Spa 860

HeLa cells grown on glass coverslips were washed with PBS, fixed with 3.7% paraformaldehyde for 5–10 min. Fixed cells were then permeabilized in PBS-0.5% BSA- 0.1% Triton solution for 5–10 min and incubated in blocking solution (PBS-0.5%BSA) for 60 min at room temperature. The permeabilized cells were then incubated for 1 h at room temperature with mouse monoclonal anti-HA antibodies and rabbit polyclonal anti-calnexin antibodies (SPA-860; Stressgen biotechnologies) diluted in blocking buffer. After PBS washing, the cells were incubated with fluorescent secondary antibodies, Fluoprobes 488 Donkey anti Mouse IGG (INTERCHIM) and Rhodamine Red_X_conjugated Donkey anti-Rabbit IgG (Jackson ImmunoResearch) for 1 h at room temperature then washed several times with PBS and mounted in immunofluor medium (ICN Biomedicals). Labeled cells were analysed with a spectral confocal microscope, Olympus FV1000.

Rabbit antisera for GFP and RFP were made as described (Stefanovic and Hegde, 2007 ) and rabbit antisera for Tmp21 was made by immunizing rabbits with a peptide corresponding to the cytoplasmic tail (CLRRFFKAKKLIE) conjugated to keyhole limpet hemacyanin via the terminal cysteine. The PrP-A antibody was previously described (Rane et al., 2004 (link)). The antibody against TCR-α, A2B4, (gift from Carolyn Suzuki) was previously described (Suzuki et al., 1991 (link)). CHOP and XBP-1 antibodies were gifts from Linda Hendershot.
Purchased antibodies included anti-calnexin (StressGen SPA-860), anti-Grp94 (StressGen SPA-850), anti-ERGIC53 (Santa Cruz SC-66880), anti-alpha tubulin (Sigma T6199), anti-GAPDH (Santa Cruz SC-20357), Cy5-conjugated anti-rabbit IgG (Jackson ImmunoResearch 111-175-003) and Alexa 488 goat anti-mouse Ab (Invitrogen A11029).