Goat anti rabbit igg hrp antibody

Cells in each group were lysed with NP-40 lysate (Beyotime Institute of Biotechnology) including 1% phenylmethanesulfonyl fluoride (PMSF). The concentration of total proteins was quantitated by a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology). Then equal amounts (20 µg) of different proteins were loaded and separated using SDS-polyacrylamide gel electrophoresis (PAGE) followed by electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After being blocked with 5% non-fat milk at 4°C overnight, the membranes were probed with specific primary antibodies against VEGFR-2, p-VEGFR-2 (Tyr1175), ERK1/2, p-ERK1 (pT202/pY204) + p-ERK2 (pT185/pY187) (all 1:1,500 diluted); and VEGF-A, MEK1, p-MEK1 (pS298) (all 1:1,000 diluted; Abcam, Cambridge, MA, USA Abcam) at 4°C overnight. The membranes were then incubated with the secondary goat anti-rabbit IgG-HRP antibody (1:20,000 diluted; Wuhan Boster Biological Technology, Ltd., Wuhan, China) at 37°C for 40 min. The unbound antibodies in each step were washed with TBST for three times. The target bands were visualized by an enhanced chemiluminescence (ECL; Millipore), and the protein intensities were detected by Gel-Pro Analyzer software (Media Cybernetics, Inc., Bethesda, MD, USA). GAPDH was used as an internal control.

Total proteins in NCI-H1299 cells were lysed by NP-40 lysate (Beyotime Institute of Biotechnology) containing 1% phenylmethanesulfonyl fluoride (PMSF). The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology). 20 µg proteins in each sample was loaded and separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The blots were then blocked with 5% non-fat milk overnight at 4°C and incubated at 4°C overnight with the specific primary antibodies as follows: anti-VEGFR-2, anti-ERK1/2, anti-p-ERK1 (pT202/pY204)+p-ERK2 (pT185/pY187) (all 1:1,500 diluted; Abcam, Cambridge, MA, USA); anti-MEK1, anti-p-MEK1 (pS298) (both 1:1,000 diluted; Abcam). After washed with TBST for three times, the membranes were incubated with a secondary goat anti-rabbit IgG-HRP antibody (1:20,000 diluted; Wuhan Boster Biological Technology, Ltd., Wuhan, China) at 37°C for 40 min. An enhanced chemiluminescence (ECL; EMD Millipore) detection method was employed to visualize the target bands, and relative protein intensities were analyzed by Gel-Pro-Analyzer software (Media Cybernetics, Rockville, MD, USA). GAPDH was used as an internal control.