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cloning grp78 cdna (Addgene inc)

Name: cloning grp78 cdna
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Addgene inc cloning grp78 cdna

Figure 6—In vitro interaction studies between human PST peptides and <t>GRP78.</t> A: Spectrophotometric Malachite Green-phosphate assay was performed to compare the effect of 2 mmol/L PST-WT and PST-297S on inhibition of GRP78 ATPase activity. The different groups were compared using one-way ANOVA followed by Tukey multiple-comparisons test; n 5 4, one-way ANOVA, F 5 30.93, P < 0.0001. B: Human HepG2 hepatocytes were treated with 100 nmol/L PST-WT or PST-297S along with 5 mg/mL tunicamycin or 5 mg/mL tunicamycin alone for 24 h. Changes in GRP78 expression following this treatment were visualized using an immunoblot with anti-GRP78 antibody (control: anti–b-actin). C: Quantitative analysis of the immunoblot in B was performed using one-way ANOVA followed by Tukey multiple-comparisons test; n 5 5, one-way ANOVA, F 5 35.29, P < 0.0001. D: Western blot for GRP78 expression posttransfection of HEK-293 cells with increasing amounts of GRP78-overexpressing construct: lane 1, 0 mg; lane 2, 0.5 mg; lane 3, 1 mg; lane 4, 2 mg; and lane 5, 3 mg of GRP78-overexpressing construct. E: Competitive binding assay was performed to assess the ability of PST-WT or PST- 297S peptides to displace labeled [125]I-Tyr PST by adding increasing concentrations (0, 10 pmol/L through 1 mmol/L) of the PST peptides along with 100 nCi of labeled PST to 10 mg of isolated plasma membrane. Displacement was calculated as a percentage of receptors in the control samples that did not contain any competing peptides. F: Quantitative analysis of the graph in E was performed by comparing the AUC of the displacement curves of PST-WT and PST-297S using an unpaired two-tailed Student t test (n 5 4).

Cloning Grp78 Cdna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Functional Gly297Ser Variant of the Physiological Dysglycemic Peptide Pancreastatin Is a Novel Risk Factor for Cardiometabolic Disorders."

Article Title: Functional Gly297Ser Variant of the Physiological Dysglycemic Peptide Pancreastatin Is a Novel Risk Factor for Cardiometabolic Disorders.

Journal: Diabetes

doi: 10.2337/db21-0289

Figure 6—In vitro interaction studies between human PST peptides and GRP78. A: Spectrophotometric Malachite Green-phosphate assay was performed to compare the effect of 2 mmol/L PST-WT and PST-297S on inhibition of GRP78 ATPase activity. The different groups were compared using one-way ANOVA followed by Tukey multiple-comparisons test; n 5 4, one-way ANOVA, F 5 30.93, P < 0.0001. B: Human HepG2 hepatocytes were treated with 100 nmol/L PST-WT or PST-297S along with 5 mg/mL tunicamycin or 5 mg/mL tunicamycin alone for 24 h. Changes in GRP78 expression following this treatment were visualized using an immunoblot with anti-GRP78 antibody (control: anti–b-actin). C: Quantitative analysis of the immunoblot in B was performed using one-way ANOVA followed by Tukey multiple-comparisons test; n 5 5, one-way ANOVA, F 5 35.29, P < 0.0001. D: Western blot for GRP78 expression posttransfection of HEK-293 cells with increasing amounts of GRP78-overexpressing construct: lane 1, 0 mg; lane 2, 0.5 mg; lane 3, 1 mg; lane 4, 2 mg; and lane 5, 3 mg of GRP78-overexpressing construct. E: Competitive binding assay was performed to assess the ability of PST-WT or PST- 297S peptides to displace labeled [125]I-Tyr PST by adding increasing concentrations (0, 10 pmol/L through 1 mmol/L) of the PST peptides along with 100 nCi of labeled PST to 10 mg of isolated plasma membrane. Displacement was calculated as a percentage of receptors in the control samples that did not contain any competing peptides. F: Quantitative analysis of the graph in E was performed by comparing the AUC of the displacement curves of PST-WT and PST-297S using an unpaired two-tailed Student t test (n 5 4).

Figure Legend Snippet: Figure 6—In vitro interaction studies between human PST peptides and GRP78. A: Spectrophotometric Malachite Green-phosphate assay was performed to compare the effect of 2 mmol/L PST-WT and PST-297S on inhibition of GRP78 ATPase activity. The different groups were compared using one-way ANOVA followed by Tukey multiple-comparisons test; n 5 4, one-way ANOVA, F 5 30.93, P < 0.0001. B: Human HepG2 hepatocytes were treated with 100 nmol/L PST-WT or PST-297S along with 5 mg/mL tunicamycin or 5 mg/mL tunicamycin alone for 24 h. Changes in GRP78 expression following this treatment were visualized using an immunoblot with anti-GRP78 antibody (control: anti–b-actin). C: Quantitative analysis of the immunoblot in B was performed using one-way ANOVA followed by Tukey multiple-comparisons test; n 5 5, one-way ANOVA, F 5 35.29, P < 0.0001. D: Western blot for GRP78 expression posttransfection of HEK-293 cells with increasing amounts of GRP78-overexpressing construct: lane 1, 0 mg; lane 2, 0.5 mg; lane 3, 1 mg; lane 4, 2 mg; and lane 5, 3 mg of GRP78-overexpressing construct. E: Competitive binding assay was performed to assess the ability of PST-WT or PST- 297S peptides to displace labeled [125]I-Tyr PST by adding increasing concentrations (0, 10 pmol/L through 1 mmol/L) of the PST peptides along with 100 nCi of labeled PST to 10 mg of isolated plasma membrane. Displacement was calculated as a percentage of receptors in the control samples that did not contain any competing peptides. F: Quantitative analysis of the graph in E was performed by comparing the AUC of the displacement curves of PST-WT and PST-297S using an unpaired two-tailed Student t test (n 5 4).

Techniques Used: In Vitro, Inhibition, Activity Assay, Expressing, Western Blot, Control, Construct, Competitive Binding Assay, Labeling, Isolation, Clinical Proteomics, Membrane, Two Tailed Test

Figure 7—A schematic representation of the plausible mechanistic basis for increased cardiometabolic disease risk associated with the PST 297Ser allele. Occurrence of a nonsynonymous genetic variation within the PST region results in the PST p.Gly297Ser variant. PST- 297S peptide differs from PST-WT in its secondary structure (especially a-helical content). Structural differences between PST-WT and PST-297S peptides and their consequent differential interactions with GRP78 and IR may cause enhanced potencies of the PST-297S peptide for inhibition of insulin-stimulated glucose uptake and activation of the gluconeogenesis pathway. These and associated cellular/ molecular processes may elevate the levels of plasma glucose, HbA1c, and catecholamines in the carriers of the PST 297Ser allele, thereby increasing their risk for T2D, HTN, CAD, and MS.

Figure Legend Snippet: Figure 7—A schematic representation of the plausible mechanistic basis for increased cardiometabolic disease risk associated with the PST 297Ser allele. Occurrence of a nonsynonymous genetic variation within the PST region results in the PST p.Gly297Ser variant. PST- 297S peptide differs from PST-WT in its secondary structure (especially a-helical content). Structural differences between PST-WT and PST-297S peptides and their consequent differential interactions with GRP78 and IR may cause enhanced potencies of the PST-297S peptide for inhibition of insulin-stimulated glucose uptake and activation of the gluconeogenesis pathway. These and associated cellular/ molecular processes may elevate the levels of plasma glucose, HbA1c, and catecholamines in the carriers of the PST 297Ser allele, thereby increasing their risk for T2D, HTN, CAD, and MS.

Techniques Used: Variant Assay, Inhibition, Activation Assay, Clinical Proteomics

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eIF2α-mediated integrated stress responses verified in carotid stable and vulnerable plaques. (A) Representative specimens of stable and vulnerable plaques stained with H&E, and immunohistochemistry (IHC) of VSMCs with organelle markers, including α-SMA, PERK, XBP1, GRP78, cytochrome C (Cyt C), LAMP1, and eIF2α. (B) Quantitative analysis of the expression levels of α-SMA, PERK, XBP1, GRP78, Cyt C, LAMP1, and eIF2α (n = 15 cases in each group). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 200 μm, 1 cm, 2 cm, respectively.

Journal: Heliyon

Article Title: eIF2α-mediated integrated stress response links multiple intracellular signaling pathways to reprogram vascular smooth muscle cell fate in carotid artery plaque

doi: 10.1016/j.heliyon.2024.e26904

Figure Lengend Snippet: eIF2α-mediated integrated stress responses verified in carotid stable and vulnerable plaques. (A) Representative specimens of stable and vulnerable plaques stained with H&E, and immunohistochemistry (IHC) of VSMCs with organelle markers, including α-SMA, PERK, XBP1, GRP78, cytochrome C (Cyt C), LAMP1, and eIF2α. (B) Quantitative analysis of the expression levels of α-SMA, PERK, XBP1, GRP78, Cyt C, LAMP1, and eIF2α (n = 15 cases in each group). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 200 μm, 1 cm, 2 cm, respectively.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, blocked in 5% skin milk for 0.5 h, and incubated with the following primary antibodies: anti-PERK(D11A8; Cell Signaling Technology), anti-actin (BE0021-100; Easybio), anti-XBP1 (102256-T34; Sino Biological Inc.), anti-DRP1 (D6C7; Cell Signaling Technology), anti-phospho-DRP1 (Ser616) (pDrp1 (S616)) (D9A1; Cell Signaling Technology), anti-Tom 20 (D8T4N; Cell Signaling Technology), anti-light chain 3 (LC3; 14322-T44; Sino Biological Inc.), anti-LAMP1 (21997-1-AP; Proteintech), anti-GRP78 (HG12063-UT; Sino Biological Inc.), and anti-eIF2α (11170-1-AP; Proteintech) antibodies.

Techniques: Staining, Immunohistochemistry, Expressing

Eukaryotic translation initiation factor (eIF)-2α integrates the stress responses of multiple organelles to regulate atherosclerotic plaque progression. (A) Representative image of ER labeled with GFP-Sec61β and mitochondria stained with deep red Mitotracker in HC-VSMCs treated with DMSO (Ctrl.), thapsigargin (Tg; 5 μM), Oligo (1 μM), and combined Tg and oligo for 24 h. (B) Quantitative analysis of the percentage of cells with ER whorls and the aspect ratio in HC-VSMCs treated with DMSO (Ctrl.), Tg, Oligo, and combined Tg and Oligo (n = 30 cells per group). (C) Representative image of ER labeled with GFP-Sec61β and mitochondria stained with deep red Mitotracker in HC-VSMCs treated with DMSO (Ctrl.), Tg (5 μM), ISRIB (100 nM), Oligo (1 μM), and their combination for 24 h. (D) Quantitative analysis of the percentage of cells with ER whorls and the aspect ratio in HC-VSMCs treated with DMSO, DTT, ISRIB, Oligo, and their combination (n = 30 cells per group). (E) Western blotting analysis of GRP78, XBP1-u, XBP1-s, p-DRP1(S616), LC3-I, and eIF2α in HC-VSMCs treated with DMSO (Cntrl.), Tg, Oligo, and combined Tg and Oligo (n = 3 in each group). (F) Western blotting analysis of GRP78, XBP1-u, XBP1-s, p-DRP1(S616), LC3-I, and eIF2α in HC-VSMCs treated with DMSO, Tg, ISRIB, Oligo, and their combination (n = 3 times each group). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Journal: Heliyon

Article Title: eIF2α-mediated integrated stress response links multiple intracellular signaling pathways to reprogram vascular smooth muscle cell fate in carotid artery plaque

doi: 10.1016/j.heliyon.2024.e26904

Figure Lengend Snippet: Eukaryotic translation initiation factor (eIF)-2α integrates the stress responses of multiple organelles to regulate atherosclerotic plaque progression. (A) Representative image of ER labeled with GFP-Sec61β and mitochondria stained with deep red Mitotracker in HC-VSMCs treated with DMSO (Ctrl.), thapsigargin (Tg; 5 μM), Oligo (1 μM), and combined Tg and oligo for 24 h. (B) Quantitative analysis of the percentage of cells with ER whorls and the aspect ratio in HC-VSMCs treated with DMSO (Ctrl.), Tg, Oligo, and combined Tg and Oligo (n = 30 cells per group). (C) Representative image of ER labeled with GFP-Sec61β and mitochondria stained with deep red Mitotracker in HC-VSMCs treated with DMSO (Ctrl.), Tg (5 μM), ISRIB (100 nM), Oligo (1 μM), and their combination for 24 h. (D) Quantitative analysis of the percentage of cells with ER whorls and the aspect ratio in HC-VSMCs treated with DMSO, DTT, ISRIB, Oligo, and their combination (n = 30 cells per group). (E) Western blotting analysis of GRP78, XBP1-u, XBP1-s, p-DRP1(S616), LC3-I, and eIF2α in HC-VSMCs treated with DMSO (Cntrl.), Tg, Oligo, and combined Tg and Oligo (n = 3 in each group). (F) Western blotting analysis of GRP78, XBP1-u, XBP1-s, p-DRP1(S616), LC3-I, and eIF2α in HC-VSMCs treated with DMSO, Tg, ISRIB, Oligo, and their combination (n = 3 times each group). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Techniques: Labeling, Staining, Western Blot

Journal: Diabetes

Article Title: Functional Gly297Ser Variant of the Physiological Dysglycemic Peptide Pancreastatin Is a Novel Risk Factor for Cardiometabolic Disorders.

doi: 10.2337/db21-0289

Figure Lengend Snippet: Figure 6—In vitro interaction studies between human PST peptides and GRP78. A: Spectrophotometric Malachite Green-phosphate assay was performed to compare the effect of 2 mmol/L PST-WT and PST-297S on inhibition of GRP78 ATPase activity. The different groups were compared using one-way ANOVA followed by Tukey multiple-comparisons test; n 5 4, one-way ANOVA, F 5 30.93, P < 0.0001. B: Human HepG2 hepatocytes were treated with 100 nmol/L PST-WT or PST-297S along with 5 mg/mL tunicamycin or 5 mg/mL tunicamycin alone for 24 h. Changes in GRP78 expression following this treatment were visualized using an immunoblot with anti-GRP78 antibody (control: anti–b-actin). C: Quantitative analysis of the immunoblot in B was performed using one-way ANOVA followed by Tukey multiple-comparisons test; n 5 5, one-way ANOVA, F 5 35.29, P < 0.0001. D: Western blot for GRP78 expression posttransfection of HEK-293 cells with increasing amounts of GRP78-overexpressing construct: lane 1, 0 mg; lane 2, 0.5 mg; lane 3, 1 mg; lane 4, 2 mg; and lane 5, 3 mg of GRP78-overexpressing construct. E: Competitive binding assay was performed to assess the ability of PST-WT or PST- 297S peptides to displace labeled [125]I-Tyr PST by adding increasing concentrations (0, 10 pmol/L through 1 mmol/L) of the PST peptides along with 100 nCi of labeled PST to 10 mg of isolated plasma membrane. Displacement was calculated as a percentage of receptors in the control samples that did not contain any competing peptides. F: Quantitative analysis of the graph in E was performed by comparing the AUC of the displacement curves of PST-WT and PST-297S using an unpaired two-tailed Student t test (n 5 4).

Article Snippet: GRP78- and IR-overexpression constructs were generated by cloning GRP78 cDNA (obtained from catalog no. 32701; Addgene, Watertown, MA) and IR cDNA (a kind gift from Dr. Frederick M. Stanley [24]) into pcDNA 3.1.

Techniques: In Vitro, Inhibition, Activity Assay, Expressing, Western Blot, Control, Construct, Competitive Binding Assay, Labeling, Isolation, Clinical Proteomics, Membrane, Two Tailed Test

Journal: Diabetes

Article Title: Functional Gly297Ser Variant of the Physiological Dysglycemic Peptide Pancreastatin Is a Novel Risk Factor for Cardiometabolic Disorders.

doi: 10.2337/db21-0289

Figure Lengend Snippet: Figure 7—A schematic representation of the plausible mechanistic basis for increased cardiometabolic disease risk associated with the PST 297Ser allele. Occurrence of a nonsynonymous genetic variation within the PST region results in the PST p.Gly297Ser variant. PST- 297S peptide differs from PST-WT in its secondary structure (especially a-helical content). Structural differences between PST-WT and PST-297S peptides and their consequent differential interactions with GRP78 and IR may cause enhanced potencies of the PST-297S peptide for inhibition of insulin-stimulated glucose uptake and activation of the gluconeogenesis pathway. These and associated cellular/ molecular processes may elevate the levels of plasma glucose, HbA1c, and catecholamines in the carriers of the PST 297Ser allele, thereby increasing their risk for T2D, HTN, CAD, and MS.

Techniques: Variant Assay, Inhibition, Activation Assay, Clinical Proteomics

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