Effect of treadmill exercise and bone marrow stromal cell engraftment on activation of BDNF-ERK-CREB signaling pathway in the crushed sciatic nerve

The effect of combined approach of exercise training and bone marrow stromal cell (BMSC) engraftment on activation of brain-derived neurotrophic factor (BDNF)-extracellular signal-regulated kinase 1 and 2 (ERK1/2)-cyclic adenosine monophosphate response element-binding protein (CREB) signaling pathway after sciatic nerve injury (SNI) was investigated. Sixty male Sprague-Dawley rats divided into the normal control, nonexercise (NEX), exercise training (EX), BMSC transplantation (TP), and exercise training+BMSC transplantation (EX+TP) groups 4 weeks after SCI. Exercise training was carried out on the treadmill device at 5–10 m/min for 20 min for 4 weeks. Single dose of 5× 106 harvested BMSC was injected into the injury area of the injured sciatic nerve. In order to evaluate induction levels of BDNF-ERK1/2-CREB signaling molecules in the whole cell and nuclear cell lysates of the injured sciatic nerve, we applied Western blot analysis. BDNF was significantly increased only in EX+TP compared to NEX, EX, and TP groups. Phosphoinositide-dependent kinase-1 was more increased in EX, TP, and EX+TP groups than NEX group, but EX+TP group showed the most upregulation of phosphorylated protein kinase B compared to other groups. In addition, in the whole cell lysate, phosphorylated ERK1/2, but not activating transcription factor-3 (ATF-3) and phosphorylated CREB, was significantly increased in TP and EX+TP groups. In the nuclear cell lysate, ATF-3 and phosphorylated CREB were strongly activated in EX+TP group compared to EX group. Regular exercise training combined with BMSC engraftment would seem to be more effective in controlling activation of regeneration-related signaling pathway after SNI.

Hybridoma Screening by Antibody Capture: A High-Throughput Western Blot Assay

Antibody capture assays are often the easiest and most convenient of the hybridoma screening methods. In this procedure, proteins in solution or in a cell lysate are separated according to size by gel electrophoresis and then transferred by blotting to a nitrocellulose sheet. Antigen bound to the solid substrate is incubated with the primary antibody, and the resultant antibody–antigen complexes are detected by a horseradish peroxidase (HRP)–conjugated secondary antibody and a chemiluminescent substrate for HRP.

The Small Metal-Binding Protein SmbP Simplifies the Recombinant Expression and Purification of the Antimicrobial Peptide LL-37

(1) Background: The cathelicidin peptide LL-37 is a prominent molecule with many biological activities, including antimicrobial. Due to its importance, here, we describe the production of LL-37 tagged with SmbP, a relatively new carrier protein that improves the production of recombinant proteins and peptides in Escherichia coli. We present an alternative method for the rapid expression, purification, and antimicrobial evaluation of LL-37, that involves only one purification step. (2) Methods: A DNA construct of SmbP_LL-37 was transformed into E. coli BL21(DE3); after overnight expression, the protein was purified directly from the cell lysate using immobilized metal-affinity chromatography. SmbP_LL-37 was treated with Enterokinase to obtain the free LL-37 peptide. The antimicrobial activity of both SmbP_LL-37 and free LL-37 was determined using the colony forming unit assay method. (3) Results: SmbP_LL-37 was observed in the soluble fraction of the cell lysate; after purification with IMAC, protein gel electrophoresis, and analysis by ImageJ, it showed 90% purity. A total of 3.6 mg of SmbP_LL-37 was produced from one liter of cell culture. SmbP_LL-37 and free LL-37 both showed inhibition activity against Staphylococcus aureus and Escherichia coli. (4) Conclusions: The SmbP fusion protein is a valuable tool for producing biologically-active LL-37 peptide. The production method described here should be of interest for the expression and purification of additional cationic peptides, since it cuts the purification time considerably prior to determination of antimicrobial activity.

Identification of photocrosslinking peptide ligands by mRNA display

Photoaffinity labelling is a promising method for studying protein-ligand interactions. However, obtaining a specific crosslinker can require significant optimisation. We report a novel mRNA display strategy, photocrosslinking-RaPID (XL-RaPID), and exploit its ability to accelerate the discovery of cyclic peptides that photocrosslink to a target of interest. As a proof of concept, we generated a benzophenone-containing library and applied XL-RaPID screening against a model target, the second bromodomain of BRD3. This crosslinking screening gave two optimal candidates that selectively labelled the target protein in cell lysate. Overall, this work introduces direct photocrosslinking screening as a versatile technique for identifying covalent peptide ligands from mRNA display libraries incorporating reactive warheads.

Proteomics workflow for whole cell lysate, endosome, and lysosome fractions v2

We present a protocol for sample preparation for LC-MS analysis of whole cell lysates and for lysosomal and endosomal fractions purified by Lyso-IP and Endo-IP. Protocols for purification of lysosomes and endosomes is provided in protocol dx.doi.org/10.17504/protocols.io.byi9puh6 using cells that express endogenously tagged TMEM192-HA and stably expressing FLAG-EEA1 as descrbed in dx.doi.org/10.17504/protocols.io.byi7puhn.

Proteomics workflow for whole cell lysate, endosome, and lysosome fractions v1

We present a protocol for sample preparation for LC-MS analysis of whole cell lysates and for lysosomal and endosomal fractions purified by Lyso-IP and Endo-IP. Protocols for purification of lysosomes and endosomes is provided in protocol dx.doi.org/10.17504/protocols.io.byi9puh6 using cells that express endogenously tagged TMEM192-HA and stably expressing FLAG-EEA1 as descrbed in dx.doi.org/10.17504/protocols.io.byi7puhn.