Figure 2: Interaction of ALKBH4 with the actomyosin network and identification of actin K84 monomethylation.

(a) Association between Flag-GFP-ALKBH4 and NM II assessed in 293T cells by immunoprecipitation. 293T cells were transfected with Flag-GFPor Flag-GFP-ALKBH4 constructs as indicated and lyisted in NET buffer, which didn’t contain EDTA. In all, 2 mM ATP and 2 mM MgCl₂ were added into NET buffer freshly. WCE, whole-cell extract. (b) Association between endogenous ALKBH4 and actin assessed in MRC5 cells by co-immunoprecipitation analysis of ALKBH4 and actin using indicated antibodies. (c) WCEs from 293T cells transfected with HA-β-actin WT, K84A or K84R were immunoblotted with the indicated antibodies. (d) Detection of free and filamentous actin by actin fractionation. Semi-confluent 293T cells were treated with or without 1 μg ml⁻¹ CCD and 1 μg ml⁻¹ phalloidin for 30 min at 37 °C as indicated. Soluble free G-actin (S) and insoluble filamentous F-actin (P). (e) 293T cells were co-transfected with Flag-GFP-ALKBH4 either WT or K84R HA-β-actin as indicated and lysates were subjected to HA immunoprecipitation. Immunoprecipitates and WCE were immunoblotted with the indicated antibodies. (f) Forty-eight hours after transfection with the indicated DNA constructs, 293T cells were lysed and subjected to F-actin fractionation. Fractions were analysed by immunoblotting with the indicated antibodies. (g) Forty-eight hours after transfection with the indicated siRNAs, 293T cells were lysed and subjected to F-actin fractionation assay. Fractions were analysed by immunoblotting with the indicated antibodies. Full-length blots in all panels are presented in Supplementary Fig. S7.