Figure 7
From: Loss of chaperone-mediated autophagy is associated with low vertebral cancellous bone mass
Macroautophagy and proteasomal degradation increase in the absence of CMA. Calvarial osteoblasts were isolated from Lamp2AC global knock-out (L2ACgKO) mice and their control littermates (wild-type mice, WT). (a) Calvarial osteoblasts isolated from one mouse per genotype were plated in 6 well plates and cultured with vehicle (DMSO) or 100 μM Bafilomycin A (BafA) for 4 h. LAMP2A, LC3, and p62 protein levels were determined by western blot analysis. (b) LC3-II and p62 levels were quantified and normalized to actin levels. For LC3-II flux analysis BafA treatment of each genotype was normalized its vehicle treatment. n = 3 wells/treatment per genotype. Lines indicate mean ± STDEV. For comparisons of LC3-II/actin and p62/bactin levels p values were calculated and evaluated by Two-way ANOVA. For LC3-II flux comparison p value was calculated and evaluated by student’s t-test; *, p < 0.05. This experiment was repeated using calvarial cells isolated from another set of WT and L2ACgKO mice. These results can be found in S. Fig. 7c. (c,d) Calvarial osteoblasts isolated from one mouse per genotype were plated in 6 well plates and cultured with vehicle (DMSO) or 50 μM of proteasome inhibitor MG132 for 6 h. The levels of ubiquitinated proteins and K48-linked ubiquitinated proteins were quantified by Western blot analysis. Full-length blots can be found in S. Fig. 7.
