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. Author manuscript; available in PMC: 2022 Feb 21.
Published in final edited form as: Cell Rep. 2020 Sep 29;32(13):108172. doi: 10.1016/j.celrep.2020.108172

Figure 3. Actin R256me1 Mark Is a Conserved Nuclear Actin Mark Regulated by PRMT5.

Figure 3.

(A) Amino acid sequence alignment of actin homologs from different species showing high conservation of the R256 residue and sequence around it..

(B and C) Western blot analysis of the whole-cell extract, purified cytoplasmic actin, nuclear-actin-containing INO80 complex, cytosolic and nuclear fractions of NIH 3T3 cells using actin R256me1 antibodies, actin (C4) (loading control), and histone H3Ac (nuclear fraction marker), as indicated.

(D) Immunofluorescence confocal images of the NIH 3T3 cells, stained with actin (C4) and actin R256me1 antibodies, showing the preferential staining of nuclear actin over cytoplasmic actin by actin R256me1 antibodies. DAPI was used to stain the nucleus. Scale bar: 50 μm.

(E) Western blot analyses of the hINO80 complex purified from HEK293 cells after FLAG tagging hIno80 in the genome using antibodies as indicated.

(F and G) Western blot analyses of whole-cell extract of HEK293 cells transfected with siRNA against individual PRMTs as mentioned at the top, using actin (C4) (loading control) antibodies and actin R256me1 antibodies as indicated (F). Results shows that PRMT5 knockdown substantially affects actin the R256me1 level compared to the knockdown of other known PRMTs after adding the PRMT5 inhibitor (CMP5), using antibodies as indicated (G).

(H) Western blot analyses after in vitro methylation assay using purified recombinant PRMT5 and rabbit muscle actin (unmethylated actin substrate) using antibodies as indicated at the right. Actin (C4) is a loading control, and histone H4 methylation by PRMT5 was used as a positive control in the assay.

(I) Western blot analyses of hINO80 complex purified from HEK293 cells after FLAG tagging hIno80 in the genome using antibodies as indicated. Data presented are the representative of three independent biological repeats with similar results.