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human irs1 (Santa Cruz Biotechnology)

Name: human irs1
Brand: Santa Cruz Biotechnology
Rating: 96 (388 reviews)

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Santa Cruz Biotechnology human irs1
Human Irs1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 388 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/human irs1/product/Santa Cruz Biotechnology
Average 96 stars, based on 388 article reviews

human irs1 - by Bioz Stars, 2026-02

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(A) Immunoblotting of IRS1 phosphorylation and protein levels in the muscle of fish fed CON or PO diet (n=6). (B) IRS1 phosphorylation and protein levels were assayed by immunoblotting in fish myocytes and C2C12 myotubes treated with the indicated concentrations of PA for 12 h(n=3). (C) IRS1 phosphorylation and protein levels were measured by immunoblotting in fish myocytes and C2C12 myotubes treated with rapamycin or Torin1 treatment in the presence or absence of PA for 12 h (n=3). (D) IRS1 phosphorylation and protein levels were tested by immunoblotting in fish myocytes treated with control or MHY1485 treatment in the presence or absence of PA for 12 h (n=3). (E) Relative mRNA levels of insa , insb , irs1 and irs2 were tested by quantitative PCR in the muscle of fish fed CON or PO diet (n=4). (F) Relative mRNA levels of insa , insb , irs1 and irs2 were analyzed by quantitative PCR in fish myocytes under control or PA treatments for 12 h and 24 h (n=3). (G) Relative mRNA levels of insr , irs1 and irs2 were measured by quantitative PCR in C2C12 myotubes under control or PA treatments for 12 h and 24 h (n=3). (H) Relative mRNA levels of irs1 were analyzed by quantitative PCR in fish myocytes and C2C12 myotubes treated with control, rapamycin or Torin1 in the presence or absence of PA for 12 h (n=3). The results are presented as the mean ± SEM and were analyzed using independent t -tests (* p < 0.05, ** p < 0.01, *** p < 0.001) and Tukey’s tests (bars bearing different letters are significantly different among treatments ( p < 0.05)). See also .

Journal: bioRxiv

Article Title: Tip60-mediated Rheb acetylation links palmitic acid with mTORC1 activation and insulin resistance

doi: 10.1101/2023.08.18.553816

Figure Lengend Snippet: (A) Immunoblotting of IRS1 phosphorylation and protein levels in the muscle of fish fed CON or PO diet (n=6). (B) IRS1 phosphorylation and protein levels were assayed by immunoblotting in fish myocytes and C2C12 myotubes treated with the indicated concentrations of PA for 12 h(n=3). (C) IRS1 phosphorylation and protein levels were measured by immunoblotting in fish myocytes and C2C12 myotubes treated with rapamycin or Torin1 treatment in the presence or absence of PA for 12 h (n=3). (D) IRS1 phosphorylation and protein levels were tested by immunoblotting in fish myocytes treated with control or MHY1485 treatment in the presence or absence of PA for 12 h (n=3). (E) Relative mRNA levels of insa , insb , irs1 and irs2 were tested by quantitative PCR in the muscle of fish fed CON or PO diet (n=4). (F) Relative mRNA levels of insa , insb , irs1 and irs2 were analyzed by quantitative PCR in fish myocytes under control or PA treatments for 12 h and 24 h (n=3). (G) Relative mRNA levels of insr , irs1 and irs2 were measured by quantitative PCR in C2C12 myotubes under control or PA treatments for 12 h and 24 h (n=3). (H) Relative mRNA levels of irs1 were analyzed by quantitative PCR in fish myocytes and C2C12 myotubes treated with control, rapamycin or Torin1 in the presence or absence of PA for 12 h (n=3). The results are presented as the mean ± SEM and were analyzed using independent t -tests (* p < 0.05, ** p < 0.01, *** p < 0.001) and Tukey’s tests (bars bearing different letters are significantly different among treatments ( p < 0.05)). See also .

Article Snippet: Antibodies against Phospho-IRS1 (Y612) (#MAB7314) was purchased from RnD systems (USA).

Techniques: Western Blot, Phospho-proteomics, Control, Real-time Polymerase Chain Reaction

(A) Relative mRNA levels of irs1 were tested by quantitative PCR in the muscle of fish fed CON, OO or LO diet (n=4). (B) Relative mRNA levels of irs1 were analyzed by quantitative PCR in C2C12 myotubes under the indicated concentrations of OA or LA treatment for 12 h (n=3). The results are presented as the mean ± SEM and were analyzed using independent t -tests (* p < 0.05).

Journal: bioRxiv

Article Title: Tip60-mediated Rheb acetylation links palmitic acid with mTORC1 activation and insulin resistance

doi: 10.1101/2023.08.18.553816

Figure Lengend Snippet: (A) Relative mRNA levels of irs1 were tested by quantitative PCR in the muscle of fish fed CON, OO or LO diet (n=4). (B) Relative mRNA levels of irs1 were analyzed by quantitative PCR in C2C12 myotubes under the indicated concentrations of OA or LA treatment for 12 h (n=3). The results are presented as the mean ± SEM and were analyzed using independent t -tests (* p < 0.05).

Article Snippet: Antibodies against Phospho-IRS1 (Y612) (#MAB7314) was purchased from RnD systems (USA).

Techniques: Real-time Polymerase Chain Reaction

$(A) Relative dual luciferase activity analysis was conducted to measure the effect of ATF4, PPARα, PPARγ, TFEB and HNF1α on IRS1 promoter activity in HEK293T cells (n=3). (B) Relative dual luciferase activity analysis was conducted to measure the effect of TFEB at different concentration gradients on IRS1 promoter activity in HEK293T cells (n = 3). (C) Relative dual luciferase activity analysis was performed to test the effect of TFEB on IRS1 promoter activity with mutation of the predicted binding sites in HEK293T cells (n = 3). (D and E) The binding between TFEB and the predicted region of the IRS1 promoter was demonstrated by EMSA (D) and ChIP (E) in HEK293T cells. (F) TFEB and IRS1 protein levels were measured by immunoblotting in fish myocytes transfected with pCS2 (empty vector) or pCS2-TFEB plasmids (n = 3). (G) TFEB nuclear translocation was assayed by immunoblotting nuclear fractions of fish myocytes and C2C12 myotubes treated with control or PA treatment for 12 h (n = 3). (H) TFEB nuclear translocation was tested by immunoblotting nuclear fractions of fish myocytes and C2C12 myotubes treated with control, rapamycin or Torin1 treatment in the presence or absence of PA for 12 h (n = 3). (I) Relative mRNA levels of tfeb and irs1 were analyzed by quantitative PCR in fish myocytes treated with control or TFEB activator 1 treatment in the presence or absence of PA for 12 h (n = 3). (J) IRS1 protein levels and the phosphorylation of AKT were tested by immunoblotting in fish myocytes treated with control or TFEB activator 1 treatment in the presence or absence of PA for 12 h (n = 3). (K) AKT phosphorylation levels were measured by immunoblotting in fish myocytes and C2C12 myotubes (n = 3). Cells were pretreated with control or TFEB activator 1 treatment in the presence or absence of PA for 12 h, and then stimulated with insulin for 5 min. The results are presented as the mean ± SEM and were analyzed using independent t -tests (* p < 0.05, ** p < 0.01, *** p < 0.001) and Tukey’s tests (bars bearing different letters are significantly different among treatments ( p < 0.05)).$

Journal: bioRxiv

Article Title: Tip60-mediated Rheb acetylation links palmitic acid with mTORC1 activation and insulin resistance

doi: 10.1101/2023.08.18.553816

Figure Lengend Snippet: (A) Relative dual luciferase activity analysis was conducted to measure the effect of ATF4, PPARα, PPARγ, TFEB and HNF1α on IRS1 promoter activity in HEK293T cells (n=3). (B) Relative dual luciferase activity analysis was conducted to measure the effect of TFEB at different concentration gradients on IRS1 promoter activity in HEK293T cells (n = 3). (C) Relative dual luciferase activity analysis was performed to test the effect of TFEB on IRS1 promoter activity with mutation of the predicted binding sites in HEK293T cells (n = 3). (D and E) The binding between TFEB and the predicted region of the IRS1 promoter was demonstrated by EMSA (D) and ChIP (E) in HEK293T cells. (F) TFEB and IRS1 protein levels were measured by immunoblotting in fish myocytes transfected with pCS2 (empty vector) or pCS2-TFEB plasmids (n = 3). (G) TFEB nuclear translocation was assayed by immunoblotting nuclear fractions of fish myocytes and C2C12 myotubes treated with control or PA treatment for 12 h (n = 3). (H) TFEB nuclear translocation was tested by immunoblotting nuclear fractions of fish myocytes and C2C12 myotubes treated with control, rapamycin or Torin1 treatment in the presence or absence of PA for 12 h (n = 3). (I) Relative mRNA levels of tfeb and irs1 were analyzed by quantitative PCR in fish myocytes treated with control or TFEB activator 1 treatment in the presence or absence of PA for 12 h (n = 3). (J) IRS1 protein levels and the phosphorylation of AKT were tested by immunoblotting in fish myocytes treated with control or TFEB activator 1 treatment in the presence or absence of PA for 12 h (n = 3). (K) AKT phosphorylation levels were measured by immunoblotting in fish myocytes and C2C12 myotubes (n = 3). Cells were pretreated with control or TFEB activator 1 treatment in the presence or absence of PA for 12 h, and then stimulated with insulin for 5 min. The results are presented as the mean ± SEM and were analyzed using independent t -tests (* p < 0.05, ** p < 0.01, *** p < 0.001) and Tukey’s tests (bars bearing different letters are significantly different among treatments ( p < 0.05)).

Article Snippet: Antibodies against Phospho-IRS1 (Y612) (#MAB7314) was purchased from RnD systems (USA).

Techniques: Luciferase, Activity Assay, Concentration Assay, Mutagenesis, Binding Assay, Western Blot, Transfection, Plasmid Preparation, Translocation Assay, Control, Real-time Polymerase Chain Reaction, Phospho-proteomics

Journal: Molecular Metabolism

Article Title: Protein kinase D2 modulates hepatic insulin sensitivity in male mice

doi: 10.1016/j.molmet.2024.102045

Figure Lengend Snippet: List of used primary antibodies.

Article Snippet: Phospho-IRS1 Tyr608 (mouse)/Tyr612 (human) , Merck Millipore , 09–432 , Rabbit , 1:5000.

Techniques:

PKD2 participates in the control of IRS1 phosphorylation to modulate insulin sensitivity . A . PKD2 fl/fl and PKD2 ΔHep primary hepatocytes were stimulated with insulin (10 nM) at the indicated times. pIR and IR protein levels were analyzed by immunoblot. Total IR and Vinculin were used as loading controls. Densitometric quantification of pIR protein levels after insulin treatment. Values are mean ± SEM (n = 9). B. Western blot images showing pIRS1 Ser612 and total IRS1 protein levels from primary mouse hepatocytes treated with CRT0066101 (10 μM, 2 h). Vinculin was used as loading control. Densitometric quantification of pIRS1 Ser612 protein levels. Values are mean ± SEM (n = 10 for vehicle and n = 8 for CRT0066101). ∗∗∗p < 0.001 versus vehicle according to Student's t-test. C . Western blot images showing pIRS1 Ser616, IRS1 and pPKD1/2 protein levels from Huh7 human hepatocytes treated with CRT0066101 (5 and 10 μM, 2 h). Vinculin was used as loading control. Densitometric quantification of pIRS1 Ser616 protein levels. Values are mean ± SEM (pSer616 IRS1, n = 11; IRS1, n = 4). ∗∗∗p < 0.001 versus untreated according to One-way ANOVA with Bonferroni post hoc test. D. Western blot images showing pIRS1 Ser612, total IRS1 and pPKD1/2 protein levels from PKD2 fl/fl and PKD2 ΔHep primary hepatocytes. Vinculin was used as loading control. Densitometric quantification of pIRS1 Ser612 and total IRS1 protein levels. Values are mean ± SEM (pIRS1 Ser612, n = 6; IRS1, n = 10 for PKD2 fl/fl and n = 12 for PKD2 ΔHep hepatocytes). ∗∗∗p < 0.001 versus PKD2 fl/fl primary hepatocytes according to Student's t-test (pIRS1) and unpaired t test with Welch's correction (IRS1). E. IRS1 was immunoprecipitated from PKD2 fl/fl primary hepatocytes treated or not with CRT0066101 (10 μM, 2 h) and from PKD2 ΔHep primary hepatocytes. Total pSer residues and IRS1 levels were determined by Western blot. The arrow points pSer residues that disappear in the absence of PKD2. A representative experiment out of 3 is shown. F. PKD2 fl/fl and PKD2 ΔHep primary hepatocytes were stimulated with insulin (10 nM) at the indicated times. pIRS1 Tyr608 and total IRS1 protein levels were analyzed by immunoblot. Vinculin was used as loading controls. Densitometric quantification of pIRS1 Tyr608 protein levels after insulin treatment. Values are mean ± SEM (n = 8). ∗p < 0.05 versus PKD2 fl/fl primary hepatocytes according to Two-way ANOVA with Bonferroni post hoc test. G . Huh7 human hepatocytes were transfected either EGFP or EGFP-PKD2-CA, a constitutively active form of human PKD2 fused to EGFP protein where Ser706 and Ser710 were mutated to glutamic acid to mimic serine phosphorylation. After 24 h, cells were serum-starved and stimulated with insulin (10 nM, 5 min). IRS1 was immunoprecipitated and PKD2 in the immunocomplexes was determined by Western blot. H. Huh7 human hepatocytes were transfected either EGFP or EGFP-PKD2-CA. After 24 h, both endogenous and transfected PKD2 were immunoprecipitated with the h-PKD2 antibody and IRS1 in the immunocomplexes was determined by Western blot.

Journal: Molecular Metabolism

Article Title: Protein kinase D2 modulates hepatic insulin sensitivity in male mice

doi: 10.1016/j.molmet.2024.102045

Figure Lengend Snippet: PKD2 participates in the control of IRS1 phosphorylation to modulate insulin sensitivity . A . PKD2 fl/fl and PKD2 ΔHep primary hepatocytes were stimulated with insulin (10 nM) at the indicated times. pIR and IR protein levels were analyzed by immunoblot. Total IR and Vinculin were used as loading controls. Densitometric quantification of pIR protein levels after insulin treatment. Values are mean ± SEM (n = 9). B. Western blot images showing pIRS1 Ser612 and total IRS1 protein levels from primary mouse hepatocytes treated with CRT0066101 (10 μM, 2 h). Vinculin was used as loading control. Densitometric quantification of pIRS1 Ser612 protein levels. Values are mean ± SEM (n = 10 for vehicle and n = 8 for CRT0066101). ∗∗∗p < 0.001 versus vehicle according to Student's t-test. C . Western blot images showing pIRS1 Ser616, IRS1 and pPKD1/2 protein levels from Huh7 human hepatocytes treated with CRT0066101 (5 and 10 μM, 2 h). Vinculin was used as loading control. Densitometric quantification of pIRS1 Ser616 protein levels. Values are mean ± SEM (pSer616 IRS1, n = 11; IRS1, n = 4). ∗∗∗p < 0.001 versus untreated according to One-way ANOVA with Bonferroni post hoc test. D. Western blot images showing pIRS1 Ser612, total IRS1 and pPKD1/2 protein levels from PKD2 fl/fl and PKD2 ΔHep primary hepatocytes. Vinculin was used as loading control. Densitometric quantification of pIRS1 Ser612 and total IRS1 protein levels. Values are mean ± SEM (pIRS1 Ser612, n = 6; IRS1, n = 10 for PKD2 fl/fl and n = 12 for PKD2 ΔHep hepatocytes). ∗∗∗p < 0.001 versus PKD2 fl/fl primary hepatocytes according to Student's t-test (pIRS1) and unpaired t test with Welch's correction (IRS1). E. IRS1 was immunoprecipitated from PKD2 fl/fl primary hepatocytes treated or not with CRT0066101 (10 μM, 2 h) and from PKD2 ΔHep primary hepatocytes. Total pSer residues and IRS1 levels were determined by Western blot. The arrow points pSer residues that disappear in the absence of PKD2. A representative experiment out of 3 is shown. F. PKD2 fl/fl and PKD2 ΔHep primary hepatocytes were stimulated with insulin (10 nM) at the indicated times. pIRS1 Tyr608 and total IRS1 protein levels were analyzed by immunoblot. Vinculin was used as loading controls. Densitometric quantification of pIRS1 Tyr608 protein levels after insulin treatment. Values are mean ± SEM (n = 8). ∗p < 0.05 versus PKD2 fl/fl primary hepatocytes according to Two-way ANOVA with Bonferroni post hoc test. G . Huh7 human hepatocytes were transfected either EGFP or EGFP-PKD2-CA, a constitutively active form of human PKD2 fused to EGFP protein where Ser706 and Ser710 were mutated to glutamic acid to mimic serine phosphorylation. After 24 h, cells were serum-starved and stimulated with insulin (10 nM, 5 min). IRS1 was immunoprecipitated and PKD2 in the immunocomplexes was determined by Western blot. H. Huh7 human hepatocytes were transfected either EGFP or EGFP-PKD2-CA. After 24 h, both endogenous and transfected PKD2 were immunoprecipitated with the h-PKD2 antibody and IRS1 in the immunocomplexes was determined by Western blot.

Article Snippet: Phospho-IRS1 Tyr608 (mouse)/Tyr612 (human) , Merck Millipore , 09–432 , Rabbit , 1:5000.

Techniques: Control, Western Blot, Immunoprecipitation, Transfection

Overexpression of a constitutively active PKD2 mutant in hepatocytes promotes insulin resistance in vitro and in vivo . A . Huh7 cells were transfected either EGFP or EGFP-PKD2-CA. After 24 h, cells were serum-starved and stimulated with insulin (10 nM, 5–15 min). pAKT (Ser473 and Thr308), total AKT and EGFP levels are shown. GAPDH was used as loading control. Values are mean ± SEM (pAKT Ser473, n = 10 for 5 min and n = 7 for 15 min; pAKT Thr308, n = 6 for 5 min and n = 4 for 15 min). ∗∗∗p < 0.001 versus each EGFP condition according to Two-way ANOVA with Bonferroni post hoc test. B . Four-week-old male mice were injected via tail vein AAV-EGFP-PKD2-CA or AAV-Control (0.5 x 10 11 vp/ml). Western blot images from human PKD2 determination in liver and muscle extracts to corroborate human PKD2 overexpression in livers from AAV-EGFP-PKD2-CA mice. C. BW was monitored from week 3 to the end of the experiment at week 9 (n = 9 mice for AAV-Control and n = 10 mice for AAV-EGFP-PKD2-CA mice). D . Plasma insulin levels were determined in AAV-EGFP-PKD2-CA or AAV-Control mice in the fed state. Values correspond to mean ± SEM (n = 9 mice for AAV-Control and n = 10 mice for AAV-EGFP-PKD2-CA mice). E–G. GTT (E), ITT (F) and PTT (G) were performed in 16-h (GTT, PTT) or 4-h (ITT) fasted mice injected (i.p.) d -glucose (2 g/kg), human insulin (0.75 U/kg) or sodium pyruvate (1.5 g/kg), respectively. Graphs depict the area under the curve (AUC) from GTT, ITT and PTT. Values correspond to mean ± SEM (GTT, n = 9 mice/group; ITT, n = 8 mice for AAV-Control and n = 9 mice for AAV-EGFP-PKD2-CA mice; PTT, n = 11 mice/group). ∗p < 0.05 versus AAV-Control mice according to Student's t-test. H. Phospho-IRS1 Ser612 (black arrow) and total IRS1 protein levels determined in 4 h-fasted AAV-EGFP-PKD2-CA or AAV-Control mice. Values correspond to mean ± SEM (n = 4 mice/group). ∗p < 0.05 versus AAV-Control mice according to Student's t-test. I, Four h-fasted AAV-EGFP-PKD2-CA or AAV-Control mice were i.p. injected 0.75 U/kg human insulin for 15 min and pIRS1 Tyr608 protein levels in liver were analyzed by immunoblot. Values are mean ± SEM (n = 7 mice for AAV-Control and n = 8 mice for AAV-EGFP-PKD2-CA mice). ∗p < 0.05 versus AAV-Control according to Student's t-test. J-K. pAKT (Ser473 and Thr308) protein levels were analyzed by immunoblot in liver (J) and muscle (K) extracts from mice treated as in I. Total AKT, α-Tubulin (J) and Vinculin (K) were used as loading controls. Densitometric quantification of pAKT (Ser473 and Thr308) protein levels after insulin treatment. Values are mean ± SEM (liver, n = 5 mice/group for pAKT Ser473 and n = 4 mice for AAV-Control and n = 5 mice for AAV-EGFP-PKD2-CA mice for pAKT Thr308; muscle, n = 5 mice/group). ∗p < 0.05, ∗∗∗p < 0.001 versus AAV-Control according to Student's t-test.

Journal: Molecular Metabolism

Article Title: Protein kinase D2 modulates hepatic insulin sensitivity in male mice

doi: 10.1016/j.molmet.2024.102045

Figure Lengend Snippet: Overexpression of a constitutively active PKD2 mutant in hepatocytes promotes insulin resistance in vitro and in vivo . A . Huh7 cells were transfected either EGFP or EGFP-PKD2-CA. After 24 h, cells were serum-starved and stimulated with insulin (10 nM, 5–15 min). pAKT (Ser473 and Thr308), total AKT and EGFP levels are shown. GAPDH was used as loading control. Values are mean ± SEM (pAKT Ser473, n = 10 for 5 min and n = 7 for 15 min; pAKT Thr308, n = 6 for 5 min and n = 4 for 15 min). ∗∗∗p < 0.001 versus each EGFP condition according to Two-way ANOVA with Bonferroni post hoc test. B . Four-week-old male mice were injected via tail vein AAV-EGFP-PKD2-CA or AAV-Control (0.5 x 10 11 vp/ml). Western blot images from human PKD2 determination in liver and muscle extracts to corroborate human PKD2 overexpression in livers from AAV-EGFP-PKD2-CA mice. C. BW was monitored from week 3 to the end of the experiment at week 9 (n = 9 mice for AAV-Control and n = 10 mice for AAV-EGFP-PKD2-CA mice). D . Plasma insulin levels were determined in AAV-EGFP-PKD2-CA or AAV-Control mice in the fed state. Values correspond to mean ± SEM (n = 9 mice for AAV-Control and n = 10 mice for AAV-EGFP-PKD2-CA mice). E–G. GTT (E), ITT (F) and PTT (G) were performed in 16-h (GTT, PTT) or 4-h (ITT) fasted mice injected (i.p.) d -glucose (2 g/kg), human insulin (0.75 U/kg) or sodium pyruvate (1.5 g/kg), respectively. Graphs depict the area under the curve (AUC) from GTT, ITT and PTT. Values correspond to mean ± SEM (GTT, n = 9 mice/group; ITT, n = 8 mice for AAV-Control and n = 9 mice for AAV-EGFP-PKD2-CA mice; PTT, n = 11 mice/group). ∗p < 0.05 versus AAV-Control mice according to Student's t-test. H. Phospho-IRS1 Ser612 (black arrow) and total IRS1 protein levels determined in 4 h-fasted AAV-EGFP-PKD2-CA or AAV-Control mice. Values correspond to mean ± SEM (n = 4 mice/group). ∗p < 0.05 versus AAV-Control mice according to Student's t-test. I, Four h-fasted AAV-EGFP-PKD2-CA or AAV-Control mice were i.p. injected 0.75 U/kg human insulin for 15 min and pIRS1 Tyr608 protein levels in liver were analyzed by immunoblot. Values are mean ± SEM (n = 7 mice for AAV-Control and n = 8 mice for AAV-EGFP-PKD2-CA mice). ∗p < 0.05 versus AAV-Control according to Student's t-test. J-K. pAKT (Ser473 and Thr308) protein levels were analyzed by immunoblot in liver (J) and muscle (K) extracts from mice treated as in I. Total AKT, α-Tubulin (J) and Vinculin (K) were used as loading controls. Densitometric quantification of pAKT (Ser473 and Thr308) protein levels after insulin treatment. Values are mean ± SEM (liver, n = 5 mice/group for pAKT Ser473 and n = 4 mice for AAV-Control and n = 5 mice for AAV-EGFP-PKD2-CA mice for pAKT Thr308; muscle, n = 5 mice/group). ∗p < 0.05, ∗∗∗p < 0.001 versus AAV-Control according to Student's t-test.

Article Snippet: Phospho-IRS1 Tyr608 (mouse)/Tyr612 (human) , Merck Millipore , 09–432 , Rabbit , 1:5000.

Techniques: Over Expression, Mutagenesis, In Vitro, In Vivo, Transfection, Control, Injection, Western Blot

PKD2 ΔHep primary hepatocytes from HFD-fed mice exhibit improved insulin sensitivity compared to PKD2 fl/fl hepatocytes. A . Primary mouse hepatocytes from PKD2 fl/fl mice were treated with 600 μM PA in 5.5 mM glucose DMEM supplemented with 2.5% FBS and 0.5% free-FA BSA for 2 and 4 h , and pPKD1/2 protein were determined. Vinculin was used as loading control. Densitometric quantification of pPKD1/2 protein levels. Values are mean ± SEM (n = 9–13). ∗∗∗p < 0.001 versus time 0 according to One-way ANOVA with Bonferroni post hoc test. B. Protein levels of pPKD2, pIRS1 Ser612 and total IRS1 in primary hepatocytes isolated from CHD- and HFD-fed mice. Vinculin was used as loading control. Densitometric quantification of pPKD2, pIRS1 Ser612 and total IRS1 protein levels. Values are mean ± SEM (pPKD2 and pIRS1 Ser612, n = 7 for CHD hepatocytes and n = 6 for HFD hepatocytes; IRS1, n = 7 for CHD hepatocytes and n = 4 for HFD hepatocytes). ∗∗p < 0.01 versus CHD hepatocytes according to Student's t-test. C. Prkd2 (left) and Prkd3 (right) mRNA levels of primary mouse hepatocytes from PKD2 fl/fl and PKD2 ΔHep mice fed a CHD or HFD. Rplp0 was used for normalization. Values are mean ± SEM ( Prkd2 : CHD-PKD2 fl/fl , n = 6, CHD-PKD2 ΔHep , n = 5, HFD-PKD2 fl/fl , n = 8, HFD-PKD2 ΔHep , n = 4; Prkd3 : n = 6 for CHD-PKD2 fl/fl and n = 4 for HFD-PKD2 fl/fl , CHD- and HFD-PKD2 ΔHep ). ∗∗∗p < 0.001 versus PKD2 fl/fl hepatocytes and ### p < 0.001 versus CHD hepatocytes according to Two-way ANOVA with Bonferroni post hoc test. D. Basal protein levels of pIRS1 Ser612, IRS1 and pPKD1/2 in PKD2 fl/fl and PKD2 ΔHep primary hepatocytes isolated from HFD-fed mice. Vinculin was used as loading control. Densitometric quantification of pIRS1 Ser612 and IRS1 protein levels. Values are mean ± SEM (pIRS1 Ser612, n = 6 for PKD2 fl/fl and n = 10 for PKD2 ΔHep ; IRS1, n = 8 for PKD2 fl/fl and n = 12 for PKD2 ΔHep ). ∗∗p < 0.01, ∗∗∗p < 0.001 versus PKD2 fl/fl hepatocytes according to Student's t-test. E . PKD2 fl/fl and PKD2 ΔHep primary hepatocytes isolated from HFD-fed mice were stimulated with insulin (10 nM, 2.5–10 min). pIRS1 Tyr608, IRS1 and pAKT (Ser473 and Thr308) protein levels were analyzed by immunoblot. Total AKT and Vinculin were used as loading controls. Densitometric quantification of pIRS1 and pAKT protein levels after insulin treatment. Values are mean ± SEM (IRS1, n = 7 for PKD2 fl/fl and n = 9 for PKD2 ΔHep ; pIRS1 Tyr608, n = 7 for PKD2 fl/fl and n = 11 for PKD2 ΔHep ; pAKT Ser473, n = 9 for PKD2 fl/fl and n = 13 for PKD2 ΔHep ; pAKT Thr308, n = 8 for PKD2 fl/fl and n = 12 for PKD2 ΔHep primary hepatocytes). ∗p < 0.05, ∗∗p < 0.01 versus PKD2 fl/fl primary hepatocytes according to Two-way ANOVA with Bonferroni post hoc test.

Journal: Molecular Metabolism

Article Title: Protein kinase D2 modulates hepatic insulin sensitivity in male mice

doi: 10.1016/j.molmet.2024.102045

Figure Lengend Snippet: PKD2 ΔHep primary hepatocytes from HFD-fed mice exhibit improved insulin sensitivity compared to PKD2 fl/fl hepatocytes. A . Primary mouse hepatocytes from PKD2 fl/fl mice were treated with 600 μM PA in 5.5 mM glucose DMEM supplemented with 2.5% FBS and 0.5% free-FA BSA for 2 and 4 h , and pPKD1/2 protein were determined. Vinculin was used as loading control. Densitometric quantification of pPKD1/2 protein levels. Values are mean ± SEM (n = 9–13). ∗∗∗p < 0.001 versus time 0 according to One-way ANOVA with Bonferroni post hoc test. B. Protein levels of pPKD2, pIRS1 Ser612 and total IRS1 in primary hepatocytes isolated from CHD- and HFD-fed mice. Vinculin was used as loading control. Densitometric quantification of pPKD2, pIRS1 Ser612 and total IRS1 protein levels. Values are mean ± SEM (pPKD2 and pIRS1 Ser612, n = 7 for CHD hepatocytes and n = 6 for HFD hepatocytes; IRS1, n = 7 for CHD hepatocytes and n = 4 for HFD hepatocytes). ∗∗p < 0.01 versus CHD hepatocytes according to Student's t-test. C. Prkd2 (left) and Prkd3 (right) mRNA levels of primary mouse hepatocytes from PKD2 fl/fl and PKD2 ΔHep mice fed a CHD or HFD. Rplp0 was used for normalization. Values are mean ± SEM ( Prkd2 : CHD-PKD2 fl/fl , n = 6, CHD-PKD2 ΔHep , n = 5, HFD-PKD2 fl/fl , n = 8, HFD-PKD2 ΔHep , n = 4; Prkd3 : n = 6 for CHD-PKD2 fl/fl and n = 4 for HFD-PKD2 fl/fl , CHD- and HFD-PKD2 ΔHep ). ∗∗∗p < 0.001 versus PKD2 fl/fl hepatocytes and ### p < 0.001 versus CHD hepatocytes according to Two-way ANOVA with Bonferroni post hoc test. D. Basal protein levels of pIRS1 Ser612, IRS1 and pPKD1/2 in PKD2 fl/fl and PKD2 ΔHep primary hepatocytes isolated from HFD-fed mice. Vinculin was used as loading control. Densitometric quantification of pIRS1 Ser612 and IRS1 protein levels. Values are mean ± SEM (pIRS1 Ser612, n = 6 for PKD2 fl/fl and n = 10 for PKD2 ΔHep ; IRS1, n = 8 for PKD2 fl/fl and n = 12 for PKD2 ΔHep ). ∗∗p < 0.01, ∗∗∗p < 0.001 versus PKD2 fl/fl hepatocytes according to Student's t-test. E . PKD2 fl/fl and PKD2 ΔHep primary hepatocytes isolated from HFD-fed mice were stimulated with insulin (10 nM, 2.5–10 min). pIRS1 Tyr608, IRS1 and pAKT (Ser473 and Thr308) protein levels were analyzed by immunoblot. Total AKT and Vinculin were used as loading controls. Densitometric quantification of pIRS1 and pAKT protein levels after insulin treatment. Values are mean ± SEM (IRS1, n = 7 for PKD2 fl/fl and n = 9 for PKD2 ΔHep ; pIRS1 Tyr608, n = 7 for PKD2 fl/fl and n = 11 for PKD2 ΔHep ; pAKT Ser473, n = 9 for PKD2 fl/fl and n = 13 for PKD2 ΔHep ; pAKT Thr308, n = 8 for PKD2 fl/fl and n = 12 for PKD2 ΔHep primary hepatocytes). ∗p < 0.05, ∗∗p < 0.01 versus PKD2 fl/fl primary hepatocytes according to Two-way ANOVA with Bonferroni post hoc test.

Article Snippet: Phospho-IRS1 Tyr608 (mouse)/Tyr612 (human) , Merck Millipore , 09–432 , Rabbit , 1:5000.

Techniques: Control, Isolation, Western Blot

PKD2 deficiency in hepatocytes potentiates hepatic insulin responses after HFD administration in male mice . A . Eight-week-old PKD2 fl/fl and PKD2 ΔHep male mice were fed a HFD for 14 weeks. Basal Ser612 IRS1 phosphorylation and total IRS1 levels were analyzed . Vinculin was used as loading control. Densitometric quantification of pIRS1 Ser612 and total IRS1 protein levels. Values are mean ± SEM (pIRS1, n = 6 for PKD2 fl/fl mice and n = 4 for PKD2 ΔHep mice; IRS1, n = 13 for PKD2 fl/fl mice and n = 10 for PKD2 ΔHep mice). ∗p < 0.05 versus PKD2 fl/fl mice according to Student's t-test. B-C. Four h-fasted HFD-fed PKD2 fl/fl or PKD2 ΔHep mice were i.p. injected 0.75 U/kg human insulin for 15 min. In B , pIR, pIRS1 Tyr608, pAKT (Ser473 and Thr308) protein levels were analysed by immunoblot in liver extracts. Total IR, AKT and Vinculin were used as loading controls. Densitometric quantification of pIR, pIRS1 Tyr608, pAKT (Ser473 and Thr308) protein levels after insulin treatment. Values are mean ± SEM (pIR, pAKT Ser473 and pAKT Thr308, n = 11 for PKD2 fl/fl mice and n = 15 for PKD2 ΔHep mice; pIRS1 Tyr608, n = 7 for PKD2 fl/fl mice and n = 10 for PKD2 ΔHep mice). ∗p < 0.05, ∗∗p < 0.01 versus PKD2 fl/fl mice according to Student's t-test. In C , pGSK3α/β, pFOXO1 and pS6 protein levels were analyzed by immunoblot in liver extracts. Total GSK3α/β, FOXO1, S6 and Vinculin were used as loading controls. Densitometric quantification of pGSK3α/β, pFOXO1 and pS6 protein levels after insulin treatment. Values are mean ± SEM (pGSK3α/β: n = 11 for PKD2 fl/fl mice and n = 13 for PKD2 ΔHep mice; pFOXO1, n = 5 for PKD2 fl/fl mice and n = 7 for PKD2 ΔHep mice; pS6, n = 6). ∗p < 0.05, ∗∗p < 0.01 versus PKD2 fl/fl mice according to Student's t-test. D. Glycogen, total and free glucose levels in livers from CHD- and HFD (14 weeks)-fed PKD2 fl/fl and PKD2 ΔHep mice in the fed state. Values are mean ± SEM (for glycogen and total glucose, CHD n = 9, HFD n = 11 for PKD2 fl/fl mice and n = 8 for PKD2 ΔHep mice; for free glucose, CHD n = 9, HFD n = 11 for PKD2 fl/fl mice and n = 9 for PKD2 ΔHep mice). ∗∗p < 0.01 versus PKD2 fl/fl mice, ##p < 0.01, ###p < 0.001 versus CHD condition, according to Two-way ANOVA with Bonferroni post hoc test. E. Protein levels of pIR and pAKT (Ser473 and Thr308) of muscle extracts from HFD-fed PKD2 fl/fl or PKD2 ΔHep mice treated as in b. Total IR, AKT and Vinculin were used as loading controls. Densitometric quantification of pIR and pAKT (Ser473 and Thr308) protein levels after insulin treatment. Values are mean ± SEM (n = 3 for PKD2 fl/fl mice and n = 5 for PKD2 ΔHep mice).

Journal: Molecular Metabolism

Article Title: Protein kinase D2 modulates hepatic insulin sensitivity in male mice

doi: 10.1016/j.molmet.2024.102045

Figure Lengend Snippet: PKD2 deficiency in hepatocytes potentiates hepatic insulin responses after HFD administration in male mice . A . Eight-week-old PKD2 fl/fl and PKD2 ΔHep male mice were fed a HFD for 14 weeks. Basal Ser612 IRS1 phosphorylation and total IRS1 levels were analyzed . Vinculin was used as loading control. Densitometric quantification of pIRS1 Ser612 and total IRS1 protein levels. Values are mean ± SEM (pIRS1, n = 6 for PKD2 fl/fl mice and n = 4 for PKD2 ΔHep mice; IRS1, n = 13 for PKD2 fl/fl mice and n = 10 for PKD2 ΔHep mice). ∗p < 0.05 versus PKD2 fl/fl mice according to Student's t-test. B-C. Four h-fasted HFD-fed PKD2 fl/fl or PKD2 ΔHep mice were i.p. injected 0.75 U/kg human insulin for 15 min. In B , pIR, pIRS1 Tyr608, pAKT (Ser473 and Thr308) protein levels were analysed by immunoblot in liver extracts. Total IR, AKT and Vinculin were used as loading controls. Densitometric quantification of pIR, pIRS1 Tyr608, pAKT (Ser473 and Thr308) protein levels after insulin treatment. Values are mean ± SEM (pIR, pAKT Ser473 and pAKT Thr308, n = 11 for PKD2 fl/fl mice and n = 15 for PKD2 ΔHep mice; pIRS1 Tyr608, n = 7 for PKD2 fl/fl mice and n = 10 for PKD2 ΔHep mice). ∗p < 0.05, ∗∗p < 0.01 versus PKD2 fl/fl mice according to Student's t-test. In C , pGSK3α/β, pFOXO1 and pS6 protein levels were analyzed by immunoblot in liver extracts. Total GSK3α/β, FOXO1, S6 and Vinculin were used as loading controls. Densitometric quantification of pGSK3α/β, pFOXO1 and pS6 protein levels after insulin treatment. Values are mean ± SEM (pGSK3α/β: n = 11 for PKD2 fl/fl mice and n = 13 for PKD2 ΔHep mice; pFOXO1, n = 5 for PKD2 fl/fl mice and n = 7 for PKD2 ΔHep mice; pS6, n = 6). ∗p < 0.05, ∗∗p < 0.01 versus PKD2 fl/fl mice according to Student's t-test. D. Glycogen, total and free glucose levels in livers from CHD- and HFD (14 weeks)-fed PKD2 fl/fl and PKD2 ΔHep mice in the fed state. Values are mean ± SEM (for glycogen and total glucose, CHD n = 9, HFD n = 11 for PKD2 fl/fl mice and n = 8 for PKD2 ΔHep mice; for free glucose, CHD n = 9, HFD n = 11 for PKD2 fl/fl mice and n = 9 for PKD2 ΔHep mice). ∗∗p < 0.01 versus PKD2 fl/fl mice, ##p < 0.01, ###p < 0.001 versus CHD condition, according to Two-way ANOVA with Bonferroni post hoc test. E. Protein levels of pIR and pAKT (Ser473 and Thr308) of muscle extracts from HFD-fed PKD2 fl/fl or PKD2 ΔHep mice treated as in b. Total IR, AKT and Vinculin were used as loading controls. Densitometric quantification of pIR and pAKT (Ser473 and Thr308) protein levels after insulin treatment. Values are mean ± SEM (n = 3 for PKD2 fl/fl mice and n = 5 for PKD2 ΔHep mice).

Article Snippet: Phospho-IRS1 Tyr608 (mouse)/Tyr612 (human) , Merck Millipore , 09–432 , Rabbit , 1:5000.

Techniques: Control, Injection, Western Blot

Desmosome proteins detected in serum following SARS-CoV-2 infection. Detection of ( A ) desmoplakin (DSP, *** P = 0.0004), ( B ) junction plakoglobin (JUP), and ( C ) desmoglein 2 (DSG2, ** P = 0.0051) protein by ELISA. Results are expressed as median concentration with interquartile range, and all groups are statistically not significant from healthy controls unless annotated.

Journal: Clinical and Experimental Immunology

Article Title: SARS-CoV-2 infection is associated with anti-desmoglein 2 autoantibody detection

doi: 10.1093/cei/uxad046

Figure Lengend Snippet: Desmosome proteins detected in serum following SARS-CoV-2 infection. Detection of ( A ) desmoplakin (DSP, *** P = 0.0004), ( B ) junction plakoglobin (JUP), and ( C ) desmoglein 2 (DSG2, ** P = 0.0051) protein by ELISA. Results are expressed as median concentration with interquartile range, and all groups are statistically not significant from healthy controls unless annotated.

Article Snippet: Human DSG2 protein (Biorbyt, UK; REF: orb624074) was diluted to a concentration of 1 μg/ml in Dulbecco’s PBS and 50 μl diluted antigen added to each well of a 96-well plate.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Desmoglein 2 IgG autoantibody concentrations are higher in patients post-severe SARS-CoV-2 infection. Detection of desmoglein 2 autoantibodies in COVID-19 cohorts by ELISA; convalescent COVID Intensive therapy unit (ITU), acute COVID ITU, and mild COVID convalescent, compared with non-COVID control cohorts; non-COVID ITU, Influenza (flu) ITU, and healthy controls ( N = 252, P < 0.0001). The flu ITU, non-COVID ITU, and mild COVID convalescent cohorts were all not statistically significant compared to healthy controls. Results are expressed as median OD (optical density) with interquartile range. Figure in brackets indicates number of sera per group. **** indicates a significant difference between groups at a P value of <0.0001.

Journal: Clinical and Experimental Immunology

Article Title: SARS-CoV-2 infection is associated with anti-desmoglein 2 autoantibody detection

doi: 10.1093/cei/uxad046

Figure Lengend Snippet: Desmoglein 2 IgG autoantibody concentrations are higher in patients post-severe SARS-CoV-2 infection. Detection of desmoglein 2 autoantibodies in COVID-19 cohorts by ELISA; convalescent COVID Intensive therapy unit (ITU), acute COVID ITU, and mild COVID convalescent, compared with non-COVID control cohorts; non-COVID ITU, Influenza (flu) ITU, and healthy controls ( N = 252, P < 0.0001). The flu ITU, non-COVID ITU, and mild COVID convalescent cohorts were all not statistically significant compared to healthy controls. Results are expressed as median OD (optical density) with interquartile range. Figure in brackets indicates number of sera per group. **** indicates a significant difference between groups at a P value of <0.0001.

Article Snippet: Human DSG2 protein (Biorbyt, UK; REF: orb624074) was diluted to a concentration of 1 μg/ml in Dulbecco’s PBS and 50 μl diluted antigen added to each well of a 96-well plate.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Control

Immunohistochemical staining of post-mortem cardiac tissue. ( A ) Non-COVID-19 post-mortem heart tissue shows uniform single-banded staining for DSG2 corresponding to the site of intercalated discs (these are indicated by black arrows). ( B ) Cardiac muscle from COVID-19 post-mortem cases showed areas of single and notable double banded staining for DSG2 (arrowed). ( C ) Post-mortem heart tissue from a non-COVID-19 patient stained for IgG deposition. ( D ) Post-mortem heart tissue from a COVID-19 patient demonstrating IgG deposition at intercalated discs. Images representative of 3 hearts from non-COVID-19 patients and 8 patients who died from COVID-19.

Journal: Clinical and Experimental Immunology

Article Title: SARS-CoV-2 infection is associated with anti-desmoglein 2 autoantibody detection

doi: 10.1093/cei/uxad046

Figure Lengend Snippet: Immunohistochemical staining of post-mortem cardiac tissue. ( A ) Non-COVID-19 post-mortem heart tissue shows uniform single-banded staining for DSG2 corresponding to the site of intercalated discs (these are indicated by black arrows). ( B ) Cardiac muscle from COVID-19 post-mortem cases showed areas of single and notable double banded staining for DSG2 (arrowed). ( C ) Post-mortem heart tissue from a non-COVID-19 patient stained for IgG deposition. ( D ) Post-mortem heart tissue from a COVID-19 patient demonstrating IgG deposition at intercalated discs. Images representative of 3 hearts from non-COVID-19 patients and 8 patients who died from COVID-19.

Article Snippet: Human DSG2 protein (Biorbyt, UK; REF: orb624074) was diluted to a concentration of 1 μg/ml in Dulbecco’s PBS and 50 μl diluted antigen added to each well of a 96-well plate.

Techniques: Immunohistochemical staining, Staining

A Western blot analysis of PTEN, p-Akt s473, t-Akt, p-IRS1 Y612, and t-IRS1 expression in BGC803 and MKN45 cells transfected with PTEN siRNAs. B Western blot analysis of IRS1, p-Akt t308, p-Akt s473, t-Akt, and PTEN expression in BGC803 and MKN45 cells infected with lentivirus expressing IRS1-specific shRNA or scramble shRNA. Cell proliferation ability detection of BGC803 and MKN45 cells after IRS1 knockdown by ( C ) CCK-8 assay, ( D ) plate clone formation assay, ( E ) soft agar clone formation assay, and ( F ) EdU assay. *** p < 0.001, ** p < 0.01, * p < 0.05.

Journal: Oncogene

Article Title: Targeting the E3 ligase NEDD4 as a novel therapeutic strategy for IGF1 signal pathway-driven gastric cancer

doi: 10.1038/s41388-023-02619-4

Figure Lengend Snippet: A Western blot analysis of PTEN, p-Akt s473, t-Akt, p-IRS1 Y612, and t-IRS1 expression in BGC803 and MKN45 cells transfected with PTEN siRNAs. B Western blot analysis of IRS1, p-Akt t308, p-Akt s473, t-Akt, and PTEN expression in BGC803 and MKN45 cells infected with lentivirus expressing IRS1-specific shRNA or scramble shRNA. Cell proliferation ability detection of BGC803 and MKN45 cells after IRS1 knockdown by ( C ) CCK-8 assay, ( D ) plate clone formation assay, ( E ) soft agar clone formation assay, and ( F ) EdU assay. *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The human IRS1-shRNA-1, human IRS1-shRNA-2, human NEDD4-shRNA-1, and human NEDD4-shRNA-2 were contracted into pLKO.1-TRC cloning vector (Addgene, Cambridge, MA, USA; 10878).

Techniques: Western Blot, Expressing, Transfection, Infection, shRNA, CCK-8 Assay, Tube Formation Assay, EdU Assay

A Western blot analysis of NEDD4, p-Akt s473, t-Akt, p-IRS1 Y612, t-IRS1, and PTEN protein level in BGC803 and MKN45 cells after Dox-induced silencing of NEDD4. NEDD4-con, scramble shRNA; NEDD4-sh1 and sh2, NEDD4 shRNA constructs 1 and 2. B Western blot analysis of NEDD4, p-Akt s473, t-Akt, p-IRS1 Y612, t-IRS1, and PTEN protein level in BGC803 and MKN45 cells after NEDD4 overexpression. Cell proliferation ability detection of BGC803 and MKN45 cells after NEDD4 knockdown and overexpression by ( C , D ) CCK-8 assay, ( E ) plate clone formation assay, ( F ) soft agar clone formation assay, and ( G , H ) EdU assay. I Western blot analysis of p-Akt s473 and t-Akt protein levels in BGC803 and MKN45 cells after treatment with Heclin (10 μM) for 48 h. J Cell viability detection of cells treated with Heclin (10 μM) by CCK-8 assay. *** p < 0.001, ** p < 0.01, * p < 0.05, ns > 0.05.

Journal: Oncogene

Article Title: Targeting the E3 ligase NEDD4 as a novel therapeutic strategy for IGF1 signal pathway-driven gastric cancer

doi: 10.1038/s41388-023-02619-4

Figure Lengend Snippet: A Western blot analysis of NEDD4, p-Akt s473, t-Akt, p-IRS1 Y612, t-IRS1, and PTEN protein level in BGC803 and MKN45 cells after Dox-induced silencing of NEDD4. NEDD4-con, scramble shRNA; NEDD4-sh1 and sh2, NEDD4 shRNA constructs 1 and 2. B Western blot analysis of NEDD4, p-Akt s473, t-Akt, p-IRS1 Y612, t-IRS1, and PTEN protein level in BGC803 and MKN45 cells after NEDD4 overexpression. Cell proliferation ability detection of BGC803 and MKN45 cells after NEDD4 knockdown and overexpression by ( C , D ) CCK-8 assay, ( E ) plate clone formation assay, ( F ) soft agar clone formation assay, and ( G , H ) EdU assay. I Western blot analysis of p-Akt s473 and t-Akt protein levels in BGC803 and MKN45 cells after treatment with Heclin (10 μM) for 48 h. J Cell viability detection of cells treated with Heclin (10 μM) by CCK-8 assay. *** p < 0.001, ** p < 0.01, * p < 0.05, ns > 0.05.

Article Snippet: The human IRS1-shRNA-1, human IRS1-shRNA-2, human NEDD4-shRNA-1, and human NEDD4-shRNA-2 were contracted into pLKO.1-TRC cloning vector (Addgene, Cambridge, MA, USA; 10878).

Techniques: Western Blot, shRNA, Construct, Over Expression, CCK-8 Assay, Tube Formation Assay, EdU Assay

A–C Western blot analysis of NEDD4, p-Akt s473, t-Akt, p-IRS1 Y612, t-IRS1, and PTEN protein level in AGS cells after silencing of IRS1 and NEDD4 and overexpressing NEDD4. Cell proliferation ability detection of AGS cells after IRS1 and NEDD4 knockdown and NEDD4 overexpression, respectively, by ( D–F ) CCK-8 assay, ( G , H ) plate clone formation assay, and ( I–K ) EdU assay. L Western blot analysis of p-Akt s473 and t-Akt in AGS cells after treatment of Heclin(10 μM) for 48 h. M Cell viability detection of AGS treated with Heclin (10 μM) by CCK-8 assay. *** p < 0.001, ** p < 0.01, * p < 0.05, ns > 0.05.

Journal: Oncogene

Article Title: Targeting the E3 ligase NEDD4 as a novel therapeutic strategy for IGF1 signal pathway-driven gastric cancer

doi: 10.1038/s41388-023-02619-4

Figure Lengend Snippet: A–C Western blot analysis of NEDD4, p-Akt s473, t-Akt, p-IRS1 Y612, t-IRS1, and PTEN protein level in AGS cells after silencing of IRS1 and NEDD4 and overexpressing NEDD4. Cell proliferation ability detection of AGS cells after IRS1 and NEDD4 knockdown and NEDD4 overexpression, respectively, by ( D–F ) CCK-8 assay, ( G , H ) plate clone formation assay, and ( I–K ) EdU assay. L Western blot analysis of p-Akt s473 and t-Akt in AGS cells after treatment of Heclin(10 μM) for 48 h. M Cell viability detection of AGS treated with Heclin (10 μM) by CCK-8 assay. *** p < 0.001, ** p < 0.01, * p < 0.05, ns > 0.05.

Article Snippet: The human IRS1-shRNA-1, human IRS1-shRNA-2, human NEDD4-shRNA-1, and human NEDD4-shRNA-2 were contracted into pLKO.1-TRC cloning vector (Addgene, Cambridge, MA, USA; 10878).

Techniques: Western Blot, Over Expression, CCK-8 Assay, Tube Formation Assay, EdU Assay

A , B Western blot analysis of NEDD4, PTEN, p-Akt s473, t-Akt, p-IRS Y612, and t-IRS1 protein level in BGC803 and MKN45 cells infected with lentivirus expressing scramble shRNA (control), shNEDD4-1 and shNEDD4-2 combined with PTEN siRNA. C , D CCK-8 assay and ( E , F ) EdU assay showing proliferation ability of the indicated cells. *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05.

Journal: Oncogene

Article Title: Targeting the E3 ligase NEDD4 as a novel therapeutic strategy for IGF1 signal pathway-driven gastric cancer

doi: 10.1038/s41388-023-02619-4

Figure Lengend Snippet: A , B Western blot analysis of NEDD4, PTEN, p-Akt s473, t-Akt, p-IRS Y612, and t-IRS1 protein level in BGC803 and MKN45 cells infected with lentivirus expressing scramble shRNA (control), shNEDD4-1 and shNEDD4-2 combined with PTEN siRNA. C , D CCK-8 assay and ( E , F ) EdU assay showing proliferation ability of the indicated cells. *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05.

Article Snippet: The human IRS1-shRNA-1, human IRS1-shRNA-2, human NEDD4-shRNA-1, and human NEDD4-shRNA-2 were contracted into pLKO.1-TRC cloning vector (Addgene, Cambridge, MA, USA; 10878).

Techniques: Western Blot, Infection, Expressing, shRNA, CCK-8 Assay, EdU Assay

Correlations between IRS1 expression and clinical characteristics in patients with GC.

Journal: Oncogene

Article Title: Targeting the E3 ligase NEDD4 as a novel therapeutic strategy for IGF1 signal pathway-driven gastric cancer

doi: 10.1038/s41388-023-02619-4

Figure Lengend Snippet: Correlations between IRS1 expression and clinical characteristics in patients with GC.

Article Snippet: The human IRS1-shRNA-1, human IRS1-shRNA-2, human NEDD4-shRNA-1, and human NEDD4-shRNA-2 were contracted into pLKO.1-TRC cloning vector (Addgene, Cambridge, MA, USA; 10878).

Techniques: Expressing

A Representative images of IGF1R, NEDD4, and p-Akt s473 expression in adjacent non-tumor tissues and primary GC tissues detected by IHC staining. B IHC scores of IGF1R, NEDD4, and p-Akt S473 expressions in adjacent non-tumor tissues and primary GC tissues. C Analysis of IGF1 and NEDD4 expressions in normal tissue and GC tissues from Gastric datasets (GSE3468354 and GSE27342, N, normal gastric mucosa, n = 80; T, gastric tumor, n = 80). D Analysis of IGF1R expression in GC stratified by N-cadherin expression and normal tissues, NEDD4 expression in GC stratified by N-cadherin expression and normal tissues, and NEDD4 expression in GC stratified by IGF1 expression and normal tissues in TCGA database. E Protein expression correlation of IGF1R, NEDD4, and p-Akt s473 in GC tissues. F Kaplan–Meier plots of OS among GC patients with different expressions of IGF1, IGF1R, IRS1, and NEDD4. G Overall survival rate of STAD from the TCGA database was analyzed according to the mRNA levels of IGF1 and NEDD4, NEDD4 and N-cadherin. IGF1-High/NEDD4-High ( n = 103); IGF1-High/NEDD4-Low ( n = 82); IGF1-Low/NEDD4-High ( n = 82); IGF1-Low/NEDD4-Low ( n = 103). NEDD4-High/N-cadherin-High ( n = 111); NEDD4-High/N-cadherin-Low ( n = 74); NEDD4-Low/N-cadherin-High ( n = 74); NEDD4-Low/N-cadherin-Low ( n = 111). *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05.

Journal: Oncogene

Article Title: Targeting the E3 ligase NEDD4 as a novel therapeutic strategy for IGF1 signal pathway-driven gastric cancer

doi: 10.1038/s41388-023-02619-4

Figure Lengend Snippet: A Representative images of IGF1R, NEDD4, and p-Akt s473 expression in adjacent non-tumor tissues and primary GC tissues detected by IHC staining. B IHC scores of IGF1R, NEDD4, and p-Akt S473 expressions in adjacent non-tumor tissues and primary GC tissues. C Analysis of IGF1 and NEDD4 expressions in normal tissue and GC tissues from Gastric datasets (GSE3468354 and GSE27342, N, normal gastric mucosa, n = 80; T, gastric tumor, n = 80). D Analysis of IGF1R expression in GC stratified by N-cadherin expression and normal tissues, NEDD4 expression in GC stratified by N-cadherin expression and normal tissues, and NEDD4 expression in GC stratified by IGF1 expression and normal tissues in TCGA database. E Protein expression correlation of IGF1R, NEDD4, and p-Akt s473 in GC tissues. F Kaplan–Meier plots of OS among GC patients with different expressions of IGF1, IGF1R, IRS1, and NEDD4. G Overall survival rate of STAD from the TCGA database was analyzed according to the mRNA levels of IGF1 and NEDD4, NEDD4 and N-cadherin. IGF1-High/NEDD4-High ( n = 103); IGF1-High/NEDD4-Low ( n = 82); IGF1-Low/NEDD4-High ( n = 82); IGF1-Low/NEDD4-Low ( n = 103). NEDD4-High/N-cadherin-High ( n = 111); NEDD4-High/N-cadherin-Low ( n = 74); NEDD4-Low/N-cadherin-High ( n = 74); NEDD4-Low/N-cadherin-Low ( n = 111). *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05.

Article Snippet: The human IRS1-shRNA-1, human IRS1-shRNA-2, human NEDD4-shRNA-1, and human NEDD4-shRNA-2 were contracted into pLKO.1-TRC cloning vector (Addgene, Cambridge, MA, USA; 10878).

Techniques: Expressing, Immunohistochemistry

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