Polyvinylidene difluoride pvdf membrane

Protein pellets from the TRI Reagent® extraction were dissolved in 2% SDS containing 8 M urea or cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Merck, R0278) containing protease inhibitors (Merck, P0044). Samples (∼10 µg total protein) were separated by polyacrylamide gel electrophoresis using Bolt^™ or NuPAGE^™ 12%, Bis-Tris, 1.0 mm, Mini Protein Gels (Thermo Fisher Scientific, NW00127BOX or NP0349BOX). Following electrophoresis, protein was transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, 88520). Membranes were then rinsed with PBS, fixed for 20 min with 4% paraformaldehyde in PBS and then washed (4 × 5 min) with PBST [PBS containing 0.01% (v/v) Tween^™-20]. Membranes were then incubated with block solution (PBST containing 2% (w/v) bovine serum albumin (BSA) and 0.005% (w/v) thiomersal for 1 h and were then incubated with rabbit monoclonal antibodies against α-synuclein and β-actin overnight diluted in block solution (Table 1). Protein bands were visualized by 3,3′-diaminobenzidine/peroxidase reaction or with enhanced chemiluminescence (ECL) plus reagents (Merck, GERPN2232), digitized with a ChemiDoc^™ MP imaging system (Biorad) and analysed using ImageJ v1.53u (RRID:SCR_003070, https://imagej.net/). A detailed protocol is available via this link: dx.doi.org/10.17504/protocols.io.bp2l69pqdlqe/v1.

Cell lines were harvested in log-phase growth, seeded at a density of 3 × 10⁵cells/mL (THP-1) or 5 × 10⁵cells/mL (OCI-AML3) and incubated with the indicated drugs for up to 48 h, as indicated. Cells were lysed in the presence of protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA). Whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Inc., Rockford, IL, USA) and immunoblotted as previously described24 (link)26 (link)30 (link)31 (link). Immunoreactive proteins were visualized using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE, USA), as described by the manufacturer. Western blots were repeated at least three times and one representative cropped blot is shown.

To investigate protein levels related to osteogenic, chondrogenic, and adipogenic differentiation or to autophagy in in vitro experiments, we performed Western blot analyses. Protein extraction was performed using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. A BCA assay (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine protein concentrations. Protein samples were diluted in 4x Laemmli's sample buffer (Bio-Rad, CA, US), heated for 5 min at 95°C, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE), using a mini-PROTEAN® TGX™ Precast gradient 4-20% gel (Bio-Rad, CA, US), followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). PVDF membranes were probed with the primary and secondary antibodies reported in Table 1. Signals were detected using a chemiluminescence reagent (ECL, Millipore, Burlington, MA, USA) according to the manufacturer's instructions. Images were acquired by a LAS4000 Digital Image Scanning System (GE Healthcare, Little Chalfont, UK). Densitometric analysis was performed by using ImageQuant software (GE Healthcare, Little Chalfont, UK), and the relative protein band intensity was normalized to β-actin.

Proteins were extracted by using Ripa buffer (Thermo Fisher Scientific, Waltham, MA,USA) and concentrations were calculated with BCA assay (Thermo Scientific, Waltham, MA, USA) as previously reported [22 ]. Protein samples were diluted in Laemmli’s sample buffer (Biorad, CA, US), heated for 5 min at 95 ◦C, and separated by sodium dodecyl sulfate − polyacrylamide gel electrophoresis (SDS PAGE), followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). PVDF membranes were probed with the primary (antiMyoD (MA1-41017; Thermo Scientific, Waltham, MA, USA), β ACTIN (BA3R; Thermo Scientific, Waltham, MA, USA); and secondary antibodies ( Anti-mouse (Cell Signaling, 7076). Signals were detected with a chemiluminescence reagent (ECL, Millipore, Burlington, MA, USA) and Images were recorded using a LAS4000 Digital Image Scanning System (GE Healthcare, Little Chalfont, UK). Densitometric analysis was performed as we previously reported [24 (link)].