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. 2020 Mar 16;111(4):1266–1278. doi: 10.1111/cas.14348

Figure 3.

Figure 3

ERK phosphorylates zinc finger protein 148 (ZNF148) at Ser306. A, Gastrointestinal stromal tumor (GIST)‐T1 cells with SDHB depletion were treated with or without EGF (100 ng/mL) for 1 h. The immunoprecipitates were treated with CIP (10 units), followed by immunoblotting analysis. B, Purified GST‐Forkhead box M1 (FOXM1) protein was mixed with cellular extracts from SDHB‐depleted GIST cells treated with EGF (100 ng/mL) for 1 h (left panel). Purified GST‐ZNF148 protein was mixed with cellular extracts from SDHB‐depleted GIST cells treated with EGF (100 ng/mL) for 1 h. C, In vitro phosphorylation analyses were performed by mixing purified active ERK with the indicated purified GST‐ZNF148 proteins in the presence of [γ‐32P] ATP. D, Ser306 of ZNF148 is evolutionarily conserved in the indicated species (left panel). In vitro phosphorylation analyses were performed by mixing the purified active ERK with the indicated purified GST–ZNF148 proteins in the presence of [γ‐32P] ATP (right panel). E, GIST‐T1 cells with SDHB depletion were expressed with WT MEK or MEK1 Q56P constitutively active mutant and WT ERK or ERK K52R kinase‐dead mutant. F, GIST‐T1 cells expressed with Flag‐tagged WT ZNF148 or ZNF148 S306A were treated with or without EGF for 1 h. G, Purified GST‐ZNF148 protein was mixed with or without purified active ERK prior to the incubation with His‐FOXM1 protein. H, GIST‐T1 cells with SDHB depletion, ZNF148 depletion and reconstituted expression reconstituted expression of WT ZNF148 or ZNF148 S306A were treated with or without EGF (100 ng/mL) for 12 h. Q‐PCR (left panel) or immunoblotting analyses (right panel) were performed. I, GIST‐T1 cells with SDHB depletion, ZNF148 depletion and reconstituted expression of WT ZNF148 or ZNF148 S306A were treated with or without EGF (100 ng/mL). Cell invasion assays were performed. In A‐H, immunoblotting analyses were performed using the indicated antibodies. In H and I, the values are presented as mean ± SEM (n = 3 independent experiments), ** represents P < 0.01 between the indicated groups