Bis tris gels 4 12

For immunoblot analysis, harvested cells were lysed on ice for 30 minutes with radioimmunoprecipitation assay buffer (RIPA). Lysates were then centrifuged for clearing and protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Pierce, ThermoFisher #23225). Samples were then heated at 95°C for 5 minutes. Electrophoresis was performed using bis-Tris gels 4-12% (Invitrogen) and then samples were transferred to polyvinylidene difluoride (PVDF; Millipore) membranes. For probing, the following antibodies were used: E-cadherin (1:1000; rabbit monoclonal, Cell signaling, Danvers, MA, #3195), and GAPDH (1:1000; rabbit monoclonal, Cell signaling, Danvers, MA, #5174). Detection was performed using Western Lightning Plus ECL enhanced chemiluminescent substrate (Perkin-Elmer Inc., #NEL103001EA) according to manufacturer’s instructions.

For immunoblot analysis, harvested cells were lysed on ice for 30 minutes with radioimmunoprecipitation assay buffer (RIPA) (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl). Lysates were then cleared by centrifugation and protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Thermo). Electrophoresis was performed using bis-Tris gels 4–12% (Invitrogen) and then samples were transferred to polyvinylidene difluoride (PVDF; Millipore) membranes. Detection was performed using Western Lightning Plus ECL enhanced chemiluminescent substrate (Perkin-Elmer) according to the manufacturer’s instructions. See supplemental methods for the information of antibodies used.
For QPCR, RNA was extracted from activated lymphocytes using an RNAeasy purification kit (Qiagen) and complementary DNA was synthesized (Bio-Rad). Target Primer sets below used were used with Sybr-Green (Bio-Rad) to measure relative transcript levels. Ubiquitin was used as a house keeping gene. ΔΔCT was used to calculate relative fold change.