Journal: bioRxiv

Article Title: Stabilization of the open conformation of Insulin-Regulated Aminopeptidase by a novel substrate-selective small molecule inhibitor

doi: 10.1101/2024.06.04.597268

Figure Lengend Snippet: Panel A , representative chromatograms of the oxytocin trimming assay: Reversed-phase HPLC chromatograms of oxytocin control and oxytocin incubated with 10 nM IRAP for 30 min at 37 °C. Panel B , titration of compound 3 inhibits trimming of oxytocin, but with a very weak IC 50 estimated to be over 100 μM. Panel C . titration of compound 3 inhibits trimming of the 10mer linear antigenic peptide precursor with the sequence LDRASFIKNL, but with a very weak IC 50 estimated to be 34 μM. Panel D , titration of compound 3 inhibits trimming of the 9mer linear antigenic epitope with the sequence YTAFTIPSI. Data for panels B, C were fitted to a variable slope log(inhibitor) versus response model and data for panel D were fitted to a biphasic dose-response curve using GraphPad Prism 8.0. Panel E , Michaelis-Menten analysis of oxytocin digestion by IRAP in the presence or absence of 100 μM compound 3. Panel F , calculated parameters k cat and K M based on the data shown in Panel E. Error bars are calculated from independent replicates and only shown if are significantly wider than the size of the data point (n=2 for panels B,C,E and F; n=4 for panel D).

Article Snippet: Linear peptide trimming of the extended antigenic peptide precursor LDRASFIKNL (JPT Peptide Technologies GmbH, derived from the epitope DRASFIKNL from Collagen alpha-2(VI) chain ) and antigenic peptide YTAFTIPSI (derived from the Gag-Pol polyprotein of human immunodeficiency virus ) was performed similarly to oxytocin trimming.

Techniques: Incubation, Titration, Sequencing

Journal: eLife

Article Title: Comment on ‘YcgC represents a new protein deacetylase family in prokaryotes’

doi: 10.7554/eLife.37798

Figure Lengend Snippet: ( A ) SDS-PAGE analysis of proteins used in this study. All proteins were expressed and purified as GST-fusion proteins. In terms of YcgC and YcgC S200A the GST-tag was removed by TEV protease during the purification steps. Staining of the gel was done by coomassie brilliant blue (CBB). For molecular masses see figure legend for . ( B ) Analytical size exclusion chromatography on a S200 10/300 GL column shows that YcgC WT and YcgC S200A as well as CobB and the corresponding catalytically inactive variant CobB H110Y display an almost identical elution profile. Moreover, RutR proteins show a nearly identical elution profile in analytical SEC runs indicating that RutR acetylation at K52 and K62 does not interfere with protein folding or its oligomeric state. ( C ) RutR-His 6 AcK52 and AcK62 are quantitatively acetylated. Shown are SDS-PAGE and immunoblot analyses of all RutR-His 6 proteins used in this study. Staining for AcK using an anti-acetyl-L-lysine antibody revealed a strong signal for RutR AcK52 and AcK62, whereas no signal was obtained for RutR WT. As loading control anti-His 6 staining was performed. ( D ) ESI-MS data show the quantitative and homogenous incorporation of acetyl-L-lysine into RutR. Shown is the deconvoluted spectrum on the true mass scale after software transformation yielding one single peak and the corresponding molecular mass as indicated. Expected mass non-acetylated RutR: 24567.5 Da; acetylated RutR: 24609.5 Da.

Article Snippet: After addition of 10 mM N-(ε)-acetyl-L-lysine (Chem-Impex International Inc.) and 20 mM nicotinamide (NAM) cells were grown for further 30 min before protein expression was induced by adding 300 µM IPTG.

Techniques: SDS Page, Purification, Staining, Size-exclusion Chromatography, Variant Assay, Western Blot, Software, Transformation Assay

Journal: International Journal of Molecular Sciences

Article Title: Myogenic Potential of Extracellular Matrix Derived from Decellularized Bovine Pericardium

doi: 10.3390/ijms22179406

Figure Lengend Snippet: Expression of myogenic markers, analyzed by Western blot. C2C12 myoblasts were incubated either on uncoated (CTR) or dECM pre-coated wells and induced to differentiate in differentiation medium. At different time points the contents of α-smooth muscle actin ( A ), myogenin ( B ), and MHC ( C ) were measured by Western blots of whole cell lysates and the intensity of the bands quantified, relative to the housekeeping proteins vinculin or tubulin used to normalize. Representative Western blots out of the three for each condition are shown. Statistics were determined by t -test. ** p < 0.01, *** p < 0.001.

Article Snippet: Results were normalized with the housekeeping proteins vinculin and tubulin (anti-vinculin, anti-tubulin 1:1000; Merck Millipore, Milan, Italy).

Techniques: Expressing, Western Blot, Incubation