Figure 2: CTIF forms a complex with DCTN1 and eEF1A1 and is localized to the aggresome upon UPS impairment.
From: Misfolded polypeptides are selectively recognized and transported toward aggresomes by a CED complex
(a) Silver staining of immunoprecipitates of FLAG-CTIF in HEK293T cells. The indicated bands were excised and subjected to LC-MS/MS. Identified proteins in each band are specified. Immunoglobulin heavy chain is marked with an asterisk (*). MW, molecular weight. (b) IPs of endogenous CTIF. Extracts of HEK293T cells untreated or treated with MG132 for 12 h were obtained and then treated with RNase A before IP. IPs were performed using either α-CTIF antibody or nonspecific rabbit IgG (rIgG). Western blotting of samples before and after IPs was performed using the indicated antibodies. Threefold serial dilutions of total-cell extracts were loaded in the four left-most lanes. n=2. (c) IPs of endogenous CTIF using the extracts of HEK293T cells depleted of eEF1A1 or DCTN1. The cells were treated with MG132 for 12 h before harvesting. Total-cell extracts were treated with RNase A. n=2. (d) RNA IPs of endogenous CTIF. As in b, except that the extracts of HeLa cells stably expressing misfolded CFTR-ΔF508 were not treated with RNase A. Western blotting (upper) of CTIF and qRT–PCR of CFTR-ΔF508 mRNAs (lower) were performed using samples from either before or after IP. The levels of CFTR-ΔF508 mRNAs were normalized to the levels of GAPDH mRNA. The relative ratio of normalized CFTR-ΔF508 mRNAs obtained from the IPs using rIgG was arbitrarily set to 1.0. Columns and error bars represent the mean and s.d. of three independent transfections and qRT–PCRs. n=3. **P<0.01. (e) Immunostaining for CFTR-ΔF508 protein (green), CFTR-ΔF508 mRNA (red) and GAPDH mRNA (yellow). n=2. Scale bar, 10 μm.
