Fig. 3

Validation of TRAP clients by western blot and quantitative RT-PCR. The color coding follows Fig. 1. a–c HeLa cells were depleted of TRAP- or Sec61-complex using two different TRAPB-targeting siRNAs, or SEC61A1-UTR-targeting siRNA, or treated with a non-targeting (control) siRNA, and the consequences of complex depletion were analyzed by quantitative RT-PCR and western blots for TRAPβ and TRAP client candidates. a Quantitative RT-PCR data represent the mean mRNA values relative to control and the corresponding dot plots for nine replicates for each siRNA from three independent experiments. b, c Quantitative western blot data represent the mean protein levels (normalized to ß-actin) relative to control and standard errors of the mean (s.e.m.) for five to six (b) or three (c) independent experiments. c Silencing phenotypes were rescued by the indicated complementation and analyzed by western blot. In the case of TRAPβ, the upper band represents the tagged protein and the numbers refer to the sum of tagged and un-tagged protein. d Control fibroblasts (co) as well as TRAP-deficient fibroblasts from CDG patients with mutations in TRAPG (G, one patient) or TRAPD (D, two patients) genes²⁷ were analyzed by quantitative proteomics