Elh gpc1 1

Ligands were analyzed by Western blotting or ELISA, HDAC6 activity was assessed using a homogenous fluorescence release HDAC deacetylase assay. For each assay, samples from 3 independent experiments were analyzed. Equal number of EVs were solubilized and subjected to analysis as published previously [20 ,27 ]. In short, after samples (1.7 × 10¹⁰ EVs) were separated by electrophoresis on 4–20% Criterion^™ TGX^™ Precast Gels (Bio-Rad Laboratories, Inc), and proteins transferred to PVDF membranes, membranes were probed with primary antibodies against annexin-A2 (1:1000; ab41803, Abcam) or fibronectin (1:1000; 610077, BD Bioscience) followed by visualization with horseradish peroxidase-conjugated secondary antibodies and Clarity^™ Western ECL Blotting Substrate (Bio-Rad Laboratories) with chemiluminescent detection, using 10 s exposure times [27 ]. The glypican 1 ELISA was carried out according to manufacturer instructions (ELH-GPC1–1, Ray-BioTech) using a microplate reader, analyzing 1.38 × 10¹¹ EVs per sample as published previously [27 ]. HDAC activity was measured with a homogenous fluorescence release HDAC deacetylase assay, incubating EVs (1 × 10⁹ per reaction) with (AMC)-K(Ac)GL-Ac substrate to assess class I HDACs (HDACs 1, 2, 3, 6 and 10) in the presence of MS-275 to block HDAC 1, 2 and 3 activities [33 (link)] and activity was calculated as reported [34 (link)].

The glypican 1 ELISA was carried out according to manufacturer instructions (ELH-GPC1-1, RayBioTech). EVs pellets were dissolved in ice cold PBS buffer by passing the sample through a syringe tip (20x). Equal numbers of EVs (1.38 × 10¹¹) were added to each well of a 96 well plate and incubated overnight to enhance binding. Measurements were obtained at 450 nm using the microplate reader.