Ab18199

Proteins were extracted from cells using ice-cold RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors and phosphase inhibitors. The protein concentrations were measured by BCA method. Then 20 μg of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a 0.45 μm PVDF membrane. The membrane was blocked with 5% skim milk for 1 h and incubated with primary antibodies overnight. After washing with PBS, blots were incubated with secondary antibodies. Images were acquired by enhanced chemiluminescence. GAPDH antibody was used as a control. Results were treated with gray analysis by Gel-Pro Analyzer (United States Biochemical, Cleveland, OH). The semi-quantitative analysis was performed according to the relative expression of objective protein and GAPDH.
The primary antibodies used in this study were as follow: anti-CAV1 (1:1000, Rabbit polyclonal antibody, ab18199, Abcam, Cambridge, MA, USA); anti-AKT (1:500, Mouse monoclonal antibody, #9272, Cell Signaling Technology, Beverly, MA, USA); anti-p-AKT (1:1000, Mouse monoclonal antibody, Ser473, 66,444–1-Ig, Proteintech, Manchester, UK); anti-Cyclin D1 (1:1000, Rabbit monoclonal antibody, ab16663, Abcam); anti-P70 (1:1000, Rabbit monoclonal antibody, ab184551, Abcam); anti-GAPDH (1:500, Mouse monoclonal antibody, ab8245, Abcam).

FITC-conjugated lectins from A. integrifolia (Jacalin; FL-1151) and D. stramonium (DSA; FL-1181) were purchased from Vector Laboratories (Burlingame, CA, USA) and FITC-conjugated lectins from A. caudatus (ACA; 21,510,290) were purchased from GlycoMatrix (Dublin, OH, USA). Colloidal gold-conjugated lectins Jacalin-15 nm gold (GP-6301–15), ACA-10 nm gold (GP-8201–10) and DSA-5 nm gold (GP-5701–5) were purchased from EY Laboratories (San Mateo, CA, USA). Lectin inhibitory molecule bovine submaxillary mucin for ACA, galactose for jacalin and chitin hydrolysate for DSA were also purchased from Vector Laboratories. Rabbit polyclonal anti-uroplakin (UP) primary antibody was a kind gift from Prof. Dr. Tung-Tien Sun, Department of Cell Biology, New York University Medical School (Wu et al. 1990 (link)). The following rabbit polyclonal antibodies were also used: anti-flotillin (ab41927, Abcam), anti-clathrin (610,499, BD Transductions), anti-caveolin (ab18199, Abcam) and anti-dynamin 2 (ab65556, Abcam). Goat anti-rabbit IgG AlexaFluor 488 (A11008, Invitrogen) and goat anti-rabbit IgG AlexaFluor 555 (A21428, Invitrogen) secondary antibodies were used. Vectashield mounting medium with DAPI, a nuclei marker, was obtained from Vector Laboratories.