Journal: Molecular & Cellular Proteomics : MCP

Article Title: Molecular Display of the Animal Meta-Venome for Discovery of Novel Therapeutic Peptides

doi: 10.1016/j.mcpro.2024.100901

Figure Lengend Snippet: Metavenom Kunitz type inhibitor domains can potentiate MRGPRX4. A , volcano plot of animal venom, metavenome, and secretome libraries binding to HEK293-MRGPRX4 cells versus HEK293 cells. Each point is a unique peptide in the AV, MV, and secretome libraries. B , heat map showing selectivity of fold-change values of the six peptide candidates across the HEK293-MRGPRX families. C , MSA analysis and motif discovery with MEME Suite identifies conserved amino acids shared among the candidate hits ( left ). Structural alignment of the hits using AlphaFold ( right ). D , flow cytometric analysis of ERR1712142|105-166 cell binding. MRGPRX4-overexpressing cells and parental HEK293 cells were incubated with ERR1712142|105-166 fused with a FLAG-tag. Following incubation, the cells were stained with Alexa Fluor 647-labeled anti-FLAG antibodies to quantify the amount of specifically bound candidate ligand via flow cytometry. An Empty control, which expressed irrelevant recombinant protein, was included as an additional negative control to ensure the specificity of the binding measurement. E , flow cytometric analysis of cell binding of TFPI ( left ), SPINT2 ( middle ), and APP751 ( right ). MRGPRX4-overexpressing cells and parental HEK293 cells were incubated with TFPI, SPINT2, or APP751 fused with a His-tag. Following incubation, the cells were stained with Alexa Fluor 647-labeled anti-His antibodies. MFI, median fluorescence intensity. Data are shown as mean ± SD of 5 (in D ) or 3 (in E ) biological replicates. Statistical comparisons use two-tailed unpaired Student’s t tests. ns, nonsignificant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. F , dose-response curves of TFPI ( left ) and SPINT2 ( right ) in the presence of 200 μM UDCA using the MRGPRX4 PRESTO-Tango assay. Each point is one replicate. RLU, relative luminescence unit. G , dose-responses curves of UDCA in the presence of 0.2 μM TFPI ( left ) or 0.8 μM SPINT2 ( right ) in the MRGPRX4 PRESTO-Tango assay. Data are shown as mean ± SD of three technical replicates. MSA, multiple sequence alignment; SPINT2, serine peptidase inhibitor, Kunitz type 2; TFPI, tissue factor pathway inhibitor; UDCA, ursodeoxycholic acid.

Article Snippet: The cells were then incubated with ERR1712142 (fused with FLAG-tag), 30 nM TFPI (Acro Biosystems, Cat No. TFI-H5226, fused with His-tag), 30 nM SPINT2 (Sino Biological, Cat No. 10324-H08H, fused with His-tag) and 30 nM APP751 (a 751 amino-acid isoform of amyloid-beta precursor protein) (BioLegend, Cat No. 842601, fused with His-tag) prepared in 1% PBSA for 4 h at 4 °C on an end-to-end rotator.

Techniques: Binding Assay, Incubation, FLAG-tag, Staining, Labeling, Flow Cytometry, Control, Recombinant, Negative Control, Fluorescence, Two Tailed Test, Sequencing

Journal: Frontiers in Oncology

Article Title: Characterizing the secretome of EGFR mutant lung adenocarcinoma

doi: 10.3389/fonc.2023.1286821

Figure Lengend Snippet: Protein candidates identified from microarray gene expression analysis, listed by gene symbol, sorted by adjusted p value (p adj< 0.05; Benjamini-Hochberg adjustment).

Article Snippet: MDK and SPINT2 were quantified using the Human MDK ELISA Kit (Invitrogen, EH319RB) and SPINT2 (HAI-2) Human ELISA Kit (Invitrogen, EH319RB and EHSPINT2) according to kit instructions.

Techniques: Microarray, Expressing

Journal: Frontiers in Oncology

Article Title: Characterizing the secretome of EGFR mutant lung adenocarcinoma

doi: 10.3389/fonc.2023.1286821

Figure Lengend Snippet: Survival analysis of protein candidates identified from filtering pipeline. Overall patient survival was analyzed (log rank test, median split) for EGFR mutant tumors, n=125 and EGFR wild type tumors, n=68 from the NCBI GEO GSE 31219 dataset. (A) ENO1. (B) PFKP (C) RAC1 (D) SPINT2 (E) MDK. n.s. non-significant.

Article Snippet: MDK and SPINT2 were quantified using the Human MDK ELISA Kit (Invitrogen, EH319RB) and SPINT2 (HAI-2) Human ELISA Kit (Invitrogen, EH319RB and EHSPINT2) according to kit instructions.

Techniques: Mutagenesis

Journal: Frontiers in Oncology

Article Title: Characterizing the secretome of EGFR mutant lung adenocarcinoma

doi: 10.3389/fonc.2023.1286821

Figure Lengend Snippet: ELISA analysis of selected protein candidates in secretome conditioned media. Mean values ± SD are shown, experiment performed in technical duplicate (Student’s unpaired T-test, two tailed). (A) MDK (B) GDF15 (C) SPINT2. ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. non-significant.

Article Snippet: MDK and SPINT2 were quantified using the Human MDK ELISA Kit (Invitrogen, EH319RB) and SPINT2 (HAI-2) Human ELISA Kit (Invitrogen, EH319RB and EHSPINT2) according to kit instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test