Sbp tag sb19 c4

Cells seeded onto cover slips in 6-well plates were fixed in 4% paraformaldehyde solution for 10 min with gentle rocking. Fixed cells were washed three times in PBS, incubated in blocking buffer (PBS/2% BSA/0.2% Triton X-100) for 20 min and incubated with primary antibody diluted in blocking buffer at 4°C overnight. The following primary antibodies were used: E-cadherin (3195: Cell Signaling), V5 (R96025; ThermoFisher), SBP tag (SB19-C4; Santa Cruz), GM130 (ab31561; Abcam), DYKDDDDK Tag (14793: Cell Signaling), BBF2H7/CREB3L2 (MABE1018: Millipore). Following primary antibody incubation, cells were washed three times in PBS and incubated with Alexa Fluor 488- or 568-conjugated secondary antibody (Life Technologies) diluted in blocking buffer for 1 h, followed by three PBS washes and mounting of slides using DAPI Fluoromount G (Southern Biotech). For Phallodin immunofluorescence, rhodamine phallodin was diluted in blocking buffer and incubated at 4°C overnight, cells were then washed three times in PBS and mounted. Images were taken using an Olympus FV10i LIV laser scanning confocal microscope.