Isolation of mRNP complex by THOC5 as bait. pNTAP (TAP-Vector, [CBP] calmodulin-binding peptide; [SBP] streptavidin-binding peptide) and pNTAP carrying myc-tagged THOC5 (TAP-THOC5) (A) were transfected into MEF THOC5 flox/flox. After 24 h, RNP complex was isolated using streptavidin Sepharose. (B) Cell extract (input) and binding fractions with streptavidin Sepharose (binding) were analyzed by immunoblotting using THOC5-, Actin-, THOC1-, and GAPDH-specific antibodies. (C) The same samples were analyzed: Gigyf2-, Prkcbp1-, c-Myc-, actin-, or (37°C) Hsp70-specific RT-PCR. In addition, MEF THOC5 flox/flox cells were transfected as in B and were then incubated for 2 h at 42°C. After isolation of RNP, samples were applied for Hsp70 (42°C) RT-PCR. (D) The depletion of THOC5 causes down-regulation of the Hsp70 gene export under heat shock conditions. MEF THOC5 flox/flox cells were infected with Ad-GFP or Ad-GFP-Cre virus for 4 d. Cells were incubated for 2 h at 37°C or 42°C, and RNAs were then isolated from cytoplasmic fraction followed by Hsp70- or actin-specific RT-PCR.
