Immunoblot Analysis Following Blue-Native (BN) PAGE BN-PAGE analysis was performed on crude membrane isolates prepared from 253T-NT cells stably transfected with empty vector, wild type CEACAM1-4S and each of the single and double glycine mutants described above. Cell pellets were thawed on ice and lysed following 10 passages through a 20-gauge needle. Lysates were centrifuged at 3,500 rpm for 15 min at 4uC and MgCl 2 and benzonase (Sigma Aldrich) were added to the supernatant to give final concentrations of 2 mM and 1 unit/ml, respectively. After incubation at room temperature for 30 min, membranes were centrifuged at 17,000 rpm for 15 min at 4uC. Blue-native (BN) PAGE electrophoresis was performed using the Native Page Novex Bis-Tris gel system (Invitrogen). Membrane pellets were solubilized on ice for 30 min in 16 Native Page sample buffer (Invitrogen) containing 1% dodecyl-ß-D-maltoside (Invitrogen) and protease inhibitors. After a 30 min incubation, the samples were centrifuged at 20,0006 g for 20 min at 4uC to remove insoluble material. A Bradford assay (Bio-Rad, Hercules, CA, USA) was performed to determine protein concentration. One ml of 5% NativePage G-250 sample additive (Invitrogen) was added to each sample, and the samples and a Native Mark protein ladder (Invitrogen) were loaded into the wells of a 4-16% Bis-Tris gel (Invitrogen). After running at 150 V for approximately 30 min, the cathode buffer was changed from 0.02% to 0.002% G-250 and electrophoresis at 150 V was continued for an additional 120 min. BN-PAGE gels were immunoblotted onto PVDF membranes (Bio-Rad). Transfer was performed using Bio-Rad's semi-dry transfer apparatus for 1 hour at 25 V. After transfer, membranes were incubated in 8% acetic acid for 15 min, rinsed with water and air-dried. PVDF membranes were re-hydrated with methanol, rinsed with water and blocked in 5% milk overnight at 4uC. Blots were labeled with antibodies as previously described [24] .