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. 2023 Jun 2;9(22):eadg4993. doi: 10.1126/sciadv.adg4993

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Fig. 1. — (A) ULK1 interacts with LDHA. Flag-tagged Atg13, LDHA, or LDHB were coexpressed with HA-ULK1 individually, and immunoprecipitation was performed using anti-Flag beads. Samples were analyzed by anti-HA Western blot. (B) Interaction assay of ULK1 and LDHA by yeast two-hybrid assay. pGBKT7-Atg13, pGBKT7-LDHA, or pGBKT7-LDHB and pGADT7-ULK1 were cotransformed into the yeast strain MJ109 and grown on the agar plate without leucine and tryptophan (YPD/−Leu/−Trp). Atg13 as positive control. (C) Interaction assay of ULK1 and endogenous LDHA. ULK1-HA was expressed in HEK293T cells. Immunoprecipitation was performed using anti-HA beads and analyzed by Western blot with antibody to LDHA. (D) Antibody specificity was analyzed by dot blot assay with a LDHA Ser¹⁹⁶ phosphorylation antibody. +P peptide represent phosphorylation modification of LDHA Ser¹⁹⁶. −P peptide represent nonphosphorylation modification of LDHA Ser¹⁹⁶. (E) Antibody specificity test. LDHA^WT-Flag or LDHA^S196A-Flag was expressed in HEK293T cells. Immunoprecipitation was performed by anti-Flag beads, and LDHA Ser¹⁹⁶ phosphorylation levels were analyzed by Western blot with phospho-LDHA Ser¹⁹⁶ antibody. (F) LDHA Ser¹⁹⁶ phosphorylation assay. HEK293T cells were treated with EBSS medium, mTOR inhibitor rapamycin, and ULK1 inhibitor MRT68921 for 4 hours. LDHA Ser¹⁹⁶ phosphorylation was analyzed by phospho-LDHA Ser¹⁹⁶ antibody. (G) LDHA Ser¹⁹⁶ phosphorylation assay in ULK1-KO MEF cells. MEF cells and ULK1-KO MEF cells were transfected with vector or ULK1-HA and were cultured in complete medium or EBSS medium for 3 hours. LDHA Ser¹⁹⁶ phosphorylation was analyzed by phospho-LDHA Ser¹⁹⁶ antibody. (H) Autophagy assay in LDHA knockdown control cells and ULK1-KO cell (followed by expression of either LDHA^WT, LDHA^S196A, or LDHA^S196D). (I) LDHA Ser¹⁹⁶ phosphorylation assay in vitro. ULK1 was purified by immunoprecipitation from HEK293T cells, and its kinase activity in vitro was analyzed using purified LDHA^WT-Flag and LDHA^S196A-Flag from E. coli as substrate. LDHA Ser¹⁹⁶ phosphorylation was analyzed by anti–phospho-LDHA Ser¹⁹⁶ and anti–phospho-Ser/Thr antibody.