The use of the in situ proximity ligation assay for validating S6 kinase 1 CoAlation under oxidative stress

Aim. To verify CoAlation of ribosomal protein S6 kinase 1 (S6K1) in the HEK293/Pank1b cells exposed to oxidative stress using proximity ligation assay (PLA). Methods. In situ proximi ty ligation assay. Results. S6 kinase 1 undergoes CoA covalent modification in cellular response to oxidative stress. Conclusions. The previously developed mass spectrometry-based methodology allowed us to identify over 2100 CoAlated proteins in the cells exposed to oxidative or metabolic stress. Many protein kinases were found to be CoAlated, including S6K1. In this study, the proximity ligation assay was used to demonstrate a significant increase of S6K1 CoAlation under oxidative stress compared to untreated cells

Monoclonal antibodies to Coenzyme A.

Aim. To generate monoclonal antibodies to Coenzyme A (CoA) with antigenic epitope specificity different to previously developed anti-CoA mAb (1F10) by introducing alternative approach for antigen preparation animal immunization. Methods. Cross-linking of CoA to Keyhole Limpet Hemocyanin (KLH) and to Bovine serum albumin (BSA) using reversible chemical crosslinker SPDP. Hybridoma technique. Western blot analysis. Immunoprecipitation. ELISA. Results. Monoclonal antibody (A11) that specifically recognizes CoA in various immunoassays and has the same antigenic epitope specificity as existing anti-CoA mAb (1F10) has been generated. Conclusions. Application of different ways for CoA conjugation to the carrier proteins to create an antigen for immunization does not affect the antigenic epitope specificity of generated anti-CoA antibodies most probably due to the existence of only one immunogenic epitope in CoA molecule

Substituted Mo-V(Ti)-Te(Ce)-oxide M2 Catalysts for Propene Ammoxidation

One of the most effective propane to acrylonitrile ammoxidation catalyst is comprised of the two phases M1 (orthorhombic) Mo7.5V1.5NbTeO29 and M2 (pseudo-hexagonal) Mo4V2Te2O20. Under reaction conditions, the two phases work in symbiosis with each other where M1 is the paraffin activating component and M2 is the olefin activating component. Since the catalytic improvement of either phase should result in an enhancement of the overall acrylonitrile yield, controlled substitution of certain elements in either or both phases might result in the desired improvement. Our current study concentrates on the partial substitutions of V with Ti and Te with Ce in the M2 phase. Ti substitution results in a considerable propene activity improvement, whereas the selectivity to acrylonitrile is unaffected. Substitution with Ce, on the contrary, substantially improves the selectivity to acrylonitrile. Also, a minor improvement of the activity is notable. The acrylonitrile selectivity improvement is a result of better NH3 utilization and comes at the expense of reduced acrolein make. XRD reveals that all of the substituted compositions retain the M2 structure and essentially are monophasic. XANES recordings show for the bulk that the Mo is 6+, the V is 4+, or 4+ and 5+ when Ce is present, the Ti is 4+, the Ce is 3+, and the Te 4+ with some 6+ also present. According to the ESR data, in the M2 with Ce (7Te/3Ce) only 21% of the V is 4+, the remainder being 5+, which tentatively can be explained by the existence of some cation vacancies in the hexagonal channels. HRTEM imaging reveals little if any differences between the materials, all have the typical pseudo-hexagonal habit of the M2 phase and expose a 1-2 nm thick surface layer without any apparent long-range ordering. XPS data show that all catalysts, including the base, are highly enriched at the surface with Te at the expense of other metals. The 7Te/3Ce composition exhibits also substantial Ce surface enrichment. Moreover, the valences of the cations at the surface differ from the bulk in that for all fresh catalysts V is 5+ and Te is 6+ on the surface. Characterization by XPS of catalysts used in propene ammoxidation, reveals reduction of Te and, except when Ce is present, also Mo. Therefore, it might be inferred that the surfaces of the catalysts studied here are comprised essentially of one or a few monolayers of TeMoO or TeCeMoO on an interacting M2 crystalline base