cTAGE5 interacts with Sec12 at ER exit sites. (A) Protein A beads conjugated with (lanes 1 and 3) or without (lane 2) cTAGE5 CC1 antibody were incubated with (lanes 2 and 3) or without (lane 1) HeLa cell lysates. Beads were washed, and proteins retained by beads were analyzed by SDS-PAGE followed by silver staining or Western blotting with TANGO1, cTAGE5 CC1, and Sec12 (clone 7A10) antibodies. HC, heavy chain. (B) HeLa cells were transfected with control or cTAGE5 siRNA (1,825). After 48 h, cell lysates were prepared and immunoprecipitated with the cTAGE5 CC1 antibody conjugated to protein A beads. Cell lysates and immunoprecipitants were resolved on SDS-PAGE and subject to Western blotting with TANGO1, cTAGE5 CC1, and Sec12 (clone 7A10) antibodies. (C) Protein G beads were untreated or conjugated with Sec12 antibody (clone 6B3) and then incubated with HeLa cell lysates. Beads were washed, and proteins retained by beads were analyzed by SDS-PAGE followed by Western blotting with cTAGE5 CC1 and Sec12 (clone 7A10) antibodies. (D) HeLa cells fixed with cold methanol were subject to staining with Sec12 (clone 6B3) antibody and cTAGE5 CT, TANGO1, or Sec16 antibody. Insets show magnification of the indicated regions. The Mander’s colocalization coefficient of Sec12 and cTAGE5, TANGO1, or Sec16 is shown in the bottom of the merged insets. n = 6. Means ± SEM. IB, immunoblot; IP, immunoprecipitation. Bars, 5 µm.