Supplementary Figure 1: Proteomics profiling of EMT-induced changes in membrane proteins.

(a) For stable isotope labeling, the HMLE-Twist cells and control HMLE-vector cells were maintained in the same medium, except that the medium for one of the two cell types contained the C13N15 Arg and Lys amino acids (heavy) instead of the standard C12N14 Arg and Lys (light). A replicate experiment was subsequently performed in which the light/heavy labels were swapped in order to reduce noise from proteins present in the culture medium. Following culture, the HMLE-vector and HMLE-Twist cells were mixed in equal numbers; a membrane protein fraction was prepared from the mixture (panel b) and subjected to tryptic digestion. The resulting peptides were further resolved by off-gel electrophoresis and analyzed by LC-MS/MS (liquid chromatography-tandem mass spectrometry). (b) A flow chart of the membrane protein fractionation strategy. (c) Western blot detecting protein enrichment and contamination in different fractions. E-cadherin and N-cadherin, plasma membrane proteins; Large T antigen, nuclear protein; β-actin, cytosolic and cytoskeletal protein. Numbers on the gels indicate molecular weight markers in kDa.