LC-MS Analysis

Detection of type III secretion system genes

Gel documentation and image analysis

Formaldehyde gel electrophoresis

LTT assay

Electrochemical Measuremen

15 mM CaCl2.2H2O 250 mM KCl 55 mM MnCl2.4H2O pH was adjusted to 6.7 with 1 N KOH. MnCl2needsto beaddedseparately,drop by drop with stirring, tothe buffer. PIPES goes into solutionwhenpH is greater than 6.7. The solution, after pH adjustment to 6.7 was filter-sterilized and stored at -20ºC.Reagents for yeast transformation:1 M Lithium acetate (LiOAc)50% Polyethylene glycol10 mg/ml Carrier DNADimethylsulfoxide (DMSO)

INOUE transformation buffer:For bacterial DH5α ultra-competent cells preparation10 mM PIPES (free acid)

Transformation-related solutions

Binding Buffer (10X)

Blocking buffer: 2% BSA

Permeabilization buffer: 0.2% Triton X100

Fixative : 4% Formaldehyde

For Immunofluorescence

Running Buffer

Ethylene Glycol Tetraacetic acid (EGTA), pH 7.0ComponentsFinal concentrationFor 50 mlEGTA0.1M1.902gH2Oq.sThe pH is adjusted to 7.0 using 10M NaOH

Extraction buffer

10XBinding buffer

Agarose gel

Nuclear lysis buffer (without protease inhibitors

Permeabilisation buffer: 0.2% Triton X100

Stripping buffer

Blocking buffer

TBS-T

Transfer buffer

(f) Running buffer

(e) Stacking polyacrylamide gel

(d) Resolvingpolyacrylamide gel

(c) 6X Protein loading buffer (Lammeli buffer)

(b) Celllysis buffer B(For IB)

Cell lysis bufferA(For IP)

II. For Immunoprecipitation(IP)and Immunoblotting(IB)

(b) Tris Buffered Saline (TBS)