Anti vinculin e1e9v xp

Primary antibody dilutions were prepared at 1:1000 to 1:500 in 5% BSA or dry milk in TBS with 0.05% v/v Tween. Protein samples prepared directly from lysate were prepped in 5X SDS sample buffer. Westerns were run in BioRad equipment in SDS buffer, using BioRad gradient gels, at 175 V for 42 min. Precision plus dual color protein marker (BioRad) was used in parallel to samples. Proteins were wet-transferred onto nitrocellulose membranes (0.2 µM, BioRad) at 100 V for 1 h. Membranes were blocked in 5% BSA or milk for 1 h RT depending on antibodies to be used. Membranes were incubated with primary antibodies overnight at 4° and washed for a total of 30 min in TBST before adding secondary diluted to 1:10,000 through 1:5,000 in 5% milk. Antibodies used in immunoblots are as follow: Anti-USP15 (Proteintech 14354-1-AP) used at 1:500-1:1000, Anti-Vinculin E1E9V XP (Cell Signaling Technologies #13901) used at 1:1000, Anti-KEAP1 P586 (Cell Signaling Technologies #4678) used at 1:500, Anti-NRF2 (Cell Signaling Technologies #12721) used at 1:500, anti-GAPDH D16H11 XP (Cell Signaling Technologies #5174) used at 1:1000, anti-pan-Actin (Cell Signaling Technologies #4968) used at 1:1000.

Cells were lysed at 4 °C using ice cold lysis buffer containing 30 mM Tris/HCl pH 6.7, 5% glycerol, 2.5% β-mercaptoethanol, and 1% SDS. Protein extracts were analyzed by SDS-PAGE and western blotting. Enhanced chemiluminescence (ECL) signals were detected as described before [38 (link)]. The following antibodies were used for western blot: anti-AXL C89E7 (Cell Signaling Technology, Boston, MA, USA), anti-CD68 (Cell Signaling Technology, Boston, MA, USA), anti-DICER1 clone D38E7 (Cell Signaling Technology, Boston, MA, USA), anti-SPRY4 (GeneTex, Irvine, CA, USA), pan-AGO clone 2A8 (Millipore, Overijse, Belgium), anti-vinculin E1E9V XP (Cell Signalling Technology, Boston, MA, USA), anti-alpha-tubulin (Santa Cruz, Heidelberg, Germany). HRP-labelled secondary antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).