Figure 3.
Enhanced ubiquitin-proteasome activity in livers from young CMA-deficient mice. (A) Immunoblot (IB) for K48-linked ubiquitinated proteins of liver homogenates from 4-month-old control (Ctr) and Albumin-Cre:L2Af/f (L2AKO) mice. Right: densitometric quantification, n = 10. (B) IB of Ctr and L2AKO mouse livers incubated for 2 h in the presence/absence of lactacystin (lacta). Right: densitometric quantification, n = 4. (C) Proteasome activity against fluorogenic substrates specific for trypsin- or chymotrypsin-like activities measured in the presence or absence of ATP in liver homogenates from Ctr and L2AKO mice, n = 6. (D) IB for subunits of the proteasome core (20S) or regulatory (19S) particles of 4-month-old Ctr and L2AKO mice livers. (E) Densitometric quantification of blots as in D, n = 3. (F) IB of isolated lysosomes active for CMA isolated from livers of 4-month-old Ctr and L2AKO mice treated or not with leupeptin 2 h prior to tissue dissection. (G, H) Cell viability of primary hepatocytes isolated from Ctr and L2AKO mice 24 h after treatment with the indicated concentrations of lactacystin alone (G) or in combination with leupeptin (H), n = 3. All values are expressed as mean ± SEM. Differences are significant for *P < 0.05, aaccumulation of p62, and ***P < 0.001.