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. 2008 Nov 24;587(Pt 2):443–460. doi: 10.1113/jphysiol.2008.163162

Figure 3. Calsequestrin 1, CSQ2 and SERCA1 in segments of individual muscle fibres.

Figure 3

A, entire constituents of a segment of an individual EDL fibre (extreme left) and a soleus fibre (SOL, extreme right) were separated on a 10% SDS-PAGE gel, and CSQ1 and CSQ2 detected by Western blotting (anti-CSQ1 and 2). The blot was reprobed for SERCA1, and also for actin as an indicator of the relative amount of fibre added. Post-transfer Coomassie Blue staining for MHC (top panel) also confirmed that approximately equal amounts of the EDL and SOL fibre were run. Calsequestrin 1 content was much greater in the EDL fibre than in the SOL fibre, whereas CSQ2 was present only in the SOL fibre. B, Western blot comparing CSQ1 and CSQ2 contents in segments of 7 SOL (I) fibres and 2 SOL (II) fibres, calibrated by indicated amounts of purified CSQ1 (5–15 ng, rabbit) and CSQ2 (2–10 ng, human); (anti-CSQ1 and 2 and anti-CSQ1). The relative amount of fibre present is indicated by MHC stain (top panel). Fibre type was determined beforehand from the contractile response of the fibre segment to pSr 5.3 solution (see Methods and Fig. 4). The SOL (II) fibres contained more CSQ1 than SOL (I) fibres, and had a high density of SERCA1 but no CSQ2. C, relative amounts of CSQ1 in individual fibres from different muscles. Amounts of CSQ1 were derived from the standard curve for purified CSQ1 on the same gel, normalized by MHC content and expressed as a percentage of the mean value for EDL fibres on the same gel. In two SOL (II) cases (crossed circles) there were no EDL fibres on the same gel (shown in B), and values were first normalized to the mean of SOL (I) fibres on that gel and then rescaled by the ratio of mean values of EDL to SOL (I) fibres. The SOL (I) fibres were significantly different from EDL, RG (II) and SOL (II), and the SOL (II) and RG (II) fibres were significantly different from EDL fibres; one-way ANOVA with Newman–Keuls post hoc analyses. D, 8% gel Stains-All of 30 μg of protein loaded for EDL (2nd lane), soleus (3rd lane) and heart muscle preparations (4th lane; see Methods). Purified CSQ1 and CSQ2 (5 μg each) were loaded in the same lane. The sizes of two different molecular weight markers, applied to either side of the gel, are indicated (MWt).