Fig 2: Means (± SEM) of the Western blot analysis (n = 8): HSP90 (a), HSP60 (b), OGT (c), NF-kB p65 (d), BCKDH (e), p-AMPK (f), p-mTOR (g), p-4EBP1 (h), IDE (i), p-AKT (j), p85 (k). Different letters represent significant differences (p < 0.05). GAPDH is loading control. Groups: control (rest, without gavage), vehicle (water), L-isoleucyl-leucine (lle-Leu), L-leucyl-isoleucine (Leu-lle), L-valyl-leucine (Val-Leu), L-leucyl-valine (Leu-Val) and whey protein hydrolysate (WPH).

Fig 3: GLP-2 activates mTORC1 in a rapamycin-sensitive manner. (A and B) Representative Western blot analysis of phosphorylated S6 and Hsp90 (loading control) in full thickness jejunal segments from overnight fasted C57BL/6J mice injected intraperitoneally (i.p.) with vehicle (PBS), GLP-2 (53 nmol/kg = 200 µg/kg), or Ex-4 (10 nmol/kg) (A), or overnight fasted Glp2r+/+ and Glp2r-/- mice injected (i.p.) with vehicle (PBS), or GLP-2 (200 µg/kg) (B) for 1 h. Bar diagrams represent phospho-S6 levels normalized to Hsp90. Data are mean ± SEM. For panel (A) n = 4 mice per group from 2 independent experiments. For panel (B) n = 2–3 mice per group from 2 independent experiments. **p < 0.01 vehicle vs. treatment. (C) Western blot analysis of phosphorylated S6, Akt, or 4E-BP1 in jejunal mucosal scrapings of overnight fasted C57BL/6J mice treated with oral rapamycin (2 mg/kg) or vehicle. 30 min after rapamycin, mice were injected (i.p.) with vehicle or GLP-2 (200 µg/kg), followed by an oral gavage of a 10% protein mix 30 min later. Mice were sacrificed 30 min after the protein gavage. Bar diagrams represent phospho-protein expression normalized to paired total protein levels, or the intensity of the hyperphosphorylated ?-isoform band of 4E-BP1. Data are mean ± SEM, n = 3–5 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 vehicle vs. treatment. #p < 0.05 vehicle-treatment vs. rapamycin-treatment. (D) Following an overnight fast, Glp2r+/+ or Glp2r-/- mice were orally gavaged with a 10% protein mix 30 min prior to collection of mucosal scrapings from jejunum to measure phosphorylation of S6 and 4E-BP1 by immunoblotting. Bar diagrams represent phospho-S6 expression normalized to total S6 protein levels, or the intensity of the hyperphosphorylated ?-isoform band of 4E-BP1. Data are mean ± SEM, n = 4 mice per group. *p < 0.05 vehicle vs. protein gavage, #p < 0.05 Glp2r+/+vs. Glp2r-/- mice.

Fig 4: Effect of cast immobilization on phosphorylation of p70S6K1 and 4E-BP1. Phosphorylation of (A) p70S6K1 on Thr389 and (B) 4E-BP1 on Ser65 was assessed by Western blot analysis of muscle from mice that had been immobilized, fasted overnight, and then re-fed for 30 min prior to sacrifice. The results are expressed as a ratio of the signal obtained for the phosphorylated protein relative to that for the total protein. Data displayed as cast immobilized (open bars) versus contralateral non-immobilized control (closed bars). The data are presented as means ± SE of the arbitrary density values (ADV); n = 6–7 mice/group (1 Fasted/Non-Immobilized outlier removed). Groups sharing a similar letter are not significantly different (P = 0.05).

Fig 5: Regulation of eIF3 subunit accumulation in response to HCMV infection(A) NHDFs were mock (uninfected [UI]) or HCMV infected (AD169 strain; MOI = 3). At each indicated time post-infection total protein was collected, fractionated by SDS-PAGE, and analyzed by immunoblotting using the antibodies shown. GAPDH served as the loading control.(B) As in (A) except total RNA was isolated at the indicated times. qRT-PCR analysis was performed for each indicated gene (n = 3). The HCMV IE1 gene provided a control for HCMV infection. IE1 RNA levels peaked around 6 hpi and then decreased as infection progressed as described (Stamminger et al., 1991; Stenberg and Stinski, 1985). n = 3 for each gene. Error bars indicate SEM. *p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001 by Student’s t test.(C) As in (A) except cells were treated with DMSO or the mTOR active-site inhibitor, PP242 (added at 1.5 hpi; used at 2.5 µM). The change in 4E-BP1 migration from hyperphosphorylation (slow migrating) to hypophosphorylation (fast migrating) confirms PP242 activity.