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. 2021 Dec 16;298(2):101496. doi: 10.1016/j.jbc.2021.101496

Figure 2.

Figure 2

Polyoma small T expression leads to thedown-regulationof DBC1 protein levels in aPP2A-dependentmanner.A, 293T cells were transfected with pCDNA3-PyST-HA-Flag (1.5 μg). After 48 h, the cell extracts were blotted for endogenous DBC1 using anti-DBC1 antibody and PyST with anti-HA antibody. B, graphical representation of relative DBC1 expression (endogenous) in PyST-transfected cells compared with control cells. Protein expression in control cells was arbitrarily taken as one in all of the Western blotting quantifications. C, 293T cells were cotransfected with pCDNA3 PyST-HA-Flag (1.5 μg) and myc-DBC1 (1.5 μg) constructs. Cell lysates were blotted for exogenously expressed myc-DBC1 using anti-myc tag antibody and PyST using anti-HA antibody. D, graph representing relative myc-DBC1 expression in the cells transfected with empty vector (control), myc-DBC1, and myc-DBC1/PyST. The values indicate mean ± SEM; n = 3 biological replicates. E, Western blotting of pTREX PyST-HA-Flag expressing cell extracts at different time periods of doxycycline addition using anti-DBC1 for detecting DBC1 (endogenous) and anti-HA antibodies for PyST. F, effect of PyST overexpression on exogenously expressing myc-DBC1 protein levels in pTREX PyST-HA-Flag cell line at different time points as seen by Western blotting. G, effects of overexpression of PyST on DBC1 mRNA at about 24 h of doxycycline addition. After cDNA synthesis, RT-qPCR was carried out using primers for DBC1 and Actin. Fold change was calculated by ΔΔCq method, and p-value was calculated using student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. The error bars represent mean ± SEM. H, 293T cells were transfected with PyST (1.0 μg), BC1075 (1.5 μg), or D44N (1.5 μg). HA-Flag-tagged PyST (WT and mutants) was immunoprecipitated by anti-Flag antibody, and the lysates were immunoblotted with indicated antibodies. HC:heavy chain of IgG. I, 293T cells were cotransfected with myc-DBC1 (2 μg) and different PyST-HA-Flag constructs like WT PyST (1.5 μg), BC1075 (1.5 μg) (PyST: PP2A-), or D44N (1.5 μg) (PyST: HSPs-) constructs. Western blot for DBC1 and PyST was carried out using indicated antibodies. J, U2OS cells were treated with different concentrations of okadaic acid (OA) (for 16 h) to inhibit PP2A. Endogenous DBC1 was detected with DBC1 antibody. K, PyST expressing cells were treated with chloroquine (50 μM). Twenty hours posttreatment, the cell lysates were obtained and used for Western blotting. Endogenous levels of DBC1 were detected using anti-DBC1 antibody. DBC1, deleted in breast cancer 1; HSPs, heat shock proteins; PP2A, protein phosphatase 2A.