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. 2011 Aug 23;6(8):e22968. doi: 10.1371/journal.pone.0022968

Figure 4. FKRP dimer and multimer formation is not dependent on N-glycosylation.

Figure 4

Cells were transfected with pcDNA 3.1-FKRP. Forty-eight hrs after transfection cells were solubilised in lysis buffer with protease inhibitor. A) A cleared BHK-21 lysate was treated with either PNGase F or Endo H as explained in Materials and Methods and subjected to (4–12%) SDS-PAGE under reducing conditions, followed by Western blot analysis. An untreated sample served as control. B) COS-7 cells were transfected with mutant pcDNA3.1-FKRP constructs, in which either one or both asparagines (Asn) involved in N-glycosylation had been replaced with glutamine (Gln). The lysates were prepared in the presence of 5 mM NEM and subjected to either non-reducing (left panel) or reducing SDS-PAGE (330 mM DTT, RT for 30 min) (right panel), followed by Western blot analysis. Antibody FKRP207 was used for the detection of FKRP in these experiments.