Non fat milk

Cells were lysed with 1x SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% (W/V) SDS, 10% (V/V) Glycerol, 50 mM DTT, 0.01% (V/V) β-mercaptoethanol and 0.01% (W/V) bromophenol blue) and centrifuged at 20,000 x g for 10 minutes at 4°C. Cleared total cellular lysate was then separated by SDS-PAGE (Note: for the analysis of KSHV K15 protein, samples were not boiled prior to SDS-PAGE) and transferred onto 0.45 μm nitrocellulose membranes (Amersham, GE healthcare Europe GmbH, Freiburg, Germany). Membranes were then blocked with 5% (W/V) non-fat milk (Carl Roth GmbH+Co.KG, Karlsruhe, Germany) or IgG free albumin (Carl Roth GmbH&Co.KG, Karlsruhe, Germany) for 1 hour and probed with an appropriate primary antibody in blocking solution overnight, at 4°C. The next day, membranes were washed three times and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies for an hour at room temperature (RT). After a subsequent wash, signal was developed using a standard enhanced chemiluminescence (ECL) kit (#34096; Thermo Scientific, Rockford, IL, USA). Primary and secondary antibodies used for western blot are listed above (section antibodies).

Immunohistochemistry and immunoblotting were done as described previously [45 (link)], with a minor change in blocking solutions. For immunostaining, blocking was done using PBS containing 3% IgG-free BSA (Jackson Immuno Research) and 4% normal horse serum. For immunoblotting immunoprecipitation lysate, blocking was done using PBST containing 3% BSA (Roth) and 3% nonfat Milk (Roth). Immunostaining and–blotting were done with following primary antibodies: polyclonal goat anti-CB1 (Prof. Ken Mackie); polyclonal rabbit anti-CYFIP2 (Sigma); polyclonal goat anti-pGAP43 (SCBT); monoclonal mouse anti-GFP (Neuromab); polyclonal rabbit anti-NCKAP1 (Sigma); monoclonal mouse anti-Rac1 (BD Bioscience); monoclonal mouse anti-Tau1 (Merck); polyclonal rabbit anti-βIII-Tubulin (Sigma); polyclonal goat anti-WAVE1 (R&D); polyclonal rabbit anti-WAVE1 (Sigma) polyclonal rabbit anti-pospho-WAVE1 (Sigma). Actin fibres were visualized with TRITC-conjugated Phalloidin (Invitrogen).

After 24 h and 48 h, uninfected and infected HeLa cells were collected after trypsinization and resuspended in REPA buffer (50 mM Tris, 150 mM Nacl, 0.1% SDS, 0.5% Sodium Deoxycholate, 1% Triton X100, 1 mM PMSF) containing 1x protease inhibitor cocktail (Sigma) to lyse the eukaryotic cells. The lysate was then loaded onto SDS-PAGE gels and transferred to a PVDF membrane (Millipore) through Western blotting. Blots were blocked using 5% non-fat milk (Carl Roth) and probed with rabbit anti-cleaved caspase 3 antibody (1:1000) (Cell Signalling) or rabbit anti-vinculin antibody (1:1000) (Santa Cruz Biotech). After incubating with horseradish peroxidase-conjugated rabbit IgG secondary antibody (Abcam), blots were processed using the enhanced chemiluminescence (ECL) Western blotting detection system (Millipore). Images were taken using Gel Doc™ XR+ System (Bio-Rad). Bands were quantified using ImageJ software (National Institutes of Health, USA) and normalised against loading control (vinculin). Mean optical density values were plotted using GraphPad Prism 5 (Graphpad Software).