Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Brusatol in combination with lapatinib synergistically inhibited the growth of HER2-overexpressed SK-BR-3, SK-OV-3 and AU565 cells (A) SK-BR-3, SK-OV-3 and AU565 cell lines were treated with brusatol, lapatinib or brusatol plus lapatinib in a dose range for 48 h. CCK-8 assays were used to measure the cell viability. Points, mean of 3 independent CCK-8 assays; Bars, SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (B) The synergistic effect of lapatinib in combination with brusatol was evaluated on the growth of SK-BR-3, SK-OV-3 and AU565 cell line. Combination index (CI) values were calculated using the Chou-Talalay method. Drug synergy, addition, and antagonism are defined by CI values less than 1.0, equal to 1.0, or greater than 1.0, respectively.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: CCK-8 Assay

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Lapatinib plus brusatol abrogate the activation of Nrf2/HO-1 and EGFR/HER2-AKT/ERK1/2 pathways (A and B) SK-BR-3 and SK-OV-3 cells were treated with lapatinib or brusatol alone, or their combination for 24 h. The changes in Nrf2/HO-1 and EGFR/HER2-AKT/ERK1/2 signaling pathways were monitored by Western Blotting (C and D) Densitometric analysis was performed on the Western Blotting. The levels of Nrf2, HO-1, p-HER2, p-EGFR, p-AKT and p-ERK1/2 were quantified by using the software Image J. The data are expressed as the mean ± SD of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. The original blots were provided in supplementary data as Supplementary Figure S4.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Software

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Nrf2 knockdown repressed the activation of HER2 signaling pathway and sensitizes SK-OV-3 cells to lapatinib treatment (A) Effect of Nrf2 knockdown on the expression of HO-1, p-HER2, p-AKT, and p-ERK1/2 were determined after treatment with Nrf2 siRNA or scramble siRNA for 36 h (B) Effect of Nrf2 knockdown on the sensitivity to lapatinib. Cell viability was examined after lapatinib treatment for 36 h in Nrf2 siRNA or scramble siRNA-transfected cells. Cells were transfected with Nrf2 siRNA or scramble siRNA using Lipofectamine 3000 (Invitrogen) according to the supplier's instruction. Data show the mean ± SD (three independent experiments). ∗∗∗ p < 0.001. The original blots were provided in supplementary data as Supplementary Figure S5.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: Knockdown, Activation Assay, Expressing, Transfection

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Proposed model of the molecular basis of synergistic interaction between lapatinb and brusatol. Brusatol, in combination with lapatinib, may exert synergistic effects in two ways: (a) combination therapy inhibits the phosphorylation of HER receptors including EGFR and HER2, limiting the activation of their downstream pathways including PI3K-AKT signaling and Ras/Raf/MAPK signaling. (b) combination therapy modulates cell redox homeostasis by decreasing Nrf2 level and preventing the accumulation of Nrf2 in the nucleus, interfering its binding to small Maf oncogene family proteins (Mafs) and antioxidant response elements (AREs) complex, causing the inhibition of antioxidant genes such as heme oxygenase 1 (HO-1) and superoxide dismutase (SOD), thereby resulting in ROS accumulation and cell death.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: Phospho-proteomics, Activation Assay, Binding Assay, Inhibition

Journal:

Article Title: Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation * S⃞

doi: 10.1074/jbc.M802318200

Figure Lengend Snippet: cAMP synergistically enhances the activation of ErbB3 and PI3K-Akt in response to neuregulin. A, down-regulation of PI(3,4)P2-induced Akt and GSK3 phosphorylation by forskolin in human SCs. B and C, time course of forskolin's action on neuregulin-stimulated ErbB3 and Akt activation in SCs. In A–C, cells were subjected to mitogen- and serum-deprivation and then stimulated with 20 μm PI(3,4)P2 (A) or 10 nm neuregulin (B and C), with or without forskolin (2 μm) for the indicated time points. Total cell lysates were collected and analyzed by Western blotting using phospho- and total-ErbB3 and -Akt antibodies, as indicated in the figure. Bar graphics depict quantitative data from the Western blot profiles shown above. In A (lower panels), the unchanged phosphorylation of ErbB3 is shown as a control for the absence PI3K-ErbB3 cross-talk. D, effect of forskolin on neuregulin-stimulated ErbB3 activation, and Akt and GSK3 phosphorylation, in HEK293T cells transfected with expression vectors encoding for ErbB2 and ErbB3. Cells were transfected in starvation medium (DMEM without FBS) overnight and then stimulated for 1 h before collecting the cell lysates for Western blot analysis. No detectable levels of endogenous ErbB3 were seen in non-transfected HEK293T cells. Results shown in the figures are representative of at least three independent experiments performed.

Article Snippet: Total and Phospho-Akt (Ser-473 and Thr-308), -GSK3β (Ser-9), -MEK, -ERK, -ErbB2 (Tyr-1221/22, Tyr-877, and Tyr-1248) polyclonal antibodies, phospho-ErbB3 (Tyr-1289), total phosphotyrosine and phospho-PKA substrate antibodies were from Cell Signaling Technologies (Beverly, MA).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control, Transfection, Expressing

Journal:

Article Title: Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation * S⃞

doi: 10.1074/jbc.M802318200

Figure Lengend Snippet: PKA activation, but not EPAC activation, by cAMP is required and sufficient to enhance the neuregulin-stimulated phosphorylation of ErbB3 and downstream PI3K-Akt signaling. A, synergistic effect of PKA activation on neuregulin-induced ErbB3 Tyr-1289 phosphorylation and Akt activation by the use of non-selective and PKA-selective cAMP-inducing agents in human SCs. B, temporal changes of neuregulin-dependent ErbB3 phosphorylation induced by the PKA-selective analog, 6-Bnz-cAMP, and the EPAC-selective analog, 8-CPT-cAMP. C, antagonistic effect of H-89 on the synergistic increase of neuregulin-initiated ErbB3 and Akt phosphorylation in response to forskolin in SCs. D, effect of PKA- and EPAC-selective analogs on neuregulin-induced ErbB3 phosphorylation in transfected HEK293T cells overexpressing ErbB2 and ErbB3. E, effect of PKA catalytic subunit overexpression on ErbB3 phosphorylation in HEK293T cells expressing ErbB2+ErbB3. Experiments were performed essentially as described in Fig. 2 (legend). Cells were stimulated for 4 h (A–C), 1 h (D–E), or the indicated time points (B) and analyzed by Western blotting using the antibodies indicated in the figure. Analogs of cAMP were used at 200 μm each. In C, cells were preincubated with H-89 (5 μm) for 30 min before hormonal stimulation. In E, the phosphorylation of cellular PKA substrates (lower panel) is shown as a control for the effect of PKA catalytic subunit (PKA) overexpression. Note that neuregulin triggers ErbB3 phosphorylation but no detectable PKA activity in transfected HEK293T cells. NT, non-transfected cells.

Article Snippet: Total and Phospho-Akt (Ser-473 and Thr-308), -GSK3β (Ser-9), -MEK, -ERK, -ErbB2 (Tyr-1221/22, Tyr-877, and Tyr-1248) polyclonal antibodies, phospho-ErbB3 (Tyr-1289), total phosphotyrosine and phospho-PKA substrate antibodies were from Cell Signaling Technologies (Beverly, MA).

Techniques: Activation Assay, Phospho-proteomics, Transfection, Over Expression, Expressing, Western Blot, Control, Activity Assay

Journal:

Article Title: Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation * S⃞

doi: 10.1074/jbc.M802318200

Figure Lengend Snippet: PKA activation, but not EPAC activation, by cAMP is required and sufficient to enhance the neuregulin-stimulated phosphorylation of ErbB2 and downstream MEK-ERK signaling. A, effect of forskolin on the neuregulin-stimulated tyrosine auto-phosphorylation of ErbB2 in HEK293T cells overexpressing ErbB2+ErbB3. The phosphorylation of ErbB2 on Tyr-1248, Tyr-1221/1222, and Tyr-877 (upper panels) was assessed in cells stimulated for 1 h with neuregulin alone or in combination with forskolin, as stated in the figure. The phosphorylation of MEK and ERK was determined in paralleled samples (lower panels). B and C, synergistic enhancement of neuregulin-induced ErbB2 tyrosine phosphorylation and MEK-ERK activation by non-selective and pathway-selective cAMP-stimulating agents in human SCs (B) and HEK293T cells overexpressing ErbB2 and ErbB3 (C). D, involvement of PKA in the synergistic enhancement of ErbB2 phosphorylation in human SCs. All experimental conditions were identical to those described in Fig. 3.

Article Snippet: Total and Phospho-Akt (Ser-473 and Thr-308), -GSK3β (Ser-9), -MEK, -ERK, -ErbB2 (Tyr-1221/22, Tyr-877, and Tyr-1248) polyclonal antibodies, phospho-ErbB3 (Tyr-1289), total phosphotyrosine and phospho-PKA substrate antibodies were from Cell Signaling Technologies (Beverly, MA).

Techniques: Activation Assay, Phospho-proteomics

Journal:

Article Title: Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation * S⃞

doi: 10.1074/jbc.M802318200

Figure Lengend Snippet: Adenosine and epinephrine synergistically enhance neuregulin-stimulated proliferation, ErbB2-ErbB3 activation, and MEK-ERK/PI3K-Akt signaling in human SCs. A, stimulation of PKA activity by adenosine and epinephrine in human SCs. Cells were treated with adenosine (200 μm), epinephrine and isoproterenol (10 μm each) for 30 min, fixed and immunostained (IF, left panel) or analyzed by Western blot (WB, right panel) using phospho-PKA substrate antibodies. Scale bar = 15 μm. DAPI, 4′,6-diamidino-2-phenylindole. B, effect of adenosine and epinephrine on DNA synthesis. Cells were stimulated with increasing concentrations of adenosine (10, 100, and 200 μm), epinephrine and isoproterenol (1, 10, and 20 μm each) in the absence and presence of neuregulin and the incorporation of [3H]thymidine was measured 48 h post-stimulation. Forskolin (2 μm) was used as a positive control. C, effect of adenosine and epinephrine on signaling pathways downstream of ErbB2-ErbB3 activation. Cells were stimulated with adenosine, epinephrine, and isoproterenol (as indicated in A), in the absence or presence of neuregulin (10 nm) for 4 h. Cells were lysed and analyzed by Western blot using the antibodies indicated in the figure.

Article Snippet: Total and Phospho-Akt (Ser-473 and Thr-308), -GSK3β (Ser-9), -MEK, -ERK, -ErbB2 (Tyr-1221/22, Tyr-877, and Tyr-1248) polyclonal antibodies, phospho-ErbB3 (Tyr-1289), total phosphotyrosine and phospho-PKA substrate antibodies were from Cell Signaling Technologies (Beverly, MA).

Techniques: Activation Assay, Activity Assay, Western Blot, DNA Synthesis, Positive Control, Protein-Protein interactions

Journal:

Article Title: Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation * S⃞

doi: 10.1074/jbc.M802318200

Figure Lengend Snippet: PKA phosphorylates ErbB2 on the highly conserved PKA consensus site Thr-686. A, sequence alignment of known ErbB/HER2 protein sequences from diverse species showing high conservation of the potential PKA phosphorylation site homologous to Thr-686 from the human ErbB2 sequence (arrow). Gray area: conserved motif ([RK/KK]-X-[T]) in the ErbB2 sequence, which matches a predicted consensus PKA phosphorylation site. B, phosphorylation of ErbB2 by PKA on Thr-686 in vitro using purified active PKA catalytic subunit (PKA active) and different concentrations of a bacterially expressed purified preparation of GST-ErbB2 intracellular domain as a substrate in the kinase reaction (left panel). Reactions were also performed in the absence and presence of a specific PKA inhibitory peptide (“Experimental Procedures”) to confirm the specificity of the kinase reaction (right panel). Reaction products were analyzed by Western blot using the antibodies indicated in the figure. C, phosphorylation of ErbB2 on Thr-686 by PKA in vivo. Where indicated, HEK293T cells were transfected with pcDNAIII-ErbB2 alone or in combination with expression vectors encoding ErbB3 and/or the active PKA catalytic subunit (PKA), as described under “Experimental Procedures.” Total lysates were analyzed by Western blot using anti-phospho ErbB2 Thr-686 antibody (upper panel) and total ErbB2 (center) and ErbB3 (lower panel) antibodies, as a control for ErbB2 and ErbB3 expression. D, stimulation of ErbB2 phosphorylation on Thr-686 by PKA-stimulating agents in SCs. Mitogen-starved SCs were treated with forskolin (2 μm) for 10 min and 4 h, or alternatively with adenosine (200 μm), epinephrine and isoproterenol (10 μm each) for 30 min. Cells were lysed and analyzed by Western blot using anti-phospho ErbB2 Thr-686 and total ErbB2 (control) antibodies. E, point mutation of Thr-686 abolishes PKA-mediated phosphorylation of ErbB2. HEK293T cells were transfected with pcDNAIII-ErbB2 wild type (-WT) or with a mutated construct carrying an alanine replacement of Thr-686 (-T686A). ErbB2 constructs were transfected alone or in combination with an expression vector encoding the catalytic subunit of PKA (PKA). Experiments were performed essentially as described in C. In C–E, lower panels show the expression of phosphorylated cellular PKA substrates as a control for the effect of the PKA-inducing treatments. Note that the induction of high levels of PKA activity by forskolin is sustained with time in SCs and clearly coincides with the phosphorylation of ErbB2 on Thr-686. NT, non-transfected cells.

Article Snippet: Total and Phospho-Akt (Ser-473 and Thr-308), -GSK3β (Ser-9), -MEK, -ERK, -ErbB2 (Tyr-1221/22, Tyr-877, and Tyr-1248) polyclonal antibodies, phospho-ErbB3 (Tyr-1289), total phosphotyrosine and phospho-PKA substrate antibodies were from Cell Signaling Technologies (Beverly, MA).

Techniques: Sequencing, Phospho-proteomics, In Vitro, Purification, Western Blot, In Vivo, Transfection, Expressing, Control, Mutagenesis, Construct, Plasmid Preparation, Activity Assay

Journal:

Article Title: Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation * S⃞

doi: 10.1074/jbc.M802318200

Figure Lengend Snippet: PKA enhances neuregulin-induced ErbB2 and ErbB3 activation and DNA synthesis through the phosphorylation of ErbB2 on Thr-686. A and B, phosphorylation of ErbB2 on Thr-686 is required for the increased neuregulin-dependent ErbB2-ErbB3 activation by PKA but not for neuregulin-induced tyrosine kinase activity of ErbB2, in transfected HEK293T cells. HEK293T cells were transfected with expression vectors encoding either ErbB2-WT or ErbB2-T686A, together with ErbB3, and stimulated for 10 min with neuregulin alone (A) or for 1 h with neuregulin, in the absence and presence of forskolin or 6-Bnz-cAMP (B). Total cell lysates were analyzed by Western blot using the activation state and total antibodies indicated in the figure. C and D, phosphorylation of ErbB2 on Thr-686 is required for the increased neuregulin-dependent S-phase entry (C) and ErbB2-ErbB3 activation (D) by PKA in SCs. SCs were transfected (“Experimental Procedures”) with either ErbB2-WT or ErbB2-T686A expression vectors and stimulated for 4 h for Western blot analysis (D) or for 48 h in the presence of [3H]thymidine for proliferation assays (C). Experimental conditions were identical to those described in previous figures. An arrowhead is shown where Western blot images from the same gel were cut and consolidated into one single image.

Article Snippet: Total and Phospho-Akt (Ser-473 and Thr-308), -GSK3β (Ser-9), -MEK, -ERK, -ErbB2 (Tyr-1221/22, Tyr-877, and Tyr-1248) polyclonal antibodies, phospho-ErbB3 (Tyr-1289), total phosphotyrosine and phospho-PKA substrate antibodies were from Cell Signaling Technologies (Beverly, MA).

Techniques: Activation Assay, DNA Synthesis, Phospho-proteomics, Activity Assay, Transfection, Expressing, Western Blot

Journal:

Article Title: Protein Kinase A-mediated Gating of Neuregulin-dependent ErbB2-ErbB3 Activation Underlies the Synergistic Action of cAMP on Schwann Cell Proliferation * S⃞

doi: 10.1074/jbc.M802318200

Figure Lengend Snippet: Cross-talk between PKA and ErbB signaling in the regulation of SC proliferation. We propose a model that explains how cAMP synergistically enhances cell proliferation in SCs. The direct phosphorylation of the intracellular domain of ErbB2 on Thr-686 is required for the cooperative action of PKA and neuregulin in the regulation of ErbB2 tyrosine kinase activity and thereby on the phosphorylation of key tyrosine sites on ErbB2 and ErbB3 linked to Ras- and PI3K-mediated signaling, respectively. We have identified adenosine and epinephrine as two candidate physiological inducers of PKA activity in SCs exerting cross-talk with neuregulin signaling at the ErbB2-ErbB3 level.

Article Snippet: Total and Phospho-Akt (Ser-473 and Thr-308), -GSK3β (Ser-9), -MEK, -ERK, -ErbB2 (Tyr-1221/22, Tyr-877, and Tyr-1248) polyclonal antibodies, phospho-ErbB3 (Tyr-1289), total phosphotyrosine and phospho-PKA substrate antibodies were from Cell Signaling Technologies (Beverly, MA).

Techniques: Phospho-proteomics, Activity Assay

Journal: Cancer science

Article Title: Epidermal growth factor-dependent enhancement of invasiveness of squamous cell carcinoma of the breast.

doi: 10.1111/j.1349-7006.2010.01527.x

Figure Lengend Snippet: Fig. 3. Expression of Her proteins, phosphorylated proteins in the epidermal growth factor (EGF) signaling pathway, and cortactin in HBC9 and other four breast cancer cell lines. HBC9 cells (squamous cell carcinoma) express EGF receptor (EGFR), but do not clearly express Her2 or Her3. EGF stimulation leads to phosphorylation of proteins in the EGF pathway (pEGFR, pMAPK, pAkt). Cortactin is expressed with and without EGF stimulation. The profile of protein expression of MDA-MB231 was same as that of HBC9 cells. The levels of cortactin in HBC9 and MDA-MB231 cells was similar to the level in MCF7, a cell line with cortactin overexpression. a-Tubulin was used as a positive control.

Article Snippet: Antiphospho-EGFR rabbit monoclonal antibody (53A5, Tyr1173), anti-phospho-Her2 ⁄ ErbB2 rabbit monoclonal antibody (6B12, Tyr1221 ⁄ 1222), anti-phospho-Her3 ⁄ ErbB3 rabbit monoclonal antibody (21D3, Tyr1289), anti-Akt rabbit monoclonal antibody, anti-phospho-Akt mouse monoclonal antibody (587F11, Ser473), anti-p44 ⁄ 42-MAPK mouse monoclonal antibody (L34F12), and anti-phospho-p44 ⁄ 42-MAPK rabbit monoclonal antibody (20G11, Thr202 ⁄ Tyr204) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Phospho-proteomics, Over Expression, Positive Control

Journal: Cancer science

Article Title: Epidermal growth factor-dependent enhancement of invasiveness of squamous cell carcinoma of the breast.

doi: 10.1111/j.1349-7006.2010.01527.x

Figure Lengend Snippet: Fig. 8. Epidermal growth factor receptor (EGFR) and cortactin expression in 12 cases of breast squamous cell carcinoma. The expression of EGFR and cortactin was investigated by immunostaining and western blot analysis. (a,b) EGFR and cortactin are stained along the membrane of the cancer cells. Cortactin is also stained faintly in the cytoplasm. (c) Each band corresponding to immunostaining is shown. The two cases do not show Her2 or Her3 expression by western blot analysis. a-Tubulin was used as a positive control.

Article Snippet: Antiphospho-EGFR rabbit monoclonal antibody (53A5, Tyr1173), anti-phospho-Her2 ⁄ ErbB2 rabbit monoclonal antibody (6B12, Tyr1221 ⁄ 1222), anti-phospho-Her3 ⁄ ErbB3 rabbit monoclonal antibody (21D3, Tyr1289), anti-Akt rabbit monoclonal antibody, anti-phospho-Akt mouse monoclonal antibody (587F11, Ser473), anti-p44 ⁄ 42-MAPK mouse monoclonal antibody (L34F12), and anti-phospho-p44 ⁄ 42-MAPK rabbit monoclonal antibody (20G11, Thr202 ⁄ Tyr204) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Immunostaining, Western Blot, Staining, Membrane, Positive Control

Journal: Virologica Sinica

Article Title: Identification of a receptor tyrosine kinase inhibitor CP-724714 inhibits SADS-CoV related swine diarrhea coronaviruses infection in vitro

doi: 10.1016/j.virs.2023.06.010

Figure Lengend Snippet: SADS-CoV infection activates HER2 cascade signaling pathways. A IPI-2I cells were infected with 10 MOI SADS-CoV at 4 ​°C for 1 ​h, and then washed with cold PBS. After washing, cells were transferred to 37 ​°C and lysed at different time points, and the changes of protein level were analyzed by Western blot. B IPI-2I cells treated with 30 ​μmol/L CP-724714 infected or non-infected with 0.5 MOI SADS-CoV, and the expression and activation levels of HER2-related Ras-Raf-Mek-Erk signaling pathway proteins were detected by Western blot at 12 ​h post-infection (hpi). C , D Protein bands were analyzed by Image J software for gray value. E The expression of HER2 in IPI-2I cells was knockdown with specific siRNA for 24 ​h, and then the cells were treated with 30 ​μmol/L CP-724714. Protein levels were assessed by Western blot assay. F IPI cells were pretreated with 10 ​μmol/L Sorafenib or Lonafarnib. After 1 ​h of treatment, 10 ​μmol/L CP-724714 was added respectively and followed infection with 0.1 MOI SADS-CoV for 12 ​h. Samples were collected for RNA quantification. Data are presented as mean ± SDs. Statistical analysis was performed by Stuendent's t -test. ∗ P ​< ​0.05, ∗∗∗ P ​< ​0.001, ∗∗∗∗ P ​< ​0.0001, ns, not significant.

Article Snippet: Antibodies related with HER2 cascade signaling pathways (HER2 rabbit polyclonal antibody: 18299-1-AP, Erk1/2 rabbit antibody: 11257-1-AP, phospho-ERK1/2 (Thr202/Tyr204) rabbit antibody: 80031-1-RR, phospho-HER2/ErbB2 (Tyr1221/1222) rabbit mAb: 2243, c-Raf rabbit antibody: 9422, phospho-c-Raf (Ser289/296/301) rabbit antibody: 9431) were obtained from Proteintech (Wuhan, China) and Cell signaling technology (MA, USA).

Techniques: Infection, Protein-Protein interactions, Western Blot, Expressing, Activation Assay, Software, Knockdown