Fig 2: Knocking down LACTBM5L+R469K can upregulate wild type p53 protein via binding and suppressing PSMB7. A) The ubiquitination of wt‐p53 was analyzed by co‐IP and western blot. B) The wild type p53 protein level after treating by lysosome inhibitors for 24 h in control group and LACTBM5L+R469K knockdown group were detected by western blot. C) Proteasome subunits catalytic activity test kit was used to test the catalytic activity of PSMB5, PSMB6, and PSMB7. D) The wild type p53 protein level of SJSA‐1 cells after treating by PSMB5, PSMB6, and PSMB7 inhibitors (Bortezomib/Ixazomib/Leupeptin) for 24 h in control group and LACTBM5L+R469K knockdown group were detected by western blot. E) U2‐OS and SJSA‐1 cells were transfected with indicated plasmid, and then subjected to co‐IP using anti‐HA antibody follow by western blot. F) The wt‐LACTB‐HA and LACTBM5L+R469K‐HA overexpression plasmids were transfected into SJSA‐1 and U2‐OS cells, and then subjected to co‐IP using anti‐HA antibody. The protein levels of HA‐tag and PSMB7 were detected by using western blot after co‐IP.

Fig 3: EIF4EBP1 activation enhances the 26S proteasomal degradation capacity in skeletal muscle. (A) Immunoblotting of 19S (PSMD1) and constitutive 20S proteasome subunit proteins (PSMA2, PSMB5, PSMB6, and PSMB7) was performed in the soluble fraction extracted from the TA of 4‐month‐old male mice following 48-h fasting (n = 4). (B) the quantification of (A). (C) the quantification of (D). (D) Active proteasome complexes were resolved by native gel electrophoresis using proteasome lysates extracted from a pool of muscle of 4-month-old male mice under fed conditions. Native gel immunoblotting of 19S (PSMD1) and constitutive 20S proteasome subunit proteins (PSMB5, PSMB7, and PSMA2) were showed (n = 4). (E) Proteasome activity analysis in the presence of ATP was performed in lysate extracted from 4-month-old male mouse muscle under fed conditions (n = 4 for 4EPB1mt-muscle group and EIF4EBP1mt-atg7mKO group; n = 3 for control group and atg7mKO group). (F) Immunoblotting of K48 linked ubiquitinated protein and muscle-specific E3 ubiquitin ligases (FBXO32, and TRIM63) was performed in the soluble fraction extracted from TA of 4-month-old male mice following 48-h fasting (n = 3). (G) the quantification of (F). For all the quantification of immunoblotting shown in (B), (C), and (G), the relative protein expression levels were normalized to control group, and data were shown as mean. Individual points corresponded to one mouse. Ponceau S was used as a loading control. Statistical significance was determined by One-Way ANOVA with Tukey’s multiple comparison test for other proteins. Only significant differences (p < 0.05) were shown. The quantification of (A) and (F) were also shown in Table S4 and (D) was shown in Table S5.