Fig. 3.

Stalled proteins interact with and regulate the HB complex. (A) Effects of depletion of LTN1, NACA, or DNAJC2 (ZUO1) on the suppression of LATS1/2 upon proteasome inhibition. MCF10A cells with the indicated depletions were incubated for the indicated times with 3 μM MG132, and the lysates were analyzed by immunoblot. (B) Effects of proteasome inhibition and depletion of LTN1 or NACA on levels of GFP-NS. One day after siRNA transfection, HeLa cells were transfected with plasmids encoding either GFP-NS or WT EGFP (this plasmid was 10-fold diluted with an empty vector). Indicated samples were treated for 4 h with 5 μM MG132. (C) The effects of LTN1 or NACA depletion on the association of GFP-NS with the HB complex were assessed by the Duolink assay. HeLa cells transfected for 1 d with one of the indicated siRNAs were transfected with a mixture of plasmids encoding GFP-NS and FLAG-Bag3 (3:1) and 1 d later were treated with MG132 or were left untreated. The cells transfected with only the GFP-NS–encoding plasmid or only the FLAG-Bag3–encoding plasmid were used as negative controls and did not produce any measurable signal. The cells grown on a glass-bottomed plate were treated according to the Duolink protocol with anti-GFP and anti-FLAG antibodies. Images of the Duolink signal and of DAPI nuclear staining were taken from at least 10 randomly chosen images with a fluorescence microscope with a 20× objective. Duolink images were quantified using NIH ImageJ software, and the data were normalized by the total number of cells in all fields chosen for each sample. The error bars represent the SD. For images of cells, see SI Appendix, Fig. S3. (D) Effects of LTN1 or NACA depletion on the association of GFP-NS with the HB complex assessed by the modified ActivSignal protocol. The cells were grown and treated as in Fig. 3B, but here the cells were plated on a plastic 96-well plate for incubation with MG132 and for a following protein–protein interaction assay (Materials and Methods). The qPCR results are presented. The results for a signal produced with the pair of anti-GFP and anti-FLAG antibodies (reflecting the amount of GFP-NS associated with FLAG-Bag3) were normalized by a signal produced with the pair of anti-Bag3 and anti-FLAG antibodies (reflecting the relative amount of FLAG-Bag3) for the same cells. The signals generated from the cells transfected only with a vector were used as a baseline. The error bars represent the SD. For the effect of LTN1 depletion (compared with control), P < 10⁻⁶; for NACA, P < 10⁻⁵.