Anti vinculin antibody

Freshly harvested cells were lysed in RIPA buffer. Protein concentrations were determined by Pierce BCA^™ Protein Assay Kit (Pierce). Proteins were separated via Mini Protean TGX stain free gel 4–15% (BioRad) and transferred to polyvinilydene difluoride membrane by using iBlot 2 PVDF Regular Stacks (Invitrogene) and iBlot system transfer system (LifeTechnologies).
Membranes were blocked in 5% BSA solution (Sigma). Primary antibodies were diluted following the manufacturer’s instructions: anti-Vinculin antibody (Cell Signaling) (1:1000) and antiCHD1 (Novus Biologicals) (1:2000).
Signals were developed by using Clarity Western ECL Substrate (BioRad) and Image Quant LAS4000 System (GEHealthCare).

Preparation of cell lysates and Western blot analysis were performed as previously described [5 (link)]. In brief, protein samples were run using SDS-PAGE and transferred to PVDF membranes. Membranes were blocked for one hour with either 5% skim milk or bovine serum albumin (BSA) at room temperature. Following blocking, the membranes were incubated with one of the following primary antibodies overnight at 4°C: anti-caspase-8 antibody (1:1000) raised in mouse (Cell Signalling, Cat. No. 9746 S, Danvers, MA, USA), anti-caspase-9 antibody (1:1000) raised in rabbit (Cell Signalling, Cat. No. 9502, Danvers, MA, USA), anti-caspase-3 antibody (1:2000) (Novus Biologicals, Cat. No. NB100-56709V2, Littleton, CO, USA), anti-β-actin antibody (1:1000) (Santa Cruz Biotechnology, Inc., Cat. No. sc-81178, Paso Robles, CA, USA), anti-p-Histone H2A.X (Ser 139) (γ-H2AX) antibody (Santa Cruz Biotechnology, Inc., Cat. No. sc-101696, Paso Robles, CA, USA), and anti-vinculin antibody (1:2000) raised in rabbit (Cell Signalling, Cat. No. 13901, Danvers, MA, USA)