HokkaidoU Japan

iGEM 2011 Team of Hokkaido University

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Preparation of T3SS E. coli

Infection assay using onion cells

Infection assay using HeLa cells

Detection of injected protein using GSK tag

Protein extraction from infected HeLa cells

SDS-PAGE and Western Blot analysis

HeLa cell lysates were subjected to SDS-PAGE, and separated proteins were transferred to an Immobilon-P membranes (Millipore). The membranes were blocked with Blocking buffer (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, 5% nonfat milk) for 1 h at room temperature. The blots were probed with Phospho-GSK-3β (Ser9) antibody (Cell Signaling Technology #9336) or GSK-3β antibody (Cell Signaling Technology #9315) diluted 1/1000 in Blocking buffer and incubated overnight at room temperature. Blots were washed three times with TTBS (20 mM Tris, 150 mM NaCl, 0.05% Tween 20) for 15 min each time. Secondary antibody (alkaline phosphatase-conjugated anti-rabbit immunoglobulin G) was diluted 1/1000 in Blocking buffer and incubated with the blots for 1.5 h at 37C. Blots were washed as described above and developed with BCIP/NBT.