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. Author manuscript; available in PMC: 2024 Nov 2.
Published in final edited form as: Mol Cell. 2023 Oct 10;83(21):3818–3834.e7. doi: 10.1016/j.molcel.2023.09.020

Figure 3. m6A modifiers of 7SK, METTL3, and ALKBH5 influence RNA Pol II serine 2 phosphorylation and transcription.

Figure 3.

(A and E) Western blots using anti-FLAG antibody demonstrate the overexpression of FLAG-METTL3 (WT and D395A) (A) and FLAG-ALKBH5 (WT and H204A) (E) in A549 cells, with actin as a loading control.

(B and F) Representative immunofluorescence images of A549 cells transfected with plasmids encoding FLAG-METTL3 (WT and D395A) (B) and FLAG-ALKBH5 (WT and H204A) (F).

(C and G) The cells were dual stained using RNA Pol II Ser2P (red) and FLAG (green) antibodies, with quantification of RNA Pol II Ser2P levels presented in (C) and (G), respectively. White arrows point to cells transfected with the plasmids.

(D and H) Western blots of METTL3 (D) or ALKBH5 (H) in A549 cells stably expressing shRNA against these proteins. Total RNA Pol II and RNA Pol II Ser2P levels are also detected, with actin as a loading control.

(I) EU incorporation and staining in A549 cells transfected with plasmids encoding FLAG-ALKBH5 (WT and H204A). White arrows point to cells transfected with the plasmids. Scale bars, 10 μm. See also Figure S2.