Figure 2.

SorL1 is a phosphoprotein. Wt MEFs were grown to confluency and equal concentrations of protein lysate were incubated with (+) or without (-) calf intestine alkaline phosphatase (CIP). Equal volumes were subsequently analyzed in parallel by immunoblotting with either; α-SorL1 antibody (BD biosciences), phosopho-serine or ProQ diamond phospho-stain (Invitrogen). A. Separation of samples by 6% SDS PAGE resulted in identification of multiple SorL1 immunopositive bands (-). Following treatment with CIP (+) (first panel) the largest molecular weight SorL1 immunopositive band was seen to decrease in molecular weight and as a consequence migrate with the two faster migrating bands. Detection with ProQ diamond phospho stain that selectively stains phosphoproteins in polyacrylamide gels detected a signal in only the untreated sample (-) as detected by UV transillumination (second panel). B. Immunoblotting of independent CIP treated lysates revealed phospho-serine immunopositive bands (right panel) with the same migratory pattern as SorL1 positive bands. To confirm that the phospho-serine antibody was cross-reacting with SorL1, the phospho-serine immunoblot was stripped and reprobed with α-SorL1 antibody