Rac1 antibody

The cells were lysed in an ice bath using a lysis buffer (50 mM Tris-Hcl + 150 mM ph7.4 Nacl + 1%Nonidet P-40). Protein concentrations were determined using a BCA protein assay kit (Thermo Scienti c Cat# 23225) with bovine serum albumin as the standard. The loading sample was prepared via mixing with the 4x Sample buffer (Thermo Scienti c Cat#NP0007) and the 10x Sample Reducing agent (Thermo Scienti c Cat#NP0009). Proteins (20 µg) were resolved on 16.5% polyacrylamide gels (p-PAGEL Cat# 2332260, ATTO, Tokyo, Japan) and transferred onto PVDF membranes (Q-Blot Kit ATTOCat#2322443). The membranes were blocked with 5% Skim milk in TBST at room temperature for 1 h. The Rac1 antibody (1:1000; Abcam Biotechnology) was incubated overnight at 4℃ and then washed and incubated with appropriate secondary antibodies (1;5000, anti-rabbit IgG, HRP-linked antibody Cat#7074S, Cell Signaling Technology, Danvers, Massachusetts, USA) at room temperature for 1 h. The results were visualized by immobilon Western chemiluminescent HRP substrate Cat (#WBKLS0500). Each band was normalized with β-Actin. Quantitative Rac1 analyses were performed using Empiria studio software (LI-COR, Nebraska, USA)