Homodimerization of RPTPα as assessed using a chemical cross-linker. (A) Distinct RPTPα constructs with different molecular weights were used to be able to discriminate between them. The long form (L) is a full length RPTPα construct with YFP fused to the C-terminus, while the short form (S) lacks almost all cytoplasmic sequences and contains CFP fused to residue 200, close to the transmembrane domain. (B) SK-N-MC cells were transiently transfected with the constructs depicted in (A). The cells were treated with the non-cell-permeable cross-linker, bis [sulfosuccinimidyl]suberate (BS3), or left untreated, as indicated. Aliquots of the total cell lysates were run on a 5% SDS-polyacrylamide gel, the gel was blotted, and the immunoblot was probed with anti-HA-tag antibody. The positions of marker proteins that were co-electrophoresed with the samples are indicated on the left (in kDa). The position of monomeric RPTPα-YFP (the long form, L), dimeric RPTPα-YFP (LL), monomeric RPTPα-200-CFP (the short form, S), dimeric RPTPα-200-CFP (SS), and of the RPTPα-YFP/ RPTPα-200-CFP heterodimer (LS) are indicated, as well as a non-specific band (NS). Lysates from BS3-treated cells that had been transfected with either RPTPα-YFP or RPTPα-200-CFP were mixed (mix), clearly demonstrating that the heterodimer (LS) only formed in cells that had been co-transfected with RPTPα-YFP and RPTPα-200-CFP.
Tertoolen et al. BMC Cell Biology 2001 2:8 doi:10.1186/1471-2121-2-8