Ab203896

2.5 × 10⁴ HeLa S3 cells were seeded on glass coverslips in 24-well plates and transfected 16 h later with 10 nM VPS35 or control siRNA. Forty-eight hours after transfection, cells were mock-infected or infected with wild-type HPV16 PsV at MOI of 150 or with DM mutant PsV containing the same number of encapsidated HcRed reporter plasmids. At 12 h.p.i., cells were fixed for 15 min at room temperature with 4% paraformaldehyde, permeabilized with 1% saponin for 30 min, and incubated with 1:400 anti-FLAG mouse antibody (Sigma, F3165), 1:100 anti-EEA1 mouse (BD, 610457) or rabbit (CST, 2411) antibody, 1:75 anti-TBC1D5 rabbit (Abcam, ab203896) or mouse (Santa Cruz, SC-376296) antibody, 1:100 anti-VPS35 goat antibody (Abcam, ab10099), or 1:120 anti-VARP rabbit antibody (Abcam, ab108216). Cells were then incubated at room temperature for 1 h with 1:200 AlexaFluor-conjugated secondary antibodies (Life Technologies). The slides were mounted as above, and images were captured using a Leica SP5 confocal microscope. In most experiments, a single Z-plane is shown. In Figure 5C, right panels, sequential Z-planes spaced by ~0.2 micron were recorded for three-dimensional reconstruction with the Imaris software package.