Figure 2. SFMBT1 stability is regulated by pVHL through EglN1 hydroxylation.

(A) Immunoprecipitaiton of cell lysates for the hydroxylated SFMBT1 from cells treated with MG132 or MG132 plus DMOG treatment.

(B) Immunoprecipitaiton and immunblots of cell lysates treated with of indicated drugs overnight.

(C) Immunoprecipitaiton and immunblots of lysates from cells transfected with indicated plasmids followed by MG132 treatment.

(D) Binding between FLAG-pVHL and HIF1α or SFMBT1 peptides.

(E-F) Immunoprecipitaiton of lysates for the hydroxylated SFMBT1 in cells transfected (E) or transduced with plasmids or lentivirus indicated (F).

(G) Effect of HA-pVHL on ubiquitination of endogenous SFMBT1.

(H) Ubiquitination level of wide type HA-SFMBT1 (WT), P651A and P106A.

(I) Immunoblots for lysates of transduced HA-SFMBT1 and P651A in UMRC2 transduced with lentivirus expressing FLAG-pVHL (FLAG-VHL-UMRC2) followed by treatment with indicated drugs.

(J) GST-pull down assay between GST (EV) or GST-EglNs (EglN1, EglN2 and EglN3) and in vitro translated (IVT) HA-SFMBT1.

(K) Capture of biotinylated HIF1α (left panel) and SFMBT1 (right panel) peptides by FLAG-VHL after in vitro hydroxylation by IVT EglN1 (wide-type) or EglN1 catalytic dead variant (H314VD316/A314VA316, EglN1-CD). HIF1α peptide was used as positive control. Capture of peptides by FLAG-pVHL indicates hydroxylation of peptides by EglN1.

(L) Hydroxylation level of endogenous SFMBT1 in cells transfected with HA-EglN1 or EgLN1-CD, followed by MG132 or MG132 plus DMOG treatment.

(M) Immunoblots of Immunoprecipitated samples to detect endogenous SFMBT1 hydroxylation in cells with EglN1 knock down (sh1) as well as these cells restored with HA-EglN1 or catalytic dead mutant (HA-EglN1-CD).

(N) Immunoblots for lysates of transduced HA-SFMBT1 and P651A protein level in FLAG-VHL-UMRC2 cells transfected with control siRNA (Ctrl) or EglN1 siRNAs (si-1, si-2 and si-3).