Skip to main content
. 2018 Jan 24;14(2):347–358. doi: 10.1080/15548627.2017.1407889

Figure 4.

Figure 4.

SQSTM1/p62 regulates colocalization of K33-linked polyubiquitinated protein to LC3 puncta. (A) Disappearing colocalization of PolyUb(K33)-FC with AsRED-SQSTM1/p62ΔUBA. MEFs were electroporated with PolyUb(K33)-FC vector and AsRED-SQSTM1/p62 (or SQSTM1/p62ΔUBA) plasmid. After 24 h, images were acquired using an FV10i confocal laser microscope. Scale bars: 20 µm. Data are representative of 2 independent experiments. (B) Affinity isolation assay showing ubiquitin chains binding to SQSTM1/p62. Ubiquitin chains bound to negative samples or GST-SQSTM1/p62. Ubiquitin chain input is shown as a control in the left lane of each GST affinity isolation experiment. Input of negative sample and GST-tagged SQSTM1/p62 is shown as a control in the left lanes. Data are representative of 3 independent experiments. (C) Modification of the interaction between K33-linked polyubiquitin chains and SQSTM1/p62 by ZRANB1. HEK293T cells were cotransfected with plasmids encoding MYC-K33Ub, FLAG-SQSTM1/p62, and siRNA against ZRANB1. After 24 h, cells were lysed and protein extracts were immunoprecipitated (IP) with FLAG antibody and immunoblotted (IB) for the indicated proteins. To confirm the knockdown efficiency of ZRANB1, cells were harvested for RT PCR 24 h after transfection. Data are representative of 2 independent experiments. Error bars indicate standard deviations. (D) Quantification of the number of punctate structures positive for PolyUb(K33)-FC and LC3 per cell. HEK 293T cells were cotransfected with PolyUb(K33)-FC vector and siRNA for SQSTM1/p62. After 20 h, cells were stained with anti-LC3 antibodies and analyzed by immunofluorescence microscopy. Immunoblots indicate the knockdown efficiency of SQSTM1/p62. In all, 30 cells from each indicated strain were analyzed. Data are representative of 3 independent experiments. *p < 0.05 in student's t-test. Error bars indicate standard deviations. (E) Schematic showing that SQSTM1/p62 puncta also colocalize with K33-linked polyubiquitin the same as K48- and K63-linked polyubiquitin. The interaction between SQSTM1/p62 and K33-linked polyubiquitin is modulated by ZRANB1. K33-linked polyubiquitin also colocalizes with LC3 puncta.