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Fig. 2 | Skeletal Muscle

Fig. 2

From: Novel γ-sarcoglycan interactors in murine muscle membranes

Fig. 2

Sarcolemmal proteins recovered from Rx (lane 6) and RIPA1 (lane 8) buffers. Gastrocnemius muscles (~ 100 mg) from either C57BL/6 (WT) mice or from mice lacking γ-sarcoglycan (Sgcg−/−) or full-length archvillin (Svil−/−) were fractionated as shown in Fig. 1; lane numbers in red correspond with Fig. 1. Blots were stained for A protein with Ponceau S and probed for the sarcolemmal proteins B dystrophin, C archvillin, and D γ-sarcoglycan (Sgcg, using our new antibody). Much actin is removed by the low-salt in buffer 1 (A, lanes 2–3); most myosin II is extracted by the high salt in Rx buffer (A, lane 6) or remains insoluble in RIPA1 buffer (A, lane 7). Blots are representative of 3 biological replicates per genotype. Fractions 1–6 are normalized to total protein; fractions 7–8 are normalized by volume to each other; total protein in fraction 8 approximates that of fraction 1. Lane 1, whole muscle extracts. Lanes 2–3, discarded washes. Lane 4, enriched membranes before extraction. Lane 5, discarded Rx pellet. Lane 6, Rx supernatants. Lane 7, discarded, insoluble RIPA1 pellets. Lane 8, RIPA1 supernatants. E Comparison of protein abundance by immunoblotting of whole tissue lysates (W) vs. RIPA1 lysates (R). Equal protein loading (based on BCA quantification) for each condition was used. DGC proteins Sgca and β-dystroglycan (β-DG) were enriched in RIPA1 extracts of wild-type C57BL/6 muscles (equivalent to lane 8 in AD), as compared with whole muscle lysates (equivalent to lane 1 in AD), whereas the sarcoplasmic reticulum marker SERCA1 and the contractile apparatus proteins myosin II and fast troponin T (TnT) were depleted. Mean band intensities from ImageStudio software are indicated below each lane and used to calculate the ratios of RIPA1 to whole muscle lysate (R/W) shown to the left of each blot

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