Extended Data Figure 5. PFKP ubiquitination and degradation.

a, Stability of PFKP analyzed by pulse chase experiments. HEK cells were pulsed with L-AHA for 12 hrs followed by a 0 or 24 hr chase period. The experiment was performed once. b, Abundance of ubiquitinated proteins and PFKP expression in HEK cells in the presence or absence of proteasome inhibitor MG132 (10 μM). Control, DMSO. Representative data from two independent experiments are shown. c, Abundance of polyubiquitinated PFKP upon ubiquitin pull-down using either control beads or beads conjugated to ubiquitin-binding protein using HEK cells. PD, pulldown. The experiment was performed once. d, Abundance of polyubiquitinated PFKP in the presence or absence of MG132 (10 μM for 3 hrs). Representative data from three independent experiments. e, Abundance of over-expressed PFKP-GFP harboring specified lysine-to-arginine (K-to-R) mutations on stiff and soft substrates. Data is normalized with respect to over-expressed wildtype (WT) PFKP-GFP on stiff substrate. Wildtype (n = 13) and each K-to-R mutants (n = 2). Data are shown as mean of two independent experiments. f-m, Abundance of PFKP-GFP harboring a lysine-to-arginine (K-to-R) mutation as indicated in cells cultured on stiff and soft substrates. Representative data from two independent experiments. n, Structure of a PFKP tetramer. Each PFKP monomer is colored differently. Arrows point to the K281 sites in each monomer. o, Enlarged structural detail of PFKP around the K281 site. Each amino acid is shown in different color. p, Abundance of over-expressed PFKL-GFP harboring K272R mutation on stiff and soft substrates. The experiment was performed once. q, Abundance of over-expressed PFKM-GFP harboring K275R mutation on stiff and soft substrates. The experiment was performed once. In a, b, d, f-m, p, q, protein abundance was normalized to the abundance of GAPDH, and in c to the abundance of β-actin.