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. 2007 Sep 4;117(9):2468–2476. doi: 10.1172/JCI30654

Figure 1. nNOS disappears from the sarcolemma during tail suspension.

Figure 1

(A) Weights of soleus (Sol) and gastrocnemius (Gc) muscles from tail-suspended (sus) wild-type mice are normalized to body weight and are expressed as percentage of ground control (gro) (n = 15/group). *P < 0.05, Student’s t test. (B) Box and whiskers plot of myofiber diameter size of soleus. Boxes represent the middle 50% of the data, lines represent the median, and whiskers represent the range. More than 200 fibers were measured on laminin-α2 chain–stained cross sections. (C) Immunofluorescent staining for nNOS and laminin-α2 chain. Transverse muscle sections from ground control mice, tail-suspended mice, and mice after reloading for 7 days (reload 7d) were stained with anti-nNOS (green) and anti–laminin-α2 chain antibodies (red). Scale bar: 50 μm. (D) Western blot using anti-nNOS antibody on subcellular fractions of muscle extracts. P indicates insoluble pellet after sequential extraction of skeletal muscle homogenates with 100 mM NaCl (S1), 500 mM NaCl (S2), and 0.5% Triton X-100 (S3). GAPDH signals in S1 and Na/K-ATPase signals in P confirmed that our fractionation was correctly done. Fractionation and western blotting were repeated 5 times, and representative data are presented. Note the slight increase of nNOS levels in S1 fraction and loss of nNOS signal in insoluble P fraction during tail suspension. (E) Quantification of nNOS signals in S1 and P fractions of muscle extracts shown in D (n = 5 /group). The signals in S1 and P fractions were normalized to GAPDH or Na/K-ATPase, respectively. Mann-Whitney, *P < 0.05. (F) Levels of nNOS mRNA in muscles from ground control mice and tail-suspended mice for 2 weeks were evaluated by real-time PCR (n = 5/group). No significant difference was found by Mann-Whitney test. (G) Immunoprecipitation with caveolin-3 antibody and immunoblot with nNOS antibody, and vice versa, for ground control and tail suspension groups. (H) Immunoprecipitation with α1-syntrophin Ab and immunoblot with nNOS antibody, and vice versa. (I) Immunoprecipitation of dysferlin antibody and immunoblot with nNOS Ab, and vice versa. In G–I, flow-through fraction was also examined by western blotting with anti–α-tubulin antibody.