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. 2019 Jun 26;317(3):C613–C625. doi: 10.1152/ajpcell.00113.2019

Fig. 2.

Fig. 2.

Quantification of matrix metalloproteinase 2 (MMP2) protein in rat skeletal [soleus (SOL) and extensor digitorum longus (EDL)] and cardiac muscle and human skeletal muscle. A: detection of mouse/rat (M/R) MMP2 pure protein (0.4, 0.2, and 0.1 ng) and rat soleus (30, 70, and 100 µg) and heart (100 µg) homogenates, with or without p-aminophenylmercuric acetate (APMA) treatment, with the antibody Ab92536. B: human vastus lateralis muscle homogenates loaded in duplicate or triplicate (40, 80, and 125 µg) and human pro-MMP2 pure protein (0.1, 0.2, and 0.4 ng). Vertical dashed lines demarcate lanes of subjects S1–S3. In A and B, Stain-Free (blue) gels for myosin, nonspecific bands, and molecular mass markers are shown. C: standard curve of MMP2 pure protein in B, derived by plotting the amount of pure pro-MMP2 (0.1, 0.2, 0.4 ng; x-axis) vs. respective relative band density of Western blot (y-axis). [Where the protein amount was examined in duplicate (0.4 ng), the average of Western band densities was plotted.] Dashed line illustrates how the band density of the 125-µg human muscle sample from subject S1 on the same gel shows it contained ~0.22 ng of MMP2, corresponding to ~24 nmol/kg muscle wet wt. au, Arbitrary units. D: individual values and means ± SD of absolute amount of MMP2 in rat soleus (n = 6), rat heart (n = 5), and human vastus lateralis (n = 3) muscle; n indicates number of independent animals/subjects, with human samples each analyzed on ≥2 separate Western blots. E: representative Western blot of MMP2 in homogenates of slow-twitch muscle (soleus; 60, 30, and 15 µg) and fast-twitch muscle (EDL; ~70 µg) from 5 different rats, along with pro- and active MMP2 pure proteins (0.1, 0.2, 0.4, and 0.8 ng), probed using Ab92536. Nonspecific bands, molecular mass markers, and Stain-Free (blue) gel are shown. F: relative amount of MMP2 in soleus (n = 7 rats) and EDL (n = 5 rats) muscle. Values are pooled data and means ± SD. Values are not significantly different (P > 0.05, by unpaired Student’s t-test). G: calpain-1 pure protein (10, 5, 2.5, and 1 ng) and rat soleus (n = 3), EDL (n = 2), and heart (n = 3) muscle and human muscle homogenates (n = 2 subjects) detected for calpain-1.