Figure 3: C/EBPδ stability is regulated by GSK-3β phosphorylation.

(a) RAW 264.7 cells were transfected with WT- or TTS/AAA-CEBPδ expression plasmids and treated ± GSK-3β inhibitor CHIR99021 (5 μM, 2 h). Cell extracts were immunoprecipitated with anti-C/EBPδ or IgG (with an aliquot of the indicated extract) and the western analysed with anti-phosphothreonine (pT) antibody. (b) Western analysis of NE from PPMs (left panel) or RAW 264.7 cells (right panel) treated with GSK-3β inhibitors CHIR99021 or BIO (6-bromoindirubin-3′-oxime) (5 μM, 2 h). β-catenin, which is known to be targeted for degradation by GSK-3β phosphorylation, served as positive control. (c) Western analysis of RAW 264.7 cells transfected with WT- or TTS/DDD-C/EBPδ expression plasmids and treated ± GSK-3β inhibitor CHIR99021 for 2 h. (d) GSK-3β phosphorylates C/EBPδ in vitro. HEK293T cells were transfected with WT- or TTS/AAA-C/EBPδ expression plasmids. C/EBPδ proteins were immunoprecipitated from cell extracts and in vitro kinase assay reactions were carried out in the presence or absence of GSK-3β. Samples were resolved by SDS–PAGE and subjected to autoradiography (top panel). Total radioactivity (bottom panel) incorporated into the C/EBPδ protein was quantified (n=3). Representative input levels of C/EBPδ are shown by western (inset). (e) RAW 264.7 cells were treated ± LPS (4 h) and cell extracts were immunoprecipitated with anti-C/EBPδ or IgG antibodies and western analysis was carried out with anti-phosphothreonine (pT) antibody. Input (2.5% of the lysate) was analysed as indicated. (f) Western analysis (top panel) of RAW 264.7 cells treated ± LPS (100 ng ml⁻¹, 4 h) followed by CHX for the indicated times, and quantification of C/EBPδ normalized to β-actin signal (bottom graph) compared with respective untreated (n=3, *P<0.05; **P<0.001; ****P<0.0001). (g) Western analysis of RAW 264.7 cells transfected with HA-GSK-3β-S9A expression plasmids treated ± LPS (4 h) as indicated. (h) Western analysis of RAW 264.7 cells pretreated with the PI3K/AKT kinase pathway inhibitor LY294002 (10 μM, 1 h) followed by LPS (100 ng ml⁻¹, 4 h) as indicated. Where applicable, data are mean±s.e.m., evaluated by two-tailed unequal variance t-test.