Journal: Scientific Reports

Article Title: Neuregulin 1 improves complex 2-mediated mitochondrial respiration in skeletal muscle of healthy and diabetic mice

doi: 10.1038/s41598-017-02029-z

Figure Lengend Snippet: NRG1 treatment increases ERBB4 activation in gastrocnemius muscle. C57BL/6JRJ (C57) control and db/db (Db) male mice were treated with vehicle (VHL; 0.9% NaCl solution; n = 8/each condition), or with NRG1 (50 μg . kg −1 ; n = 8/each condition), three days per week for eight weeks. Western blot analysis ( A ) cropped images) was used to quantify in gastrocnemius muscle samples the abundance of full length (115 kDa) ( B ) and cleaved (42 kDa) ( C ) NRG1 and the NRG1 cleavage index (the ratio between cleaved and full length NRG1) ( D ) as well as the abundance ( E ) and phosphorylation ratios ( F ) of ERBB2, ERBB3 and ERBB4. Results are the mean ± SEM (n = 8 per group) relative to the level in untreated healthy mice (C57-VHL, white bars). Diabetes (healthy vs db/db mice) and NRG1 (saline vs NRG1) effects were investigated with a 2 × 2 ANOVA. When a significant interaction was found, the Tuckey’s test was used for post-hoc multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant.

Article Snippet: The anti-porin (1/1000), -AKT (1/1000), -p-AKT Ser473 (1/1000), -p-AKT Thr308 (1/1000), -ERK (1/1000), -p-ERK, (1/1000), -AMPK (1/1000), -p-AMPK (1/1000), ACC (1/1000), -p-ACC (1/1000), -ACL (1/1000), -p-ACL (1/1000), -GLUT4 (1/1000), -ERBB3 (1/200) and -p-ERBB3 (1/200) antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: Neuregulin 1 improves complex 2-mediated mitochondrial respiration in skeletal muscle of healthy and diabetic mice

doi: 10.1038/s41598-017-02029-z

Figure Lengend Snippet: Summary of the main effects of diabetes and NRG1 treatment in gastrocnemius muscle. The abundance of ERBB receptors is increased in diabetic db/db mice. This is associated with increased phosphorylation ratio of ERBB2 and decreased phosphorylation ratios of ERBB3 and ERBB4. Similarly, phosphorylation (activation) of the metabolic regulators AMPK, ACC and ACL is reduced in diabetic mice as well as Pparb and Tfam mRNA expression level. However, mitochondrial respiration in permeabilised fibres is similar in diabetic and control healthy mice. Chronic NRG1 treatment increases ERBB4 phosphorylation and tends to decrease ERBB3 phosphorylation. Among the regulators of the mitochondrial biogenesis pathway, only Pparb mRNA expression is slightly increased by NRG1. However, in NRG1-treated mice, complex 2-mediated mitochondrial respiration and complex 2 subunit abundance are increased. Effect of diabetes: red arrows. Effect of NRG1: green arrows. Figure was produced using Servier Medical Art image bank ( http://www.servier.com/Powerpoint-image-bank ), which is licensed under a Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0/).

Article Snippet: The anti-porin (1/1000), -AKT (1/1000), -p-AKT Ser473 (1/1000), -p-AKT Thr308 (1/1000), -ERK (1/1000), -p-ERK, (1/1000), -AMPK (1/1000), -p-AMPK (1/1000), ACC (1/1000), -p-ACC (1/1000), -ACL (1/1000), -p-ACL (1/1000), -GLUT4 (1/1000), -ERBB3 (1/200) and -p-ERBB3 (1/200) antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Produced

Journal: Oncogene

Article Title: ERBB3-induced furin promotes the progression and metastasis of ovarian cancer via the IGF1R/STAT3 signaling axis

doi: 10.1038/s41388-020-1194-7

Figure Lengend Snippet: A and B , Cell lysates of NRG1 (20ng/ml) stimulation at the different timepoints were immunoblotted against ERBB receptors in OVCAR5 ( A ) and SKOV3 ( B ) cell lines. C and D , Cell lysates of NRG1 stimulation at the different time points cells were collected and immunoblotted against furin in OVCAR 5 ( C ) and SKOV3 ( D ). E and F . Cell lysates of ERBB3 inhibitor (AZD8931) treatment with or without NRG1 (20ng/ml) stimulation in the different times points were collected and immunoblotted against fruin and ERBB receptors in OVCAR 5 ( E ) and SKOV3 ( F ).

Article Snippet: We obtained, ERBB2 (#2165), p-ERBB2 (Tyr1248)(#2247), ERBB3 (#12708), p-ERBB3 (Tyr1289), IGF1R-β (#3027), GAPDH (#5174), mesothelin (#99966S), TGF-β (#3711), STAT3 (#9139), p-STAT3 (Tyr705)(#9145), p-STAT3 (Ser727)(#34911), JAK1 (#3344S), p-JAK1(Tyr1034/1035) (#74129S), JAK2 (3230S), p-JAK2 (Tyr1008) (#8082S) TYK2 (#14193S) and p-TYK2 (Tyr1054/1055) (#68790S) from Cell Signaling Technology (Danvers, MA), Furin (#sc-133142) was obtained from Santa Cruz Biotechnology.

Techniques:

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 1. huHER3-8 blocks NRG1-mediated ERBB3 activation in vitro in mutant BRAFV600E melanoma cell lines. A, A375 cells were treated with 1 mmol/L vemurafenib for 24 hours, followed by 45 minutes of huHER3-8 at the concentrations indicated. Cells were stimulated with 10 ng/mL NRG1 for 15 minutes and then lysed for Western blot analysis. B, M238, 1205Lu, and A375 cells were cultured in the presence/absence of 1 mmol/L vemurafenib for 24 hours. Cells were then treated or untreated with 10mg/mL huHER3-8 for 45 minutes before stimulation with 10 ng/mL NRG1 for 15 minutes. Cell lysates were analyzed by Western blot with the indicated antibodies.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, In Vitro, Mutagenesis, Western Blot, Cell Culture

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 2. NRG1-dependent activation of ERBB3 results in increased AKT signaling and is inhibited by huHER3-8. A, 1205Lu cells were treated with 1 mmol/L vemurafenib overnight. Cells were then treated with or without 10 mg/mL huHER3-8 for 45 minutes followed by treatment with 10 ng/mL NRG1 for 15 minutes or 24 hours, as indicated. Cell lysates were prepared for RPPA analysis. Linear RPPA scores were averaged and normalized to vemurafenib alone samples. Proteins that were regulated 1.5-fold or greater are shown. B, A375 cells were treated as in A. Proteins regulated 1.5-fold or greater are shown. C, RPPA data generated from vemurafenib-treated 1205Lu cell samples were transformed and analyzed using GSEA. Significantly enriched pathways in the presence of NRG1 were visualized in Cytoscape where pathway node sizes are indicative of normalized enrichment scores relative to each other, as indicated. Data from samples treated with NRG1 for 15 minutes and 24 hours were averaged together and normalized to vemurafenib alone samples to generate the color labeling of protein nodes shown. D, significantly enriched pathways in 1205Lu samples treated with vemurafenib, NRG1, and huHER3-8. Pathway analysis was performed as in C.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Generated, Transformation Assay, Labeling

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 3. huHER3-8 blocks NRG1-mediated ERBB3/AKT signaling in RAF-inhibited melanoma cells. A, melanoma cells were treated with/without 1 mmol/L vemurafenib for 24 hours and then 10 mg/mL huHER3-8 for 45 minutes before stimulation with 10 ng/mL NRG1 for 15 minutes. Cell lysates were analyzed by Western blot for phospho/total ERBB2, AKT, and ERK1/2. B, 1205Lu, M238, A375, WM793, WM115, and SK-MEL-28 cells were treated with 1 mmol/L vemurafenib and the presence/absence of 10 mg/mL huHER3-8 and then stimulated with 10 ng/mL NRG1 for the time period shown. Cells were lysed and lysates analyzed by Western blot with the antibodies indicated.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 7. Model for targeting ERBB3 adaptive response in mutant BRAF melanoma. Vemurafenib treatment relieves a negative feedback on ERBB3 transcription, resulting in an increase of ERBB3 at the membrane. In the presence of its ligand (NRG1), ERBB3 heterodimerizes with ERBB2 to become active. Active ERBB3 recruits PI3K, which signals to the AKT pathway promoting survival of mutant BRAF melanoma cells treated with vemurafenib. Treatment with the anti-ERBB3 monoclonal antibody huHER3-8 outcompetes NRG1 and prevents the activation of ERBB3 and AKT signaling and enhances the efficacy of vemurafenib treatment.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Membrane, Activation Assay

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 1. huHER3-8 blocks NRG1-mediated ERBB3 activation in vitro in mutant BRAFV600E melanoma cell lines. A, A375 cells were treated with 1 mmol/L vemurafenib for 24 hours, followed by 45 minutes of huHER3-8 at the concentrations indicated. Cells were stimulated with 10 ng/mL NRG1 for 15 minutes and then lysed for Western blot analysis. B, M238, 1205Lu, and A375 cells were cultured in the presence/absence of 1 mmol/L vemurafenib for 24 hours. Cells were then treated or untreated with 10mg/mL huHER3-8 for 45 minutes before stimulation with 10 ng/mL NRG1 for 15 minutes. Cell lysates were analyzed by Western blot with the indicated antibodies.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, In Vitro, Mutagenesis, Western Blot, Cell Culture

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 2. NRG1-dependent activation of ERBB3 results in increased AKT signaling and is inhibited by huHER3-8. A, 1205Lu cells were treated with 1 mmol/L vemurafenib overnight. Cells were then treated with or without 10 mg/mL huHER3-8 for 45 minutes followed by treatment with 10 ng/mL NRG1 for 15 minutes or 24 hours, as indicated. Cell lysates were prepared for RPPA analysis. Linear RPPA scores were averaged and normalized to vemurafenib alone samples. Proteins that were regulated 1.5-fold or greater are shown. B, A375 cells were treated as in A. Proteins regulated 1.5-fold or greater are shown. C, RPPA data generated from vemurafenib-treated 1205Lu cell samples were transformed and analyzed using GSEA. Significantly enriched pathways in the presence of NRG1 were visualized in Cytoscape where pathway node sizes are indicative of normalized enrichment scores relative to each other, as indicated. Data from samples treated with NRG1 for 15 minutes and 24 hours were averaged together and normalized to vemurafenib alone samples to generate the color labeling of protein nodes shown. D, significantly enriched pathways in 1205Lu samples treated with vemurafenib, NRG1, and huHER3-8. Pathway analysis was performed as in C.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Generated, Transformation Assay, Labeling

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 3. huHER3-8 blocks NRG1-mediated ERBB3/AKT signaling in RAF-inhibited melanoma cells. A, melanoma cells were treated with/without 1 mmol/L vemurafenib for 24 hours and then 10 mg/mL huHER3-8 for 45 minutes before stimulation with 10 ng/mL NRG1 for 15 minutes. Cell lysates were analyzed by Western blot for phospho/total ERBB2, AKT, and ERK1/2. B, 1205Lu, M238, A375, WM793, WM115, and SK-MEL-28 cells were treated with 1 mmol/L vemurafenib and the presence/absence of 10 mg/mL huHER3-8 and then stimulated with 10 ng/mL NRG1 for the time period shown. Cells were lysed and lysates analyzed by Western blot with the antibodies indicated.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 7. Model for targeting ERBB3 adaptive response in mutant BRAF melanoma. Vemurafenib treatment relieves a negative feedback on ERBB3 transcription, resulting in an increase of ERBB3 at the membrane. In the presence of its ligand (NRG1), ERBB3 heterodimerizes with ERBB2 to become active. Active ERBB3 recruits PI3K, which signals to the AKT pathway promoting survival of mutant BRAF melanoma cells treated with vemurafenib. Treatment with the anti-ERBB3 monoclonal antibody huHER3-8 outcompetes NRG1 and prevents the activation of ERBB3 and AKT signaling and enhances the efficacy of vemurafenib treatment.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Membrane, Activation Assay

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 2. NRG1-dependent activation of ERBB3 results in increased AKT signaling and is inhibited by huHER3-8. A, 1205Lu cells were treated with 1 mmol/L vemurafenib overnight. Cells were then treated with or without 10 mg/mL huHER3-8 for 45 minutes followed by treatment with 10 ng/mL NRG1 for 15 minutes or 24 hours, as indicated. Cell lysates were prepared for RPPA analysis. Linear RPPA scores were averaged and normalized to vemurafenib alone samples. Proteins that were regulated 1.5-fold or greater are shown. B, A375 cells were treated as in A. Proteins regulated 1.5-fold or greater are shown. C, RPPA data generated from vemurafenib-treated 1205Lu cell samples were transformed and analyzed using GSEA. Significantly enriched pathways in the presence of NRG1 were visualized in Cytoscape where pathway node sizes are indicative of normalized enrichment scores relative to each other, as indicated. Data from samples treated with NRG1 for 15 minutes and 24 hours were averaged together and normalized to vemurafenib alone samples to generate the color labeling of protein nodes shown. D, significantly enriched pathways in 1205Lu samples treated with vemurafenib, NRG1, and huHER3-8. Pathway analysis was performed as in C.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Generated, Transformation Assay, Labeling

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 3. huHER3-8 blocks NRG1-mediated ERBB3/AKT signaling in RAF-inhibited melanoma cells. A, melanoma cells were treated with/without 1 mmol/L vemurafenib for 24 hours and then 10 mg/mL huHER3-8 for 45 minutes before stimulation with 10 ng/mL NRG1 for 15 minutes. Cell lysates were analyzed by Western blot for phospho/total ERBB2, AKT, and ERK1/2. B, 1205Lu, M238, A375, WM793, WM115, and SK-MEL-28 cells were treated with 1 mmol/L vemurafenib and the presence/absence of 10 mg/mL huHER3-8 and then stimulated with 10 ng/mL NRG1 for the time period shown. Cells were lysed and lysates analyzed by Western blot with the antibodies indicated.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot

Journal: Cancer Research

Article Title: Function-Blocking ERBB3 Antibody Inhibits the Adaptive Response to RAF Inhibitor

doi: 10.1158/0008-5472.can-14-0464

Figure Lengend Snippet: Figure 7. Model for targeting ERBB3 adaptive response in mutant BRAF melanoma. Vemurafenib treatment relieves a negative feedback on ERBB3 transcription, resulting in an increase of ERBB3 at the membrane. In the presence of its ligand (NRG1), ERBB3 heterodimerizes with ERBB2 to become active. Active ERBB3 recruits PI3K, which signals to the AKT pathway promoting survival of mutant BRAF melanoma cells treated with vemurafenib. Treatment with the anti-ERBB3 monoclonal antibody huHER3-8 outcompetes NRG1 and prevents the activation of ERBB3 and AKT signaling and enhances the efficacy of vemurafenib treatment.

Article Snippet: BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc135654) antibodies were purchased from Santa Cruz Biotechnology Inc. ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), pERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Membrane, Activation Assay