Journal: Theranostics

Article Title: BCAP31 drives TNBC development by modulating ligand-independent EGFR trafficking and spontaneous EGFR phosphorylation

doi: 10.7150/thno.35383

Figure Lengend Snippet: BCAP31 interacts with EGFR and facilitates ligand-independent EGFR/AKT pathway activation. (A) Mass spectrometry (MS) analysis of BCAP31-associated proteins. The purified protein complex was resolved on SDS-PAGE, followed by silver staining; then, the bands were retrieved and analysed by MS. (B) Analyses of the identified BCAP31 interactors. The diagram depicts BCAP31 interactors as detected by MS. The network was built based on the interaction network of EGFR-associated signalling and trafficking processes in the KEGG database overlaid with MS data. (C and D) The interaction between exogenous and endogenous BCAP31 and EGFR. Co-IP assay and immunoblot analyses evaluating the BCAP31-EGFR interaction in COS-7 cells and TNBC cells. (E) Downregulation of BCAP31 significantly attenuates ligand-independent signalling. TNBC cells stably expressed shBCAP31, control shRNA, or shRES when treated with EGF or EGF combined with gefitinib. IB examinations of phosphorylated EGFR and downstream signalling are shown. (F) Re-expression of the K721A kinase-dead (EGFR KD ) EGFR rescues the ligand-independent phosphorylation of EGFR at Tyr 845 and downstream AKT signalling in EGFR-knockdown MDA-MB-231 cells expressing control shRNA but not shBCAP31. SiRNA-resistant EGFR-KD was expressed in control or shBCAP31-MDA-MB-231 cells by lentivirus-mediated infection. Endogenous EGFR was knocked down by siRNA, and whole-cell lysate was harvested for western blot analysis. (G) Tumours of MDA-MB-231 tumour xenografts were harvested, homogenized, and analysed for pEGFR (Tyr1068, Tyr845), pAKT (Ser473), total EGFR and total AKT by ELISA. Statistical significance was determined using Dunnett's test.

Article Snippet: All the ELISA kits were provided by Cell Signaling Technology, and the catalogue numbers are #7189 (Tyr845 of pEGFR), #7134 (Ser473 of pAKT), #7240 (Tyr1068 of pEGFR), and #7177 (Thr202/Tyr204 of pERK).

Techniques: Activation Assay, Mass Spectrometry, Purification, SDS Page, Silver Staining, Co-Immunoprecipitation Assay, Western Blot, Stable Transfection, shRNA, Expressing, Infection, Enzyme-linked Immunosorbent Assay

Journal: Cancer cell

Article Title: Hyperactivation of TORC1 drives resistance to the pan-HER tyrosine kinase inhibitor neratinib in HER2- mutant cancers

doi: 10.1016/j.ccell.2019.12.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pAKT (S473) , Cell Signaling Technologies , Cat# 9271.

Techniques: Amplification, Activation Assay, Sequencing, Recombinant, shRNA, Variant Assay, Software

Journal: Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia

Article Title: Dysregulation of PINCH signaling in mesial temporal epilepsy

doi: 10.1016/j.jocn.2016.10.012

Figure Lengend Snippet: Levels of phosphorylated AKT and GSK3β are significantly decreased in MTLE compared to control. (A) Representative Western blot shows decreased pAKT, pGSK3β-S9 in epilepsy (n = 7) versus control (n = 4) tissues. Arrow indicates the case with out sclerosis. Membranes containing 20 μg/lane of total protein were probed with anti-pAKT serine 473 (ser273), total AKT, pGSK3β serine 9 (ser9), pGSK3β tyrosine 216 (tyr216), total GSK3β antibodies and GAPDH antibody as a loading control. (B) Graphic representation of the ratio of pAKT to total AKT (**p = 0.0019), and pGSK3β-S9 to total GSK3β (p, 0.0001) normalized to GAPDH by unpaired t-test. No significant differences (n.s.) were observed in levels of pGSK3β at tyrosine 216 (Tyr216).

Article Snippet: Antibodies included: PINCH1, (BD, Rockville, MD, USA), pAKT serine 473 (Cell Signaling, Danvers, MA, USA), total AKT (Cell Signaling), pGSK3β-S9, pGSK3β-Y216 (Cell Signaling), total GSK3β (SCBT, Santa Cruz, CA, USA), GAPDH (1: 5,000) (SCBT), or Grb-2 (Cell Signaling).

Techniques: Western Blot

Journal: Cell Reports

Article Title: Single-Cell Analysis Identifies LY6D as a Marker Linking Castration-Resistant Prostate Luminal Cells to Prostate Progenitors and Cancer

doi: 10.1016/j.celrep.2018.11.069

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal, 736E11 pAKT , Cell Signaling , Cat#3787.

Techniques: Recombinant, Concentration Assay, Microarray, Expressing, Software

Journal: Oncotarget

Article Title: EGFRvIII does not affect radiosensitivity with or without gefitinib treatment in glioblastoma cells

doi:

Figure Lengend Snippet: A. Effect of EGFRvIII expression on EGFR signaling as determined by Western blot analysis using antibodies against (p)EGFR (Y1173), (p)AKT (T308) and (p)ERK1/2 (T202/Y204). U87MG cell served as a negative control cell line. B. Morphology of EGFRvIII−/+ DKMG and BS153 cells (phase-contrast microscopy, 100× magnification). C. Proliferation of EGFRvIII− and EGFRvIII+ DKMG and BS153 cells. The cell number was determind for up to 8 days. D. Relative clonogenicity of EGFRvIII−/+ DKMG and BS153 cells as determined by colony forming assay (pre-plating).

Article Snippet: The antibodies recognizing EGFR, pEGFR (Y1173), ERK, pERK (T202, Y204), AKT and pAKT (T308) were obtained from Cell Signaling Technology, while the anti β-Actin antibody was purchased from Sigma-Aldrich.

Techniques: Expressing, Western Blot, Negative Control, Microscopy

Journal: Oncotarget

Article Title: EGFRvIII does not affect radiosensitivity with or without gefitinib treatment in glioblastoma cells

doi:

Figure Lengend Snippet: DKMGvIII−/+ and BS153vIII−/+ cells were treated with 5 μM gefitinib. A. After 2 h incubation, phosphorylation of EGFR (Y1173), AKT (T308) and ERK1/2 (T202/Y204) was determined by Western blot analysis using phosphospecific antibodies. The detection of unphosphorylated proteins and actin served as controls. B. Proliferation of DKMGvIII−/+ and BS153vIII−/+ cells in the presence of gefitinib ( n = 2). The cell number was determind for up to 8 days. The data set from Figure was used for comparison with untreated cells. C. Relative cytotoxicity of gefitinib as determind by colony forming assay (pre-plating). The surviving fraction of gefitinib-treated cells was normalized to the plating efficiency of untreated cells.

Article Snippet: The antibodies recognizing EGFR, pEGFR (Y1173), ERK, pERK (T202, Y204), AKT and pAKT (T308) were obtained from Cell Signaling Technology, while the anti β-Actin antibody was purchased from Sigma-Aldrich.

Techniques: Incubation, Western Blot