Hrp labeled goat anti rabbit igg secondary antibody

BEAS2B cells treated with RIPA lysis buffer, were homogenized for the collection of cell supernatant, and then total proteins were extracted according to the manufacturer’s recommendations. Quantification of proteins was performed by BCA protein quantification kits (ab102536; Abcam). Then, the protein samples were separated by 12% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes. Nonspecific binding was blocked with 5% skim milk in Tris-buffered saline (TBS) at room temperature for 4 h. The membranes were then incubated with the primary antibodies (p-MYPT1, sc-377,542; total-MYPT1, sc-514,261; p-p65, sc-166,748; p-65, sc-8008; 1:1000. SANTA CRUZ BIOTECHNOLOGY, INC. RhoA, ab187027, 1:5000; TLR4, ab13556, 1:500; pCox2, ab179800; Bax, ab32503; Bad, ab32445; cleaved caspase3, ab32042; Bcl-2, ab32124; 1:1000. GAPDH, ab8245, 1:5000, abcam, England) at 4°C overnight, following which was TBS washing for three times and the incubation with an HRP-labeled goat anti-rabbit IgG secondary antibody (1:10,000, abcam, England) at room temperature for 1.5 h. The bands were visualized using an enhanced chemiluminescent detection system (Bio-Rad, Hercules, CA, USA). Band intensities were determined using the ImageJ software (National Institutes of Health, Bethesda, MA, USA).

According to our previous protocol [7 (link)], the proteins were extracted and separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). And the membranes were separately incubated with anti-ITGB3 antibody (Abcam, USA), anti-GAPDH or anti-ACTIN antibody (Abcam, USA), or HRP-labeled goat anti-rabbit IgG secondary antibody (Abcam, USA). Pictures of the film strips were taken by using the gel imaging and analysis system (LabWorksTM) (UVP, USA) and the brightness values of the strips were analyzed.