Extended Data Fig. 3: Elucidating the O-GlcNAc modification of Hexokinase 1 and 2.
From: Organization of a functional glycolytic metabolon on mitochondria for metabolic efficiency
a, Illustration of CRISPR-based approach to add eGFP tag at the C-terminal of HK1 in HEK293T cells. The strategy is based on transcript-202 (NM_000188.2) and was implemented by BioCytogen. b, Western blot analysis of the whole cell lysate (Input), used for the generation of mitochondrial and cytoplasmic fractions as shown in Fig. 3, from HEK293T cells. The whole cell lysate (Input) from CRISPR edited HEK293T cells was probed with antibodies against O-GlcNAc (RL2), GFP (HK1), OGT and tubulin (loading control), with or without OGT overexpression and Thiamet-G treatments. c, Schematic demonstrating the sequence of mitochondrial isolation and O-GlcNAc immunoprecipitation (IP) using the anti-O-GlcNAc antibody RL2 from cultured rat hippocampal neurons. d, Western blot analysis of mitochondrial fraction (Input) and O-GlcNAc IP using antibody against HK1 and ATP5B (mitochondrial marker). e, f, Western blot analysis of whole cell lysate from cortical neuron cultures. The lysate was probed with antibodies against O-GlcNAc (RL2), OGT, OGA, and tubulin (as a loading control), following treatments with Thiamet-G or OSMI-4. (f) Quantification of O-GlcNAcylation levels, normalized to tubulin. All values are presented as mean ± SEM. n = 3 independent experiments (one-tailed Mann-Whitney U test). g, eGFP tagged Hexokinase 2 (HK2) was expressed in HEK293T cells. GFP antibody was used to immunoprecipitate (IP) HK2, with or without OGT overexpression and Thiamet-G treatments. The IPs were probed with anti-GlcNAc (RL2) and anti-GFP antibodies. Whole cell lysates (Input) were probed with anti-GFP and anti-tubulin antibodies. Rabbit IgG serves as an IP control. h, Quantification of HK2 O-GlcNAcylation levels. All values are shown as mean ± SEM, unpaired one-tailed t-test. n = 3. i-k, Quantification of the expression levels of HK1-GFP (i), myc-OGA (1–400) (j) and nGFP-HA-OGA (544–706) (k) in COS-7 cells, as shown in Fig. 3d. Data are presented as a violin plot with individual data points and associated p-value. n = 42 cells, three independent experiments (Unpaired two-tailed t-test, and one-way ANOVA with post hoc Tukey’s multiple comparison test).
