primary antibodies against phosphorylated egfr  (ZenBio)

Journal: Frontiers in Pharmacology

Article Title: Oxyresveratrol as a novel ferroptosis inducer exhibits anticancer activity against breast cancer via the EGFR/PI3K/AKT/GPX4 signalling axis

doi: 10.3389/fphar.2024.1527286

Figure Lengend Snippet: ORes induces ferroptosis in breast cancer cells via the EGFR/PI3K/AKT/GPX4 signalling axis. (A) The table indicates the top 10 connectivity scores between the input gene signature and the gene signatures of compounds in the LINCS L1000 touchstone dataset. (B) The table displays the top 10 scores between the input gene signature and the gene signatures of compounds in the DGDB dataset. (C) GSEA of HTS 2 results of Osimertinib dimesylate–treated MDA-MB-231 cells. (D) GSEA of HTS 2 results of AZ-5104–treated MDA-MB-231 cells. (E) Volcano plot of the DEGs in EGFR knockdown MDA-MB-231 cells, with red and blue dots indicating upregulated and downregulated genes, respectively. Differential gene screening criteria: |FoldChange|>1.3 and p- value < 0.05. (F) Representative Western blot results. (G) Quantification of western blots. Experiments were performed in triplicate, and data are presented as mean ± SD. *** p < 0.001; ns, no significance.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies against GPX4 (Cell Signaling Technology, 59735, 1:1,000), phosphorylated EGFR (ZenBio, R26283, 1:1,000), EGFR (Selleck, A5858, 1:1,000), phosphorylated PI3K (ZenBio, 310164, 1:1,000), PI3K (ZenBio, 200900, 1:1,000), phosphorylated AKT (Cell Signaling Technology, 13038, 1:1,000), and AKT (Cell Signaling Technology, 4691, 1:1,000).

Techniques: Knockdown, Western Blot

Journal: Molecular Cancer

Article Title: Efficient Blockade of Akt signaling is a determinant factor to overcome resistance to Matuzumab

doi: 10.1186/1476-4598-10-151

Figure Lengend Snippet: Matuzumab inhibits EGFR and HER2 phosphorylation, but not Akt and MAPK phosphorylation elicited by EGF . Effects of Matuzumab (100 μg/mL) on EGF-induced activation of EGFR (Tyr 845, 992, 1045 and 1068), HER-2/ neu , Akt and ERK 1/2 on A431, Caski and C33A cells, detected by Western blotting.

Article Snippet: Primary antibodies against total and phosphorylated EGFR, HER2, Akt and MAPK (all from Cell Signaling Technology, Beverly, MA, USA) were used.

Techniques: Activation Assay, Western Blot

Journal: Molecular Cancer

Article Title: Efficient Blockade of Akt signaling is a determinant factor to overcome resistance to Matuzumab

doi: 10.1186/1476-4598-10-151

Figure Lengend Snippet: Differences between matuzumab and cetuximab regarding the modulation of EGFR down-regulation, cell proliferation and survival . (A) A431 and Caski cells were incubated with matuzumab or cetuximab (100 μg/mL, each) for 24 h in serum-free culture medium and cells were then lysed for Western blotting analysis of total EGFR. (B) Effects of matuzumab or cetuximab (100 μg/mL, each) on cell metabolic viability (MTT assay) and colony formation (clonogenic assay) of A431, Caski and C33A cells. Student's t test * P < 0.05, when compared to control cells. (C) Effects of MG132 (15 μM), a proteassomal inhibitor, in combination with cetuximab or matuzumab (100 μg/mL, each) on EGFR expression of A431 cells, detected by Western blotting. (D) Effects of MG132 (15 μM) in combination with cetuximab (100 μg/mL) on phosphorylation of EGFR (Tyr 1068), Akt and ERK1/2 on A431 and Caski cells, detected by Western blotting.

Article Snippet: Primary antibodies against total and phosphorylated EGFR, HER2, Akt and MAPK (all from Cell Signaling Technology, Beverly, MA, USA) were used.

Techniques: Incubation, Western Blot, MTT Assay, Clonogenic Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits hepatitis B virus infection and replication

doi: 10.1016/j.jbc.2022.102097

Figure Lengend Snippet: 10M-D42AN inhibited actin polymerization. A , phalloidin staining of actin filaments in HepG2 cells treated with siRNA targeting DOCK11 or Ack1. The samples were observed using confocal microscopy. The scale bars represent 10 μm. B , phalloidin staining for actin filaments in HepG2 cells treated with 10M-D42AN for 24 to 48 h and then incubated in the usual medium for another 24 to 72 h. The samples were observed using confocal microscopy, and the length of actin filaments was measured. Error bars represent SE. ∗∗∗ p < 0.0005. The scale bars represent 10 μm. C , expression or phosphorylation level of Ack and WASP in HepG2 cells treated with 0 to 100 nM 10M-D42AN for 24 h. Whole-cell lysates were separated in a 3% to 8% SDS-PAGE and analyzed by Western blotting with antibodies against Ack1, pAck1, WASP, and pWASP. GAPDH is shown to verify equal loading.

Article Snippet: Equivalent amounts of protein were separated in NuPAGE 4% to 12% Bis–Tris gel (Thermo) or NuPAGE 3% to 8% Tris-acetate gel (Thermo) followed by incubation with primary antibodies against Ack1, EGFR, phosphorylated-EGFR Y845, pN-WASP (Santa Cruz), pAck1, GAPDH (Abcam, CST), Chk1, pChk1, phosphorylated-EGFR Y1045, Y1068, N-WASP (CST), and γH2AX (Merck) and with HRP-conjugated antibodies against FLAG, T7 (Novagen), and GST (Santa Cruz).

Techniques: Staining, Confocal Microscopy, Incubation, Expressing, Phospho-proteomics, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits hepatitis B virus infection and replication

doi: 10.1016/j.jbc.2022.102097

Figure Lengend Snippet: 10M-D42AN inhibited endocytosis of EGFR and Ack1. A , schematic representation of the mechanism of action of 10M-D42AN in EGFR endocytosis. Upon EGF stimulation, EGFR is phosphorylated and ubiquitinated and then interacts with Ack1 activated by Cdc42, which is activated by DOCK11. The EGFR–Ack1 complex is endocytosed into early endosomes and then degraded. On the other hand, activated Ack1 promotes the formation of actin filaments through the phosphorylation of WASP. Since 10M-D42AN inhibits Cdc42 activation, the activation of Ack1 and endocytosis of the EGFR–Ack1 complex are suppressed, although the phosphorylation of EGFR is not affected. B , expression or phosphorylation level of Ack and EGFR in Huh7 cells treated with 0 to 100 nM 10M-D42AN for 24 h and 100 ng/ml EGF for 0 to 60 min. Whole-cell lysates were separated in a 3% to 8% SDS-PAGE and analyzed by Western blotting with antibodies against Ack1, pAck1, EGFR, and EGFR (pY845, pY1045, pY1068). GAPDH is shown to verify equal loading. C , the expression level of EGFR shown in B . Data are presented as the mean ± SE pooled from three independent experiments. ∗ p < 0.05. D , immunofluorescence staining of HepG2 cells using anti-Ack1 antibody (Alexa488, green ) after treatment with 0 to 100 nM 10M-D42AN for 24 h and 100 ng/ml EGF for 60 min. Early endosome staining with CellLight Early Endosomes-RFP ( red ) is also shown. The samples were observed under a fluorescence microscope. Early endosomes colocalized with Ack1 are indicated by white arrows ; otherwise, they are indicated by white arrowheads . The scale bars represent 10 μm. E , colocalization of Ack1 and early endosomes in the cells was scored for 150 to 250 cells. The ratio under the indicated conditions is shown. Error bars represent SE. ∗ p < 0.05. SE, standard error.

Article Snippet: Equivalent amounts of protein were separated in NuPAGE 4% to 12% Bis–Tris gel (Thermo) or NuPAGE 3% to 8% Tris-acetate gel (Thermo) followed by incubation with primary antibodies against Ack1, EGFR, phosphorylated-EGFR Y845, pN-WASP (Santa Cruz), pAck1, GAPDH (Abcam, CST), Chk1, pChk1, phosphorylated-EGFR Y1045, Y1068, N-WASP (CST), and γH2AX (Merck) and with HRP-conjugated antibodies against FLAG, T7 (Novagen), and GST (Santa Cruz).

Techniques: Phospho-proteomics, Activation Assay, Expressing, SDS Page, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: International Journal of Medical Sciences

Article Title: EGF Contributes to Hypertrophy of Human Ligamentum Flavum via the TGF-β1/Smad3 Signaling Pathway

doi: 10.7150/ijms.76077

Figure Lengend Snippet: Immunohistochemical staining of EGF, pEGFR, TGF-β1 and pSmad3 in LF tissue. A EGF, pEGFR, TGF-β1 and pSmad3 in the LSCS group and Non-LSCS group. B Statistical analysis of EGF, pEGFR, TGF-β1 and pSmad3 levels. The scale bar indicates 50 μm, **** indicates P<0.0001.

Article Snippet: After antigen retrieval and blocking treatment, primary antibodies against EGF (1: 200, Abcam, Cambridge, UK), phosphorylated EGFR (pEGFR) (1: 100, Cell Signaling Technology, Danvers, MA), TGF-β1(1: 100, Abcam), phosphorylated Smad3 (pSmad3) (1: 200, Cell Signaling Technology, Danvers, MA), collagen I (1: 100, Abcam), and collagen III (1: 100, Abcam) were applied overnight at 4°C.

Techniques: Immunohistochemical staining, Staining

Journal: International Journal of Medical Sciences

Article Title: EGF Contributes to Hypertrophy of Human Ligamentum Flavum via the TGF-β1/Smad3 Signaling Pathway

doi: 10.7150/ijms.76077

Figure Lengend Snippet: Protein expression levels of EGF, pEGFR, EGFR, TGF-β1, pSmad3, Smad3, collagen I and collagen III in LF tissue. The levels of EGF, pEGFR, TGF-β1, pSmad3, collagen I and collagen III in the LSCS group were significantly higher than those in Non-LSCS group. The level of phosphorylated EGFR represents the degree of EGFR activation. When the level of total EGFR is unchanged and the level of phosphorylated EGFR is decreased, it indicates that EGFR activation is decreased or inhibited, and vice versa. Likewise, the level of phosphorylated Smad3 represents the degree of Smad3 activity.

Article Snippet: After antigen retrieval and blocking treatment, primary antibodies against EGF (1: 200, Abcam, Cambridge, UK), phosphorylated EGFR (pEGFR) (1: 100, Cell Signaling Technology, Danvers, MA), TGF-β1(1: 100, Abcam), phosphorylated Smad3 (pSmad3) (1: 200, Cell Signaling Technology, Danvers, MA), collagen I (1: 100, Abcam), and collagen III (1: 100, Abcam) were applied overnight at 4°C.

Techniques: Expressing, Activation Assay, Activity Assay

Journal: International Journal of Medical Sciences

Article Title: EGF Contributes to Hypertrophy of Human Ligamentum Flavum via the TGF-β1/Smad3 Signaling Pathway

doi: 10.7150/ijms.76077

Figure Lengend Snippet: Protein expression levels of pEGFR, EGFR, TGF-β1, pSmad3, Smad3, collagen I and collagen III in LF cells after exposure to different concentrations of exogenous EGF with or without the EGFR inhibitor erlotinib. Expression of pEGFR, TGF-β1, pSmad3, collagen I and collagen III in ligamentum flavum cells was significantly increased in a dose-dependent manner after exposed to various concentrations of exogenous EGF without erlotinib, erlotinib significantly attenuated EGF-induced increases in pEGFR, TGF-β1 pSmad3, collagen I and collagen III expression.

Article Snippet: After antigen retrieval and blocking treatment, primary antibodies against EGF (1: 200, Abcam, Cambridge, UK), phosphorylated EGFR (pEGFR) (1: 100, Cell Signaling Technology, Danvers, MA), TGF-β1(1: 100, Abcam), phosphorylated Smad3 (pSmad3) (1: 200, Cell Signaling Technology, Danvers, MA), collagen I (1: 100, Abcam), and collagen III (1: 100, Abcam) were applied overnight at 4°C.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Liver-Specific Overexpression of Prostasin Attenuates High-Fat Diet-Induced Metabolic Dysregulation in Mice

doi: 10.3390/ijms22158314

Figure Lengend Snippet: LTg mice activated MMP14 cascades similar to the Tet-on HepG2 cells. ( a ) Western blotting analysis and quantification of matrix metalloproteinase (MMP) 14 cascade in the liver of the WT/LTg mice under feeding HFD after overnight fast (14 h) (n = 4 per group). A solid arrowhead; pro-MMP14, an open arrowhead; activated MMP14, #; nonspecific bands. Phospho-EGFR and -ERK were normalized to total EGFR and ERK, respectively. Other proteins were normalized to β-actin. Values are shown as the mean ± SEM; * p -value < 0.05 ( t test). ( b ) The MMP14 activity of the liver of WT/LTg mice after overnight fast (14 h) was measured (n = 4 per group). Values are shown as the mean ± SEM; ** p -value < 0.01 ( t test). ( c ) RT-PCR of MMP14 and tissue inhibitor of metalloprotease (TIMP) 2 genes in the liver of the WT/LTg mice under feeding HFD after overnight fast (14 h) (n = 5–6 per group). Values are shown as the mean ± SEM; * p -value < 0.05 ( t test).

Article Snippet: Protein lysates were subjected to Tris-Glycine gel electrophoresis and probed with primary antibodies directed against hPRSS8 (1:1000, BD Transduction Laboratories, Franklin Lakes, NJ, USA), Akt, Akt phosphorylation (Ser473), ITGB1, EGFR, EGFR phosphorylation (Tyr1068), ERK, β-actin (1:1000, Cell Signaling, Danvers, MA, USA), ERK phosphorylation (Thr202/Tyr204) (1:2000, Cell Signaling), MMP14 (1:2000, Abcam, Cambridge, Cambridgeshire, England), and CD36 (1:1000, Novus Biologicals, Centennial, CO, USA).

Techniques: Western Blot, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Liver-Specific Overexpression of Prostasin Attenuates High-Fat Diet-Induced Metabolic Dysregulation in Mice

doi: 10.3390/ijms22158314

Figure Lengend Snippet: LKO mice inactivated MMP14 cascades. Western blotting analysis and quantification of MMP14 cascade in the Flox/LKO mice under feeding HFD after overnight fast (14 h) (n = 4 per group). A solid arrow head; pro-MMP14, an open arrow head; activated MMP14, #; nonspecific bands. Phospho-EGFR and -ERK were normalized to total EGFR and ERK, respectively. Other proteins were normalized to β-actin. Values are shown as the mean ± SEM; ** p -value < 0.01 and *** p -value < 0.001 ( t test).

Article Snippet: Protein lysates were subjected to Tris-Glycine gel electrophoresis and probed with primary antibodies directed against hPRSS8 (1:1000, BD Transduction Laboratories, Franklin Lakes, NJ, USA), Akt, Akt phosphorylation (Ser473), ITGB1, EGFR, EGFR phosphorylation (Tyr1068), ERK, β-actin (1:1000, Cell Signaling, Danvers, MA, USA), ERK phosphorylation (Thr202/Tyr204) (1:2000, Cell Signaling), MMP14 (1:2000, Abcam, Cambridge, Cambridgeshire, England), and CD36 (1:1000, Novus Biologicals, Centennial, CO, USA).

Techniques: Western Blot

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: DARPP-32 is upregulated in gefitinib-resistant cells. a-b Human NSCLC HCC827P (a) and PC9P (b) cells treated with 10nM gefitinib at indicated days were immunoblotted with antibodies against phosphorylated EGFR (p-EGFR), total EGFR (T-EGFR), phosphorylated ERBB2 (p-ERBB2), total ERBB2 (T-ERBB2), phosphorylated ERBB3 (p-ERBB3), total ERBB3 (T-ERBB3), DARPP-32, and α-tubulin (loading control). c-d HCC827P, HCC827GR (c), PC9P, PC9GR2, and PC9GR3 (d) cells treated with 100nM gefitinib were lysed and antibody-reactive protein bands were detected using anti-p-EGFR, EGFR, p-ERBB2, ERBB2, p-ERBB3, ERBB3, DARPP-32, and α-tubulin antibodies. Immunoblotting experiments were repeated independently at least three times, and a representative experimental result is shown.

Article Snippet: Permeabilized cells were incubated with primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200) diluted in SignalStain® Antibody Diluent (Cell Signaling Technology).

Techniques: Western Blot

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: Gefitinib blocks EGFR phosphorylation in EGFR-mutated human NSCLC cells. a-b HCC827P (a) and PC9P (b) cells were lysed in RIPA buffer and phosphorylated EGFR (p-EGFR), total EGFR (T-EGFR), phosphorylated ERBB2 (p-ERBB2), total ERBB2 (T-ERBB2), phosphorylated ERBB3 (p-ERBB3), total ERBB3 (T-ERBB3), DARPP-32, and α-tubulin (loading control) proteins were detected by immunoblotting of cell lysates.

Article Snippet: Permeabilized cells were incubated with primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200) diluted in SignalStain® Antibody Diluent (Cell Signaling Technology).

Techniques: Western Blot

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: Overexpression of DARPP-32 isoforms increases p-ERBB3 expression. a Human lung cancer cell line PC9P treated with vehicle (UT) or 100nM gefitinib (T) were transduced with retrovirus containing control (LacZ)-, DARPP-32- or t-DARPP-overexpressing clones and immunofluorescence experiments were performed using primary antibodies against p-ERBB3 (green) and p-EGFR (red). Nuclei were stained with DAPI (blue). b-c Average red (b) and green (c) fluorescence intensity of 6-10 random microscopic fields for each sample was reported. Experiments were repeated at least three times. Scale bar, 20 μm. Bar graphs indicate mean ±SEM (n=3). *P<0.05 and **P<0.01, 2-way unpaired t-test.

Article Snippet: Permeabilized cells were incubated with primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200) diluted in SignalStain® Antibody Diluent (Cell Signaling Technology).

Techniques: Over Expression, Expressing, Transduction, Clone Assay, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: Expression of p-ERBB3 is controlled by DARPP-32. a PC9GR3 cells were transduced with lentivirus containing control (LacZ) or DARPP-32 shRNAs. Cells treated with vehicle (UT) or 100nM gefitinib (T) were fixed, permeabilized, and incubated with primary antibodies that detect p-ERBB3 (green) and p-EGFR (red) proteins. DAPI-stained nuclei were represented in blue color. b-c Expression of p-EGFR (b) and p-ERBB3 (c) was reported by calculating average fluorescence intensity of 6-10 random microscopic fields for each sample. Each circle on a graph represents an independent experiment. Scale bar, 20 μm. Results represent mean ±SEM (n=3). *P<0.05 and **P<0.01, 2-way unpaired t-test.

Article Snippet: Permeabilized cells were incubated with primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200) diluted in SignalStain® Antibody Diluent (Cell Signaling Technology).

Techniques: Expressing, Transduction, Incubation, Staining, Fluorescence

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: DARPP-32 physically associates with EGFR and ERBB3. a-b EGFR-mutated human NSCLC HCC827P (a) and PC9P (b) cells transduced with retrovirus encoding control (LacZ)-, DARPP-32- or t-DARPP-overexpressing clones were immunoprecipitated using antibodies against FLAG (detects both DARPP-32 isoforms), and ERBB3. Immunoprecipitated protein complexes and total cell lysates (input) were immunoblotted using anti-EGFR, ERBB3, FLAG, and α-tubulin antibodies. c-d Human lung adenocarcinoma HCC827P, HCC827GR (c), PC9P, PC9GR2, and PC9GR3 (d) cells were lysed and immunoprecipitated with anti-DARPP-32 (recognizes endogenous DARPP-32 and t-DARPP) and anti-ERBB3 antibodies. Immunoprecipitated lysates along with total cell lysates were separated on SDS-PAGE followed by immunoblot analysis using antibodies against EGFR, ERBB3, DARPP-32, and α-tubulin. Immunoprecipitation experiments were repeated at least three times.

Article Snippet: Permeabilized cells were incubated with primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200) diluted in SignalStain® Antibody Diluent (Cell Signaling Technology).

Techniques: Transduction, Clone Assay, Immunoprecipitation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: Depletion of DARPP-32 reduces p-EGFR to p-ERBB3 heterodimer formation. a Proximity ligation assays (PLA) were performed in PC9GR3 cells stably transduced with control (shLacZ) or DARPP-32 shRNAs (shDP32) using antibodies against phosphorylated ERBB3 and EGFR following 24h incubation with either vehicle (UT) or 100 nM gefitinib (T). The images show a maximum intensity projection of the raw image based on 10 z-planes. PLA signals are shown in red and the DAPI-stained nuclei in blue. Scale bar, 20 μm. b Total number of PLA signals per cell were reported after calculating red fluorescence signals of 6-10 random microscopic fields for each group. Each circle on a graph represents an independent experiment. Bar graphs represent mean ±SEM of three independent experiments. *P<0.05, 2-way unpaired t-test.

Article Snippet: Permeabilized cells were incubated with primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200) diluted in SignalStain® Antibody Diluent (Cell Signaling Technology).

Techniques: Ligation, Stable Transfection, Transduction, Incubation, Staining, Fluorescence

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: DARPP-32 activates AKT and ERK signaling in the presence of gefitinib. a Immunoblotting was performed in gefitinib-treated HCC827P and PC9P cells stably overexpressing control (LacZ), DARPP-32 or t-DARPP using antibodies against phosphorylated AKT (p-Akt), total AKT (Akt), phosphorylated ERK (p-Erk1/2), total ERK (Erk1/2), DARPP-32, and α-tubulin (loading control). b Gefitinib-resistant human lung cancer cell lines, HCC827GR, PC9GR2, and PC9GR3, were transduced with lentivirus containing LacZ or DARPP-32 shRNAs and treated with 100nM gefitinib for 24h. Cell lysates were separated and antibody-reactive protein bands were detected using anti-p-AKT, AKT, p-ERK, ERK, DARPP-32, and α-tubulin antibodies. Three independent immunoblotting experiments have been performed and representative results from one experiment have been shown.

Article Snippet: Permeabilized cells were incubated with primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200) diluted in SignalStain® Antibody Diluent (Cell Signaling Technology).

Techniques: Western Blot, Stable Transfection, Transduction

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: DARPP-32 silencing inhibits EGFR TKI refractory tumor growth in vivo a Luciferase-labeled human HCC827GR cells transduced with control (LacZ) or DARPP-32 shRNA were injected into the left thorax of SCID mice (n=7 mice per group), imaged for luminescence, administered 25mg/Kg gefitinib on indicated days. b Quantification of tumor growth was reported by determining the difference in relative luciferase units (RLU) before and after drug treatment. c Immunohistochemistry was performed using monoclonal Ki-67 antibody on formalin-fixed, paraffin-embedded lung tissue (n=3 mice per group) obtained from human lung tumor xenograft model. For evaluation of the morphology, slides were stained with hematoxylin and eosin (H&E) dye. Scale bar, 20 μm.

Article Snippet: Permeabilized cells were incubated with primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200) diluted in SignalStain® Antibody Diluent (Cell Signaling Technology).

Techniques: In Vivo, Luciferase, Labeling, Transduction, shRNA, Injection, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Staining

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: Expression of DARPP-32, p-EGFR, and p-ERBB3 proteins is elevated in EGFR TKI resistant lung adenocarcinoma. a Tumor tissue was biopsied before EGFR TKI treatment (i.e. baseline) and following EGFR TKI resistance (i.e. progressive disease after first-line gefitinib or erlotinib therapy) from lung adenocarcinoma patients with EGFR activating mutations (n=30 patients in each group). Paired baseline (top) and EGFR TKI resistance (bottom) lung tumor specimens were immunostained for DARPP-32, phosphorylated EGFR (p-EGFR), total EGFR, p-ERBB3, and total ERBB3. b IHC score was calculated by multiplying the staining intensity score (0-3) by the percent of positive tumor cells. Each circle on the plots represents single patient. *P<0.05, **P<0.01, ****P<0.0001,2-way unpaired t-test

Article Snippet: Permeabilized cells were incubated with primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200) diluted in SignalStain® Antibody Diluent (Cell Signaling Technology).

Techniques: Expressing, Staining

Journal: Antiviral Research

Article Title: Inhibition of herpes simplex virus by myricetin through targeting viral gD protein and cellular EGFR/PI3K/Akt pathway

doi: 10.1016/j.antiviral.2020.104714

Figure Lengend Snippet: Involvement of EGFR/PI3K/Akt signaling pathway in the anti-HSV actions of myricetin . (A-C) HSV-2 (MOI = 1.0) infected cells were treated with or without myricetin (2.5, 5, 10, 20 μM) for 2 h, and then the phosphorylation of EGFR (A), PI3K (B), and Akt (C) was evaluated by western blot. Blots were also probed for GAPDH and α-tubulin proteins as loading controls. The result shown is a representative of three separate experiments. (D-F) Quantification of immunoblot for the ratio of p-EGFR (D), p-PI3K (E), p-Akt (F) protein to GAPDH or tubulin, respectively. The ratio for non-infected cells (M) was assigned values of 1.0 and the data presented as mean ± S.D. (n = 3). Significance: ##p < 0.01 vs. normal control group (Mock); *p < 0.05, **p < 0.01 vs. virus control group (HSV). (G) HSV-2 (MOI = 1.0) infected cells were treated with or without myricetin (2.5, 5, 10, 20 μM) for 2 h, and then the total expression levels of EGFR, PI3K, and Akt were evaluated by western blot. The result shown is a representative of three separate experiments. (H) Quantification of immunoblot for the ratio of EGFR, PI3K, Akt protein to α-tubulin, respectively. The ratio for non-infected cells (Mock) was assigned values of 1.0 and the data presented as mean ± S.D. (n = 3).

Article Snippet: After being blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 (v/v) and 5% BSA (w/v) at RT for 2 h, the membranes were rinsed and incubated at 4 °C overnight with primary antibodies against phosphorylated or non-phosphorylated EGFR, PI3K, Akt antibodies, or anti-α-tubulin and GAPDH antibodies (Cell Signaling Technology, Danvers, USA) as control.

Techniques: Infection, Western Blot, Expressing

Journal: Antiviral Research

Article Title: Inhibition of herpes simplex virus by myricetin through targeting viral gD protein and cellular EGFR/PI3K/Akt pathway

doi: 10.1016/j.antiviral.2020.104714

Figure Lengend Snippet: The inhibition of EGFR/PI3K/Akt pathway by myricetin may be related to its inhibition of gD protein . (A) Non-infected Vero cells were treated with or without myricetin (2.5, 5, 10, 20 μM) for 2 h, and then the phosphorylation of EGFR, PI3K, and Akt was evaluated by western blot. Blots were also probed for α-tubulin proteins as loading controls. The result shown is a representative of three separate experiments. (B) Plots quantifying the immunoblots (as ratios to tubulin) for p-EGFR, p-PI3K and p-Akt proteins. The ratios for non-treated cells (Mock) were assigned values of 1.0 and the data presented as mean ± S.D. (n = 3). (C) Vero cells were treated with or without gD proteins in the presene or absence of myricetin (5, 10, 20 μM) for 2 h, and then the phosphorylation of EGFR, PI3K, and Akt was evaluated by western blot. Blots were also probed for GAPDH proteins as loading controls. The result shown is a representative of three separate experiments. (D) Quantification of immunoblot for the ratio of p-EGFR, p-PI3K, p-Akt protein to GAPDH, respectively. The ratio for non-treated control cells (Mock) was assigned values of 1.0 and the data presented as mean ± S.D. (n = 3). Significance: ##p < 0.01 vs. normal control group (Mock); *p < 0.05, **p < 0.01 vs. gD treated control group (gD). (E and F) HSV (MOI = 1.0) infected Vero cells were treated with myricetin (2.5, 5, 10, 20 μM), and incubated at 37 °C for 8 h. After that, total RNA was extracted for real-time RT-PCR assay of HSV-1 (E) and HSV-2 (F) gD mRNAs and cellular β-actin mRNA. The relative amounts of HSV gD mRNAs were determined using the comparative (2 -ΔΔCT ) method. RNA levels for non-treated virus control cells (HSV) were assigned values of 1.0. Values are means ± SD (n = 3). Significance: *p < 0.05, **p < 0.01 vs. virus control group.

Article Snippet: After being blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 (v/v) and 5% BSA (w/v) at RT for 2 h, the membranes were rinsed and incubated at 4 °C overnight with primary antibodies against phosphorylated or non-phosphorylated EGFR, PI3K, Akt antibodies, or anti-α-tubulin and GAPDH antibodies (Cell Signaling Technology, Danvers, USA) as control.

Techniques: Inhibition, Infection, Western Blot, Incubation, Quantitative RT-PCR