Supplemental Data
Files in this Data Supplement:
- supplemental figure 1 (.pdf, 646 KB) - Supplemental Figure 1. The HA-tagged ERdj4 cellular localization is comparable ERdj4-GFP. A. MDCK cell transiently transfected with 3HA-DSLmERdj4, fixed, and stained with anti-HA and anti-rabbit Alexa 488 secondary antibody. Cells were imaged on a Zeiss Axiovert 200 widefield microscope with a 63X oil NA 1.4 objective and imaged with a Retiga 2000 camera. The staining pattern is typical ER network of interconnected tubules and nuclear envelope. B. ER localization is confirmed with co-staining of COS-1 cells with the ER marker anti-BiP (27). Cells were imaged with a Nikon Eclipse Ti-S microscope at 20X and with a Nikon DS Qi1 Mc camera. Scale bar = 10 μm.
- supplemental figure 2 (.pdf, 201 KB) - Supplemental Figure 2. Resolution of signal peptide cleavage by SDS-PAGE. In Figure 4D, the ERdj4SS+10-GFP-KDEL reporter migrates similarly to PrlSS+10-GFP-KDEL. Here, we sought to demonstrate the capability of our 12% Tricine SDS-PAGE gels to clearly resolve small differences in protein sizes. All of the reporters illustrated in Fig. 4B were transiently transfected into cells, lysed, separated on SDS-PAGE, and immunoblotted with anti-GFP. Bands are shown in order of increasing molecular weights. ERdj4 SS+10 migration is comparable to predicted size differences. The predicted differences of 2 (Prl SS+2), 15 (Prl SS+10 and ERdj4 SS+10), and 31 amino acids (C1 P450 1-29-GFP) (note: no KDEL sequence at the COOH terminus) for the mature signal cleaved or uncleaved reporter products relative to GFP-KDEL are visually distinguishable in the "combined exposures" immunoblot. Because ERdj4 SS+10-GFP-KDEL consistently expressed at very low levels, it was only visible at very long film exposures. The short and long immunoblot exposures are displayed below the combined (separate exposures indicated by lines in combined image) exposure blot.