Figure 4. ZFYVE27 assembles into SDS-sensitive and –resistant dimer/tetramer.

(A) The protein lysates from NIH-3T3 cells overexpressing either E2-ZFYV27^WT or c-Myc-ZFYVE27^WT were subjected to immunoblot with ZFYVE27 specific antibody. Asterisks denote the formation of SDS-resistant dimers corresponding to E2-ZFYV27^WT (∼ 92 kDa) and c-Myc-ZFYVE27^WT (∼ 120 kDa). Note: In pCS2-myc vector, six c-Myc epitope tags are fused in tandem therefore resultant c-Myc-ZFYVE27^WT protein is larger in size (∼55 kDa) as compared to E2-ZFYVE27^WT protein (∼46 kDa). (B) The NIH-3T3 cells transiently transfected with E2-ZFYV27^WT were solubilized in 1% Big-CHAPS and size-fractionated by 5-30% sucrose gradient centrifugation. The resultant fractions were analyzed by immunoblot with ZFYVE27 antibody; asterisks denote the formation of SDS-resistant dimers. The mobility of molecular weight markers in the sucrose gradient are indicated on the top.