Immunoblotting confirms the specificity of LC3 and p62 immunohistochemistry.

A. Wt (ATG7+/+) and autophagy-deficient (ATG7−/−) mouse embryonic fibroblasts (MEFs) were used as positive control. In wt MEFs, 8 h treatment with 30 µM chloroquine (CQ) increased the level of LC3-II and p62 proteins compared to the untreated control (UT); LC3-I was barely detectable in either sample. In autophagy-deficient MEFs, the level of LC3-I and p62 was high at baseline and did not change following CQ treatment; LC3-II was undetectable in both samples. GAPDH was used as a loading control. B. In subjects from the autophagic myopathy group, LC3-II and p62 protein level was increased relative to subjects from either normal or drug-treated control groups. LC3-I protein level was equally high in all samples, suggesting that this isoform is not detected by LC3 immunohistochemistry. Each lane contains sample from a different study subject, with subject ID numbers indicated on top. GAPDH was used as a loading control.