in restraining endothelial cell permeability to maintain vascular homeostasis

doi: 10.1007/s12079-022-00704-z

Figure Lengend Snippet: Tyrosine phosphorylation and internalization of VEGFR2 are upregulated in MIG6 knockdown ECs. ( a, c ) MIG6 knockdown in HUVECs increases VEGFR2 phosphorylation on Tyr951 and Tyr1054/9 ( a) , and Tyr1175 and Tyr1214 ( c ) in response to VEGFA (50 ng/ml) at 5 and 10 min. ( b , d ) Tyrosine phosphorylation of VEGFR2 shown in ( a ) and ( c ) was analyzed and normalized by total VEGFR2. Fold induction relative to the control is shown as the mean ± SEM ( n = 3 for b and d ). ( e ) Antibody feeding assay of VEGFR2 internalization (red fluorescence) in response to VEGFA (20 ng/ml) for 30 min in ECs treated with siControl and siMIG6 ( n = 6–7). ( f ) Quantification of internalized VEGFR2 fluorescence in ( e ). The internalized fluorescence intensity was analyzed and normalized by the surface level of VEGFR2. Data are presented as mean ± SEM. One-way ANOVA with Sidak multiple comparison test ( b , d ) and unpaired Student’s t-test ( f ) were used for statistical analyses. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, NS: not statistically significant

Article Snippet: The cDNAs encoding human MIG6 and VEGFR2 in pCMV3 vector were obtained from SinoBiological (Beijing, China) and subcloned into pGEX-4 T-1 vector (GE Healthcare Life Sciences).

Techniques: Feeding Assay, Fluorescence

Journal: Journal of Cell Communication and Signaling

Article Title: Critical role of mitogen-inducible gene 6 in restraining endothelial cell permeability to maintain vascular homeostasis

doi: 10.1007/s12079-022-00704-z

Figure Lengend Snippet: MIG6 knockdown activates PLCγ1-Ca 2+ -eNOS signaling and VEGFA-induced increase of permeability by MIG6 knockdown is attenuated by PLCγ1 and eNOS inhibitors. ( a ) Western blot analysis of PLCγ1 phosphorylation on Tyr783 in siControl and siMIG6-treated ECs in response to VEGFA (50 ng/ml) at 10 min. ( b ) PLCγ1 phosphorylation on Tyr783 was analyzed and normalized by total PLCγ1 ( n = 4). ( c ) Intracellular Ca 2+ level in serum-starved ECs treated with siControl and siMIG6 was determined in response to VEGFA (20 ng/ml) at various time points ( n = 3). ( d ) Western blot analysis of eNOS phosphorylation on Ser1177 in ECs treated with siControl and siMIG6, after treatment with VEGFA (50 ng/ml) for 10 min and 30 min. ( e ) eNOS phosphorylation on Ser1177 was analyzed and normalized by total eNOS ( n = 3). ( f ) Western blot analysis of eNOS phosphorylation on Ser1177 in ECs treated with siControl, siMIG6, siPLCγ1, and siMIG6 + siPLCγ1, followed by treatment with VEGFA (50 ng/ml) for 10 min and 30 min. ( g ) eNOS phosphorylation on Ser1177 was analyzed and normalized by total eNOS ( n = 3). ( h, i ) VEGFA-induced permeability was measured in siControl or siMIG6 knockdown ECs treated with a PLCγ1 inhibitor (U73122, 3 μM) for 30 min or an eNOS inhibitor (L-NAME, 300 μM) for 1 h prior to VEGFA (50 ng/ml) treatment ( n = 4 for h ; n = 5 for i ). Statistical significance was determined by one-way ANOVA with Sidak multiple comparison test ( b , e , and g) , paired Student’s t-test ( c ), and two-way ANOVA with Tukey multiple comparison test ( h, i) . Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, NS: not statistically significant

Article Snippet: The cDNAs encoding human MIG6 and VEGFR2 in pCMV3 vector were obtained from SinoBiological (Beijing, China) and subcloned into pGEX-4 T-1 vector (GE Healthcare Life Sciences).

Techniques: Permeability, Western Blot

Journal: Journal of Cell Communication and Signaling

Article Title: Critical role of mitogen-inducible gene 6 in restraining endothelial cell permeability to maintain vascular homeostasis

doi: 10.1007/s12079-022-00704-z

Figure Lengend Snippet: The balance of RAC1/RHOA GTPase activation is disrupted in MIG6 knockdown ECs. ( a, c ) Western blot analysis of RAC1-GTPase ( a) and RHOA-GTPase ( c) activity in siControl and siMIG6 knockdown ECs treated with VEGFA (50 ng/ml) for 10 and 60 min. ( b , d ) RAC1-GTPase ( b) and RHOA-GTPase ( d) activation was analyzed and normalized by total RAC1 and RHOA, respectively. ( e ) The proposed working model illustrates that MIG6 deficiency promotes VEGFA-induced vascular permeability via activation of PLCγ1 and eNOS signaling and perturbation of the balance in RAC1/RHOA activation, resulting in endothelial barrier disruption. One-way ANOVA with Sidak multiple comparison test was performed ( b , d) . Data are presented as mean ± SEM ( n = 3 for b, d ). * p < 0.05, ** p < 0.01

Article Snippet: The cDNAs encoding human MIG6 and VEGFR2 in pCMV3 vector were obtained from SinoBiological (Beijing, China) and subcloned into pGEX-4 T-1 vector (GE Healthcare Life Sciences).

Techniques: Activation Assay, Western Blot, Activity Assay, Permeability