Figure 5.

Knockdown of p125A perturbs the structural organization of the early secretory pathway. (A) HeLa cells were transfected twice with ON-Targetplus SMARTpool siRNA (Thermo Fisher Scientific) for p125A, Sec31A, Sec13, or nontarget control with a 24-h interval. 72 h after the initial transfection, cell lysates were prepared and resolved on SDS-PAGE, then subjected to immunoblot analysis using antibodies against Sec31A, p125A, Sec23A, Sec13, and actin. Molecular size markers are in kD. (B) HeLa cells silenced of p125A and Sec31A were double labeled with rabbit anti-p125A and mouse anti-Sec31A antibodies. Bar, 10 µm. (C) HeLa cells stably expressing GT-GFP were silenced of p125A (left), Sec31A (center), and Sec13 (right). The cells were then processed for indirect immunofluorescence to view GT-GFP as well as p125A, Sec31A, and Sec13, respectively. Bar, 10 µm. (D) HeLa cells stably expressing GT-GFP were transfected with nontarget, p125A-, or Sec13-specific siRNA. The dispersion and fragmentation of the Golgi marked by GT-GFP were quantified using Cell^P software (Olympus). The open bar represents cells treated with nontarget siRNA, filled bar indicates p125A knockdown cells, and striped bar shows the Sec13 knockdown cells. n = 15; *, P < 0.0001; **, P < 0.0001.