Nupage bis tris gel system

Cells were homogenized in cold RIPA buffer, and total protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Segrate, Italy). Equal amounts of proteins (30 µg) were run in 10% polyacrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) membrane. Anti-LC3 primary antibody (Sigma-Aldrich, Milan, Italy Cat# L8918, RRID: AB_1079382) and secondary HRP-labeled anti-rabbit antibody (Promega, Milan, Italy Cat# W4011, RRID: AB_430833) were used. Densitometric analysis of the bands was performed using NIH ImageJ Software V1.52a. To evaluate secreted MMP2 levels, the conditioned medium was obtained as described in [51 (link)]. Briefly, equal amounts of untreated and Pg-treated cells were seeded in serum-free medium (1 × 10⁶ cells/mL medium). After 24 h, the conditioned medium was collected, stored at −80 °C for at least 24 h, and subsequently freeze-dried. The residues were resuspended in the same volume of PBS. Next, proteins were separated by electrophoresis on a 4–12% NuPAGEbis-tris gel system (Life Technologies, Monza, Italy), electroblotted to a PVDF membrane, and finally detected using an anti-MMP2 primary antibody (Proteintech, Planegg-Martinsried, Germany Cat# 10373-2-AP, RRID: AB_2250823).

NCI-H295R cells were treated with IC50 value of trabectedin for 4 days. Whole cell lysates were prepared in ice-cold buffer with protease and phosphatase inhibitor cocktails (Roche, Milano, Italy) as previously described [25 (link)]. Fractionated cell lysates were prepared according to [47 (link)]. Equal amounts of proteins were separated by electrophoresis on a 4–12% NuPAGE Bis-Tris Gel System (Life Technologies, Milano, Italy) and electroblotted to a nitrocellulose membrane. Primary and secondary antibodies are listed in Table 4. Signal was detected and quantified with the Odyssey® CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).