Cells were transfected with small interfering RNA (siRNA) using Oligofectamine (Invitrogen) on days 1 and 2 and harvested on day 4. siRNA duplexes were synthesized by Dharmacon, the target sequences were: 5′-CCA GUC UUC UCU CGU CCU A-3′ (Tsg101), 5′-AGA GAC AAG UGG AGG UAA A-3′ (Hrs), both as previously described 18 (link), 5′-GTT CTT GCT CTA CGT CCT C-3′ (CD63), as previously described 16 (link). Negative control siRNA was used for control cells (Mammalian AllStar negative siRNA, Qiagen). Efficiency of knockdown was assessed by western blotting using anti-Hrs (Enzo Lifesciences), anti-Tsg101 (Genetex) and anti-CD63 (1B5, a generous gift from Mark Marsh, University College, London) antibodies.
For concurrent depletion and expression studies, HeLa cells were transfected with siRNA on days 1 and 2 and transfected with PMEL (a generous gift from Michael Marks, University of Pennsylvania) on day 3 using Lipofectamine 2000 reagent (Invitrogen), following manufacturer's guidelines.
For concurrent depletion and expression studies, HeLa cells were transfected with siRNA on days 1 and 2 and transfected with PMEL (a generous gift from Michael Marks, University of Pennsylvania) on day 3 using Lipofectamine 2000 reagent (Invitrogen), following manufacturer's guidelines.
Free full text: Click here
