Figure 2

a. Western blot analysis of supernatants from rSAK producing strains. Lane 1 THR 174 ~ 60ng; RB11/pFPMT Mfα⁺ - rSAK-1 (lane 2 – 10) and RB11/pFPMT Mfα⁺ - rSAK-2 (lane 11–18); 7 μL loaded. b: Glycosylation evidence for SAK: SDS-PAGE analysis of de-repression supernatants. (Pool 1) was de-repressed in YP 2% glycerol for 40 h at 30°C and 37°C. Pooled supernatants of pool 1 were treated with EndoH (lane 4 and 6). Supernatants without EndoH treatment (lane 3 and 5) have been handled with the same EndoH buffers but omitting EndoH. After EndoH treatment, samples were prepared for SDS PAGE and 21 μL each was loaded on a Criterion 4-20% precast gradient gel. Supernatants have been finally examined in a Western blot analysis for presence of SAK. SAK protein was detected by using alkaline phosphatase-conjugated mouse anti-SAK antibody. Approximately 180 ng of THR174 served as positive control.