Fig. 3.

USP28 deubiquitinates MAST1 protein. The ubiquitination and deubiquitination of ectopically expressed Myc-MAST1 were analyzed in HEK293 cells. A The HEK293 cells were transfected with Myc-MAST1 and HA-Ub in a constant amount. Flag-USP28 was transfected in an increasing concentration, followed by immunoprecipitation with Myc antibody and immunoblotting with anti-HA antibody. B The ubiquitination and deubiquitination of ectopically expressed Myc-MAST1 were analyzed by transfecting HEK293 cells with Flag-USP28 and Flag-USP28CA or treatment with DUB-inhibitor PR-619 for 48 h prior to harvest in the HEK293 cells. The cells were harvested, followed by IP with a Myc antibody and immunoblotting with an anti-HA antibody. C The ubiquitination and deubiquitination of ectopically expressed Myc-MAST1 were analyzed by transfecting HEK293 cells with sgRNAs targeting USP28. The cells were harvested, followed by IP with a Myc antibody and immunoblotting with an anti-HA antibody. A–C The relative protein expression of MAST1-(Ub)n with respect to input MAST1 was quantified using ImageJ software and represented as (MAST1-(Ub)n/MAST1) below the blot. D Sanger sequencing data showing the disruption in USP28 gene sequence in A549 (upper panel) and H1299 cells (lower panel). The effect of USP28-KO on the mRNA expression of E USP28 and F MAST1 was evaluated by qRT-PCR with specific primers. The relative mRNA expression levels are shown after normalization to GAPDH mRNA expression. Data are presented as the mean and standard deviation of three independent experiments (n = 3). A two tailed t-test was used, and P values are indicated. G Flow cytometry assay showing the expression of USP28 in mock control vs. USP28-KO in A549 cells (left panel) and H1299 cells (right panel). H Western blot analysis of the endogenous expression of USP28 and MAST1 protein in A549 and H1299 USP28-KO was evaluated. GAPDH was used as the internal loading control. I The TUBEs assay was performed to assess the ubiquitination status of the MAST1 protein in mock control and USP28-KO clones from A549 and H1299 cells. Cell lysates were immunoprecipitated with TUBEs beads, followed by immunoblotting with the indicated antibodies. J–K The effect of USP28-KO on the half-life of MAST1 in A549 cells. The mock control, USP28-KO and USP28-KO cells reconstituted with (J) Flag-USP28 and K Flag-USP28CA was treated with CHX (150 μg/mL) for the indicated time, and the cells were then harvested for western blotting with the indicated antibodies. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control