Cab39

Cells with 90% confluent were seeded into a six-well culture plate. After gene transfection or siRNA interference was performed, the cells were cultured for another 24 h. Then the cells with a concentration of 5 × 10⁴ cell/ml (500 μl/well of the cell suspension) were seeded into a 24-well culture plate. Each bottom of the 24 wells were pre-inserted a 15 mm round cover glass slide (NEST Biotechnology Co., China) and the cells were cultured for 24 h to form a confluent monolayer. The cells were washed with ice-cold PBS and fixed with 4% formaldehyde (Solarbio, Beijing, China) for about 20 min. After the permeability treatment with PBS containing 0.1% Triton X-100 (Solarbio, Beijing, China) for 15 min and blocked with 3% bovine serum albumin for 1 h at 37°C. Subsequently, the coverslips were incubated with CAB39 (1:50; Cell Signaling, USA) or GLUT1 (1:50; Proteintech) antibody at 4°C overnight. After incubation with CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) secondary antibody (SA00013-2, PTG, Beijing, China) for 1 h, the cover slips were mounted in Prolong Gold antifade reagent with DAPI (Beyotime, Shanghai, China) for 5 min, and coverslipped. Images were obtained on a confocal microscope (Carl Zeiss, Germany)

Western blot analysis was performed to quantify the amount of target protein. The Western blot has been described previously.^{16 (link)} HPV16 E6 (1:700, Abcam, Boston, MA, USA), HPV16 E7 (1:200, Bioss, Beijing, China), CAB39 (1:1000; Cell Signaling Technology, Beverly, MA, USA), PI3K(P85α) (1:1000; Proteintech, Wuhan, China), P-AKT(ser473) (1:1000; Proteintech), AKT (1:1000; Proteintech), GLUT1 (1:500; Wanleibio, China), and GAPDH (1:15000, Proteintech). After the membranes were further incubated with appropriate horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG (1:5000; Proteintech) at 37°C for 2 h, the immunosignal was detected by using ECL Western blot kit (advansta, USA). The bands were analyzed with BioImaging systems (UVP Inc., Upland, CA, USA).