Fig 2: EIF4EBP1 activation promotes the atp-independent 20S proteasomal degradation via increasing immunoproteasome subunit expression and assembly. (A) 20S Proteasome activity analysis was performed with lysates without ATP 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). (B) Immunoblotting of immunoproteasome subunit proteins immunoproteasome (PSMB8, PSMB9, and PSMB10) and 11S regulatory proteasome (PSME1, and PSME2) was performed in the soluble fraction extracted from TA of 4-month-old male mouse following 48-h fasting (n = 4). (C) the quantification of (B). (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 (n = 4). Native gel immunoblotting of immunoproteasome subunit proteins immunoproteasome (PSMB9) and 11S regulatory proteasome (PSME1/2) was presented. (E) the quantification of (D). (F) the quantification of (G). (G) Immunoblotting of proteasomal transcription factor proteins STAT3, and NFKB1 was performed in the soluble fraction extracted from TA of 4-month-old male mouse following 48-h fasting (n = 4). (H) Representative immunoblotting of STAT3, NFKB1, GAPDH (used as cytoplasmic fraction marker), and Histone H3 (used as nuclei fraction marker) was performed in nuclear fraction, cytosolic fraction, and total lysate extracted from muscles of 4-month-old male mouse following 48-h fasting. The other three accompanied immunoblotting images are presented in Fig. S4G. (I) the quantification of (H) and Fig. S4G (n = 4). For all the quantification of immunoblotting, 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 (B) and (G) were shown in Table S8, (D) was shown in Table S9, and (H) was shown in Table S10.