Azurespot software

Inhibition of AAPH-induced damage to supercoiled DNA by each of the three phenolic fractions was examined according to the method of Albishi et al. [35 (link)] with a slight modification. All reagents were dissolved in a 75 mM phosphate-buffered solution (PBS, pH 7.4). Briefly, 4 μL of supercoiled plasmid DNA solution (pBR 322 from Escherichia coli RRI, 50 μg/mL), 4 μL of each sample at different concentrations, and 4 μL of 9 mM AAPH were added sequentially to 4 μL of PBS in an Eppendorf tube. After centrifugation and incubation at 37 °C for 1 h, 4 μL of loading dye consisting of 0.25% bromophenol blue, 0.25% xylene cyanol, and 50% glycerol in distilled water was added to the reaction mixture. A 0.7% (w/v) agarose gel was prepared in 50× TAE buffer containing GelRed^TM nucleic acid stain (10 μL/100 mL of gel). Each sample was loaded, and gel electrophoresis was run (60 V) for 1 h at room temperature in the TAE buffer. DNA bands were then imaged, and band intensities were analyzed using Azurespot software (Azure Biosystems Inc, Dublin, CA, USA). Results of inhibition to supercoiled DNA damage were expressed as IC₅₀ values (mg/mL), i.e., the concentration of a sample that causes 50% inhibition.

Deacetylase assays were performed using a model acetylated peptide substrate as previously described^{64 (link)}. The concentration of the Rpd3L complex used in these assays were estimated based on SDS-PAGE band intensities of various subunits following Coomassie Brilliant Blue staining compared to known amounts of bovine serum albumin; band intensities were quantified using AzureSpot software (Azure Biosystems). Assay data were fitted using GraphPad Prism ver. 9.5.0 (Dotmatics).

Using standard procedures, clarified supernatants from homogenized muscle tissues or murine serum were analyzed by SDS-PAGE followed by western blot with the following primary antibodies: anti-myostatin prodomain AF1539 (R&D Systems); anti-mature Myostatin ab124621 (Abcam). Immunoprecipitations with SRK-015, IgG control, or GDF8-C1 antibodies from homogenized clarified muscle lysate and murine serum were carried out using the Thermo Scientific Pierce™ Co-Immunoprecipitation Kit according to the manufacturer’s specifications.
For quantitative fluorescent western blots, samples were loaded onto Any kD Mini-PROTEAN TGX Stain-Free Gels (Bio-Rad) and signal was detected by near-IR imaging (Azure c600, Azure Biosystems) and quantified using the western blot analysis tool in AzureSpot software (Azure Biosystems). Band intensity (number of positive pixels per unit area) from pro and latent Myostatin was measured and normalized to total loaded protein per lane. For all data presented, a minimum of three biological replicates were measured to generate the presented average values, and error bars on all graphs represent standard deviations. Statistical significance was determined by t-test (two-tailed, homoscedastic).

HEK293T cells were transfected with myc-tagged WT or SAAX mutant small GTPases. Ninety minutes following transfection, cells were metabolically labeled with 10 μM C15AlkOPP isoprenoid probe for 18 h. Cells were then harvested and lysed as described (58 (link)). Briefly, cell pellets were suspended and lysed by sonication in PBS + 1% SDS. Protein concentrations were determined using bicinchoninic acid Assay (Thermo Fisher Scientific, 23225), following the manufacturer’s protocol. Proteins (100 μg/100 μl) were subjected to click reaction with 25 μM TAMRA PEG₃-N₃ (BroadPharm), 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (Sigma-Aldrich), 0.1 mM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (Sigma-Aldrich), and 1 mM CuSO₄ in the dark with gentle shaking for 1 h at room temperature. Proteins were precipitated using ProteoExtract Protein Precipitation Kit (Calbiochem, cat. no. 539180), following the manufacturer’s protocol. Resulting protein pellets were suspended in Laemmli sample buffer, boiled for 5 min, run on 12% SDS-PAGE gels, and transferred to PVDF. Fluorescence and immunoblotting images were obtained using Azure c600 and analyzed using AzureSpot software (Azure Biosystems). Fluorescence optical densities of overexpressed proteins were normalized to the optical density of myc-tagged protein.

This assay was performed using the standard protocol described previously^{24 (link),27 (link)}. Briefly, 25 nM PFV intasome, 50 ng of 3 kb supercoiled (SC) plasmid DNA (pGEM-T Easy, Promega), 10 mM Bis-tris propane, pH 7.5, 110 mM NaCl, 5 mM MgSO₄, 4 μM ZnCl₂, and 10 mM DTT in 15 μl total volume were incubated at 37 °C for 5 min. The reactions were terminated by adding 0.1% SDS, 2.5 mM EDTA, 1 mg/ml proteinase K and incubated at 55 °C for an hour. The products were mixed with 5% glycerol before resolving on a 1% agarose gel in 1X TAE at 105 V for an hour. Gels were stained with 0.1 μg/mL ethidium bromide and scanned on a Sapphire Biomolecular Imager (Azure Biosystems). Unreacted SC plasmid and linear concerted integration products were quantified with AzureSpot software (Azure Biosystems) as described previously^{24 (link),27 (link)} and presented in Supplementary Fig. 1.

Purified VLP samples were mixed with lithium dodecyl sulfate (LDS) sample buffer (1× final concentration) and heated at 100 °C for 5 min. Samples were loaded onto 4–12% Bis-Tris gels (Invitrogen, Waltham, MA, USA) and transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat dry milk in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20). Primary antibodies were added to membranes and incubated overnight: anti-N (1:1000 dilution, Sino Biological, Beijing China, 40143-T62), anti-M (antibody cocktail: i. (1:250 dilution, ProSci, Fort Collins, CO, USA, 3529) and ii. (1:250 dilution, Invitrogen, PA1-41160)), or anti-S (1:1000 dilution, generated in house via immunization of mice with purified S protein (BEI Resources, NIAID, Manassas, VA, USA, NR-52308)). Membranes were washed 3 times with TBS-T and subsequently incubated for 1 h with HRP-conjugated secondary antibody: anti-rabbit (1:6000 dilution, Invitrogen, 65-6120) or anti-mouse (1:6000 dilution, Invitrogen, 62-6520). Blots were developed with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific) and imaged using an Azure Biosystems C600 Imaging System. Densitometry analysis was performed using AzureSpot software (Azure Biosystems, Dublin, CA, USA). Purified recombinant S protein was used as a standard (BEI Resources, NR-52308).