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: PLoS ONE

Article Title: Periostin Responds to Mechanical Stress and Tension by Activating the MTOR Signaling Pathway

doi: 10.1371/journal.pone.0083580

Figure Lengend Snippet: ( A ) Phalloidin detection shows cells with polarized F-actin (white arrow) following treatment with recombinant Periostin compared to vehicle control. Scale bars represent 10 μm. ( B ) Graphic represents percentage of cells with stress fiber formation (polarized F-actin) after periostin or vehicle stimuli. Results were determined by measuring fields using independent triplicates (**p<0.01) ( C ) Activation of PI3K and mTOR signaling is triggered by Periostin treatment in a dose-dependent manner, as detected by phosphorylated AKT at Threonine 308 (pAKT Thr308 ) and Serine 473 (pAKT Ser473 ) and phosphorylated S6 (pS6). Note that 50 ng/ml of Periostin is the optimal concentration for PI3K activation. GAPDH was used as a loading control.

Article Snippet: Membranes were blocked in 0.1 M Tris (pH 7.5), 0.9% NaCl and 0.05% Tween-20 (TBS-T) containing 5% nonfat dry milk and probed with anti-Periostin (Biovendor), pS6 (Dako), pAKT T308 (Abcam) and pAKT S473 (Cell Signaling) antibodies.

Techniques: Recombinant, Activation Assay, Concentration Assay

Journal: Frontiers in Oncology

Article Title: IGF2BP3 promotes progression of gallbladder carcinoma by stabilizing KLK5 mRNA in N 6 -methyladenosine-dependent binding

doi: 10.3389/fonc.2022.1035871

Figure Lengend Snippet: KLK5 rescued the abrogated GBC progression in IGF2BP3-depleted cells. (A–E) IGF2BP3-depleted GBC cells were co-transfected with PLVX-KLK5 or control plasmid. Cell growth ability was assessed by CCK8 assay (A) , colony formation assay (B) and EdU assay ( C , 100X, scale bar: 100μm). Statistical significance was analyzed based on the number of EdU stained cells (right). (D) Transwell assays were conducted to compare the migration ability in each treated group of GBC cells (100X, scale bar: 100μm). (E) Western blotting assays showed the expression of IGF2BP3, KLK5, PAR2, p-AKT1 (ser473) and AKT1 in different treatment groups, GAPDH was used as the endogenous control. Unpaired t test was used in (A, C) Data is shown as mean ± SD; ** P < 0.01 and *** P < 0.001.

Article Snippet: The targeted proteins were detected using different antibodies, including GAPDH (1:1000, Proteintech, USA), IGF2BP3 (1:1000, Proteintech, USA), AKT1 (1:1000, Cell Signaling Technology, USA), P-AKT1(ser473) (1:1000, Cell Signaling Technology, USA), KLK5 (1:1000, #abs136657, Abisin, Shanghai), PAR2 (1:1000, Abclonal Technology Co., Ltd.) and METTL3 (1:1000, Abclonal Technology Co., Ltd.).

Techniques: Transfection, Plasmid Preparation, CCK-8 Assay, Colony Assay, EdU Assay, Staining, Migration, Western Blot, Expressing

Journal: Molecular Oncology

Article Title: RASSF1A independence and early galectin‐1 upregulation in PIK3CA‐induced hepatocarcinogenesis: new therapeutic venues

doi: 10.1002/1878-0261.13135

Figure Lengend Snippet: Antibodies used for immunohistochemistry and western blots.

Article Snippet: pAKT (S308) , 1 : 1000 , Cell Signaling Technology, Inc. , 13038.

Techniques: Immunohistochemistry, Western Blot

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 (T308) , Cell Signaling Technologies , Cat# 13038.

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