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. 2019 Jul 8;12:5395–5404. doi: 10.2147/OTT.S208637

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© 2019 Li et al.

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LDHA is Lys-trimethylated on K5 in HEK293T cells.

Notes: (A) The genetic sequencing results of LDHA@K5Q showed that lysine (K5) on the peptides comprising the ATLKDQLIYNLLKEEQTPQNK sequence was mutated into glutamine (Q); (B) The genetic sequencing results of LDHA@K5R showed that lysine (K5) on the peptides comprising the ATLKDQLIYNLLKEEQTPQNK sequence was mutated into arginine (R); (C, D) After LDHA@K5Q and LDHA@K5R were transfected into HEK293T cells, the HEK293T cell lysates were immunoprecipitated with anti-LDHA antibodies, and Western blotting was performed using anti-trimethyl lysine and anti-LDHA antibodies as the primary antibodies. The molecular weight of LDHA is 36.7 kDa, however, because of its connection with GFP in the plasmid sequence, theoretically, the molecular weight of exogenous LDHA was approximately 64 kDa. (E) The results of Western blotting indicated that the mutation of lysine (K5) led to the disappearance of trimethylation on Lysine (K5), while the expression of LDHA remained unchanged (p<0.01, n=3).

Abbreviations: M, marker; K5-Q, lysine (K5) on LDHA was mutated into glutamine (Q) in HEK293T cells; K5-R, lysine (K5) on LDHA was mutated into arginine (R) in HEK293T cells; WT LDHA, The plasmid constructed on the basis of LDHA wild-type sequence (Genbank NM005566) was transfected into HEK293T cells as the control sample.