Figure 3: ALKBH4 mediates demethylation of K84me1 actin in vivo regulating actomyosin interaction.

(a) U2OS cells were simultaneously transfected with siCTRL or ALKBH4 siRNA, together with the indicated siRNA insensitive Flag-GFP-ALKBH4 constructs lysed, 48 h later cells were analysed by immunoblotting analysis using the indicated antibodies. (b) U2OS cells were transfected with WT or catalytically inactive ALKBH4 (HDH) and lysates were immunoblotted with the indicated antibodies. (c) Control Alkbh4^+/+Cre (Alkbh4^+/+) and Alkbh4^L/LCre MEF (Alkbh4^Δ/Δ) cells were lysed and analysed by immunoblotting with the indicated antibodies. (d) 293T cells were co-transfected with Flag-GFP-ALKBH4 WT or HDH and HA-β-actin as indicated and lysates were subjected to Flag immunoprecipitation and immunoblotted with the indicated antibodies. (e) 293T/GFP-NM II or 293T/GFP-ALKBH4 stable cell lysates were subjected to GFP immunoprecipitation. Immunoprecipitates and WCEs were immunoblotted with the indicated antibodies. (f) 293T cells were transfected with WT or K84R HA-β-actin as indicated and lysates were subjected to HA immunoprecipitation. Immunoprecipitates and WCEs were immunoblotted with the indicated antibodies. Quantification of signal intensity of K84R relative to WT. (g) Forty-eight hours after transfection with the indicated DNA constructs, 293T cells were treated with 20 μM AdOx (adenosine-2′, 3′-dialdehyde) for 24 h, lysed and subjected to HA immunoprecipitation. Immunoprecipitates were analysed by immunoblotting with the indicated antibodies. Full-length blots in all panels are presented in Supplementary Fig. S7.