Fig. 7: UBE4B ubiquitinates the acetylated PSAT1 for proteasomal degradation.

a UbiBrowser was used to search for E3 ligases of PSAT1. b In H1299 cells, UBE4B was overexpressed to detect PSAT1 protein by Western blot. c H1299 cells were transfected with control Vector or HA-UBE4B plasmid. The mRNA levels of UBE4B and PSAT1 were detected by qPCR. Data represented as the average of three independent experiments (mean ± SD), ns: P > 0.05, ***P < 0.001. d H1299 cells were experimented with CHX (100 μg/mL) for various time points and were transfected with HA-UBE4B. The PSAT1 protein expressions were detected by Western blot. The relative PSAT1 expression compared with that of Tubulin was quantified. Data represented as the average of three independent experiments (mean ± SD), **P < 0.01. e, f H1299 cells were co-transfected with HA-UBE4B and Flag-PSAT1. The immunoprecipitations were blotted with anti-HA or anti-Flag antibodies. g In H1299 cells, endogenous interaction between PSAT1 and UBE4B was tested using anti-HA antibodies for Co-IP. The immunoprecipitations were blotted with anti-PSAT1 and anti-HA antibodies. h–j In H1299 cells, Flag-PSAT1 and HA-UBE4B plasmids were co-transfected. Co-IP was used to detect the total, K48-linked or K63-linked ubiquitination levels of PSAT1. k H1299 cells were co-transfected with Flag-PSAT1 and HA-UBE4B. Then TSA (1 μM) was added 12 h before sample collection. The immunoprecipitations were blotted with anti-Flag or anti-HA antibodies. The relative Flag expression compared with that of HA was quantified. l H1299 cells were transfected with Flag-PSAT1 plasmid alone or co-transfected with HA-UBE4B plasmids and were treated with or without TSA (1 μM). Co-IP was used to detect the ubiquitination level of PSAT1. m Proposed model for the protein stability regulation of PSAT1 by acetylation and ubiquitination.