Quantitative Protein Expression Analysis

Cells were lysed with RIPA buffer (Thermo Scientific) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein concentration was determined using the BSA Protein Assay (Thermo Fisher Scientific). Western blots were performed using NuPAGE 4–12% Bis-Tris Protein Gels (Invitrogen) as described before [26 (link)]. Briefly, gels were run in MES buffer (Invitrogen) and wet transferred onto the nitrocellulose transfer membrane. The following primary antibodies were used: PDCD4 (Cell Signaling, 9535), and ß-actin (Cell Signaling, 3700). Goat anti-rabbit IgG (H + L) 800 CW or goat anti-mouse (H + L) 680RD was applied for 45 min at room temperature (1: 25000, LI-COR) before washing with PBS containing Tween 20. Blots were imaged using an Odyssey Infrared Imaging System Scan and quantification was carried out with the LI-COR Odyssey® scanner and software (LI-COR Biosciences).