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. 2012 Nov;11(11):1353–1361. doi: 10.1128/EC.00179-12

Fig 2.

Fig 2

Expression and localization of epitope-tagged proteins in S. salmonicida. Stably transfected S. salmonicida carrying the pSpiro-PAC-fibrillarin-3×HA and pSpiro-PAC-caltractin-3×HA episomal plasmids were fixed using 2% PFA and methanol and acetone, respectively. (A) Fixed cells were blocked with 2% BSA; they were stained using rabbit anti-HA (1:1,600) and anti-tubulin TAT1 (1:150) and detected using anti-rabbit Alexa Fluor 594 (A594) (1:250) and anti-mouse Alexa Fluor 488 (A488) (1:200), respectively. (B) Cells were stained with mouse monoclonal anti-centrin 20H5 (1:100) and rabbit anti-HA (1:1,600) and detected by anti-mouse A488 and anti-rabbit A594, respectively. The cells were mounted in VectaShield medium containing DAPI and viewed using a Zeiss 510 laser scanning confocal microscope. (A and B, upper left) Maximum intensity projection (MIP) of the A594 signal (red). (A, upper right) MIP of the A594 (red) and DAPI (blue) signals. (B, upper right) MIP of A488 (green). (A and B, lower left) MIP of A488 (green), A594 (red), and DAPI (blue). (A and B, lower right) Bright-field image from the center of the confocal Z-stacks. Fibrillarin-3×HA displays nuclear localization showing partial colocalization with DAPI but enrichment in the regions devoid of DAPI stain. Caltractin-3×HA shows colocalization to the basal body marker centrin in the triangular foci anterior to the nuclei. Scale bars, 5 μm. (C) Western blot of S. salmonicida cells using the mouse monoclonal anti-centrin (20H5) antibody. A doublet of bands is observed at ∼20 kDa. (D) Western blot of S. salmonicida transfectants expressing HA-tagged proteins using the mouse monoclonal anti-HA antibody. From left to right, shown are IFT46-3×HA transfectants (theoretical molecular mass, 30.4 kDa), caltractin-3×HA transfectants (23.4 kDa), fibrillarin-3×HA transfectants (39.1 kDa), and S. salmonicida wild-type cells. (E) Western blot of S. salmonicida transfectants expressing OLLAS-tagged proteins using the rabbit anti-OLLAS antibody. From left to right, shown are S. salmonicida wild-type cells, BiP-2×OLLAS transfectants (75.1 kDa), PDI2-2×OLLAS transfectants (44.5 kDa), and Sec61α-2×OLLAS transfectants (55.6 kDa).