Journal: Journal of the American Society of Nephrology : JASN

Article Title: Macrophage-stimulating protein is produced by tubular cells and activates mesangial cells.

doi: 10.1681/ASN.V133649

Figure Lengend Snippet: Figure 1. Western blot performed with anti–macrophage-stimulating protein (MSP) antibody (Ab) in culture supernatant of the human tubular cell line HK2, human mesangial cells (HMC), and the hepa- toma cell line HepG2. A 50 ml volume of supernatant was immuno- precipitated with anti-MSP polyclonal Ab adsorbed to protein A–sepharose 4B packed beads. Immunoprecipitates were washed with an ice-cold buffer, boiled after addition of 2 sample buffer, and the proteins were loaded onto 10% sodium dodecyl sulfate–polyacrylam- ide gel electrophoresis. Proteins were analyzed by Western blot that used a polyclonal anti-MSP Ab. The 85-kD bands represent mono- meric MSP (pro-MSP), and the 55-kD bands represent the -chain of dimeric (active) MSP. Both HK2 and HepG2 cells (used as controls) release pro-MSP that is cleaved into the dimeric form in the super- natant. No band is visible in the HMC lane.

Article Snippet: In some experiments, HMC were incubated with HK2 supernatant or with HK2 supernatant preincubated for 2 h with neutralizing goat polyclonal anti-human MSP antibody (Ab; 2 g/ml) (R&D Systems, Minneapolis, MN) without fresh medium.

Techniques: Western Blot, Nucleic Acid Electrophoresis

Journal: Frontiers in Oncology

Article Title: Aberrant RON and MET Co-overexpression as Novel Prognostic Biomarkers of Shortened Patient Survival and Therapeutic Targets of Tyrosine Kinase Inhibitors in Pancreatic Cancer

doi: 10.3389/fonc.2019.01377

Figure Lengend Snippet: Effect of TKIs on cellular signaling. (A) Western blot of BxPC3 cells treated MSP, HGF, and TKIs (5 μM BMS777607, 5 μM INCB28060, 5 μM PHA665752, 0.5 μM Tivantinib). Proteins analyzed include RON, MET, AKT, ERK1/2, phospho-AKT, and phospho-ERK1/2. The membranes were also reprobed for GAPDH as the loading control. Phosphorylated RON and MET were analyzed by western blotting after immunoprecipitation. (B) ELISA measurement of phosphorylated human RON in BxPC3 cells as above. * p < 0.01, ** p < 0.001.

Article Snippet: Recombinant human MSP (Cat# 352-MS), recombinant human HGF (Cat# 294-HG), and the DuoSet IC human phospho-RON ELISA (enzyme-linked immunosorbent assay, cat. no. DYC1947-5) kit were from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: ACS Chemical Neuroscience

Article Title: Differential Neuropeptidomes of Dense Core Secretory Vesicles (DCSV) Produced at Intravesicular and Extracellular pH Conditions by Proteolytic Processing

doi: 10.1021/acschemneuro.1c00133

Figure Lengend Snippet: MSP-MS cleavage profiles of recombinant cathepsins A, B, C, D, as well as CPE. Recombinant, purified enzymes were subjected to substrate cleavage profiling analysis by MSP-MS. Sequence logos show the preferred amino acid adjacent to the cleavage site (P1–P1′), and the frequency of cleavages at each of the peptide bonds within the 14-mer peptides of the 228 peptide library is shown for (A) cathepsin A at pH 5.5, (B) cathepsin A at pH 7.2, (C) cathepsin B at pH 5.5, (D) cathepsin B at pH 7.2, (E) cathepsin C at pH 5.5, (F) cathepsin C at pH 7.2, (G) cathepsin D at pH 5.5, (H) cathepsin L at pH 5.5, (I) carboxypeptidase E (CPE) at pH 5.5, and (J) CPE at pH 7.2.

Article Snippet: For MSP-MS assays using recombinant human proteases, all enzymes were purchased from R & D Systems.

Techniques: Recombinant, Purification, Sequencing

Journal: ACS Chemical Neuroscience

Article Title: Differential Neuropeptidomes of Dense Core Secretory Vesicles (DCSV) Produced at Intravesicular and Extracellular pH Conditions by Proteolytic Processing

doi: 10.1021/acschemneuro.1c00133

Figure Lengend Snippet: Parallel cleavage profiles of CG proteolytic activity utilized for neuropeptidome production with that of selected recombinant proteases analyzed by MSP-MS. Comparison of CG protease cleavage profiles with that of selected recombinant proteases identified in CG (by proteomics), conducted by MSP-MS analysis, was assessed at pH 5.5 (A) and pH 7.2 (B). Data show the matching cleavage profiles of endogenous CG proteolytic activity with that of purified recombinant enzymes consisting of cathepsins A, B, C, D, L, and CPE, assessed for each of the 13 cleavage sites of 14-mer peptides (228 total) of the substrate peptide library.

Article Snippet: For MSP-MS assays using recombinant human proteases, all enzymes were purchased from R & D Systems.

Techniques: Activity Assay, Recombinant, Comparison, Purification