Congo Red secretion assays were performed
as previously described.29 (link) Briefly, the
total cell and supernatant fractions were separated by two centrifugations
at 20 000g for 2 min. The cell pellet of the initial centrifugation
was taken as the total cell fraction. The pellet was resuspended in
200 μL of protein loading dye (40% glycerol, 240 mM Tris/HCl
pH 6.8, 8% SDS, 0.04% bromophenol blue, 5% beta-mercaptoethanol),
and 5 μL was loaded onto a 10% SDS-PAGE gel for analysis. Proteins
in the supernatant were precipitated with trichloroacetic acid (TCA)
(10% v/v) and resuspended in 50 μL of protein loading dye. Ten
microliters of supernatant sample was loaded onto a 10% SDS-PAGE gel
for analysis. Protein content of the pellet and supernatant fraction
were assessed by western blotting with anti-FLAG (Sigma), anti-β-lactamase
(sc-66062, Santa Cruz), or anti-MyoD (C-20, Santa-Cruz) antibodies.
For type 3 secretion expression analysis, membranes were probed with
anti-IpaB and anti-IpaD antibodies, proteins in the type 3 secretion
needle apparatus. Controls for cell lysis were conducted using anti-DnaK
(a cytoplasmic protein found in Shigella and E. coli) (Abcam ab69617).
as previously described.29 (link) Briefly, the
total cell and supernatant fractions were separated by two centrifugations
at 20 000g for 2 min. The cell pellet of the initial centrifugation
was taken as the total cell fraction. The pellet was resuspended in
200 μL of protein loading dye (40% glycerol, 240 mM Tris/HCl
pH 6.8, 8% SDS, 0.04% bromophenol blue, 5% beta-mercaptoethanol),
and 5 μL was loaded onto a 10% SDS-PAGE gel for analysis. Proteins
in the supernatant were precipitated with trichloroacetic acid (TCA)
(10% v/v) and resuspended in 50 μL of protein loading dye. Ten
microliters of supernatant sample was loaded onto a 10% SDS-PAGE gel
for analysis. Protein content of the pellet and supernatant fraction
were assessed by western blotting with anti-FLAG (Sigma), anti-β-lactamase
(sc-66062, Santa Cruz), or anti-MyoD (C-20, Santa-Cruz) antibodies.
For type 3 secretion expression analysis, membranes were probed with
anti-IpaB and anti-IpaD antibodies, proteins in the type 3 secretion
needle apparatus. Controls for cell lysis were conducted using anti-DnaK
(a cytoplasmic protein found in Shigella and E. coli) (Abcam ab69617).