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. 2019 Dec 3;16(9):1618–1634. doi: 10.1080/15548627.2019.1695399

Figure 4.

Figure 4.

The GID-complex regulates K48-dependent polyubiquitination of AMPK. (A and B) Western blot of p-PRKAA. Cells were starved for 6 h with (+) or without (-) MG132 treatment (10 μM, proteasome inhibitor). ACTB as loading control. Quantification showing relative protein level of p-PRKAA compared to ACTB in WT cells. (C) Western blot of PRKAA ubiquitination. Cells were starved for 2 h, then treated with starvation medium containing MG132 for additional 4 h. Cell lysates were immunoprecipitated by anti-PRKAA antibody and immunoblotted with TUBE (high affinity ubiquitin binding peptide). (D and E) Western blot of p-PRKAA. WT cells transfected with different ubiquitin mutants for 24 h, then starved for 6 h. ACTB as loading control. Quantification showing relative protein level of p-PRKAA compared to ACTB. Abbreviations: Ub, wild-type ubiquitin; Ub[K48], ubiquitin with one lysine residue left at position 48; UbK48R, ubiquitin with K48R mutation. Plasmids shown in Table 3. (F and G) Western blot of PRKAA and p-PRKAA turnover. Cells were starved for 24 h and simultaneously treated with CHX. ACTB as loading control. Quantification showing relative protein level compared to ACTB. WT cells starved for 1 h are set to 1.