the membrane and sandwiched between 1 piece of buffer-soaked Whatman paper from Biorad on each side. The sandwich was placed between graphite electrodes with the membrane towards the anode. The transfer was done for 12-16 hr using a voltage of 40 V in the cold room. Protein transfer was viwed using Ponceau S staining. Blot was dipped in the Ponceau S stain under shaking and washed using PBST. After transfer, the membrane was blocked with 5% non-fat milk in PBST (1X PBS with 0.1% Tween-20) for 2 hr at room temperature. The membrane was then washed thrice with PBST under shaking and incubated with the primary antibody (1:1000 dilution in PBST) for 12-16 hr. The membrane was again washed thrice under shaking with PBST and incubated with 1:20000 dilution alkaline phosphatase conjugated anti-goat IgG secondary antibody (in PBST) for 2 hr. The membrane was once again washed thrice as described above and the signal developed using ECL kit from Amersham on X-ray films in a dark room. The reactive protein bands appeared as black bands upon gentle shaking at room temperature in 1X developer solution. The reaction was stopped by dipping and shaking the film in 1X Fixer solution followed by washing in water

The protein samples separated on SDS-PAGE were transferred to PVDF (polyvinyledene difluoride) membrane (Amersham, Buckinghamshire, UK) electrophoretically by a semi-dry method using BioRad apparatus. The gel and the membrane (pre-wetted with methanol) were wetted with transfer and the gel was placed in contact with

Western blotting with anti-Rho/anti-NusG antibody