Immunofluorescence and Antibodies
At the appropriate time-point, coverslips were washed with PBS prior to fixation with 4% formaldehyde for 20 minutes. Slides were again washed with PBS prior to permeabilization (0.1% Triton-X 100) for 5 minutes and blocking (3% BSA, 1 % serum) for 20 minutes. Slides were incubated with primary antibodies suspended in blocking solution for one hour. Slides were washed with 10% blocking solution in PBS, followed by PBS. All following steps were completed in the dark. Cells were incubated in secondary antibodies, DAPI, and/or Alexa Fluor 488 Phalloidin (Invitrogen) suspended in blocking solution for 20 minutes. Slides were washed as above, and appropriate slides were incubated in a 1µg/ml preparation of BODIPY 493/503 (Thermofisher) for 30 minutes. These slides were washed with PBS, and all slides were mounted with Mowiol 4-88 (Sigma), and allowed to dry overnight before imaging.
Primary antibodies used in immunofluorescence and immunoblot experiments are as follows: Myc (9E10, Invitrogen), Myc (4A6, Millipore) COXIV (3E11, Cell Signaling Technology), Calnexin (ab22595, Abcam), Gpp130 (Poly19238, BioLegend), TGN46 (AHP500, Bio-Rad), Alpha tubulin (DM1A, Cell Signaling Technology), Beta tubulin (TBN06, Thermofisher), Transferrin Receptor (CBL47, Millipore), EEA1(clone 14, BD Biosciences), CD63 (ab59479, Abcam), Lamp1 (D2D11 XP, Cell Signaling), Anti-Xpress™(46-0528, Invitrogen), and polyclonal Sec17p serum (Haas and Wickner, 1996).