Fig. 4. Northern blot analysis of Ha-CalpM expression in lobster tissues. Total RNA (20 µg) was separated electrophoretically on a 1% agarose gel and blotted overnight. The membrane (A) was hybridized under high stringency with a DIG-labeled RNA probe synthesized from a plasmid with an insert encoding the region encompassing amino acid residues 171-332 in domain II of Ha-CalpM (Fig. 1; see Materials and methods). Three Ha-CalpM transcripts were detected (3.3, 4.0 and 6.1 kb) in cutter (Cu) and crusher (Cr) claw muscles and deep abdominal (DA) muscle (A, lanes a—c). The fast muscles expressed all three mRNAs (lanes a and c), while the slow muscle (lane b) expressed primarily the 3.3 kb mRNA. Intestine (In; lane f) showed a strong hybridization to mRNA in the 3.3-6.1 kb range, as well as to mRNAs greater than 6.1 kb. Little or no Ha-CalpM mRNA was detected in digestive gland (DG; lane d), gill (Gi; lane e), nerve cord (NC; lane g), integument (Ig; lane h), heart (Ht; lane i) or antennal gland (AG; lane j). RNA loading was determined by staining the gel with Ethidium Bromide (B). Lane M, molecular mass markers (kb) are indicated.