Light sensitive films

Cells were washed with PBS containing 1mM CaCl₂ and 0.5 mM MgCl₂, lysed in SDS-sample buffer (Life technologies) containing 4% β-mercaptoethanol (Sigma Aldrich) and denatured at 95 degrees for 10 minutes. Proteins were separated on a 4-12% gradient SDS-PAGE gel (Invitrogen) in MES buffer according to manufacturer’s protocol, and transferred to nitrocellulose membrane (Thermo Scientific Cat# 26619) in blot buffer (48 nM Tris, 39 nM Glycine, 0.04% SDS, 20% methanol). Membrane was blocked with 5% (w/v) milk (Campina) in Tris-buffered saline with Tween20 (TBST) for 60 minutes. The immunoblots were analyzed using primary antibodies incubated overnight at 4 degrees, washed three times with TBST, incubated with secondary antibodies linked to HRP (Dako, Aligent Technologies) and washed three times with TBST. HRP signals were visualized by enhanced chemiluminescence (ECL, cat# 32106, Thermo Scientific) for actin antibody and Super ECL (Thermo Scientific) for RhoJ antibody and light sensitive films (Fuji Film). Mouse monoclonal antibodies against RhoJ (cat# WH0057381M1) and actin (cat# A3853) were purchased from Sigma. Secondary HRP-conjugated goat anti-mouse (cat# P0447) antibody was purchased from Dako.

Cells were washed in ice-cold PBS and lysed on ice in NP40 lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 10 mM MgCl₂, 1% NP-40, 10% Glycerol), supplemented with protease inhibitor cocktail (Sigma-Aldrich). Cell lysates were cleared by centrifugation, heated at 95ºC in SDS sample buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue), and proteins were resolved by SDS-PAGE. Thereafter, proteins were transferred to nitrocellulose membranes (GE Healthcare Amersham) and aspecific binding was blocked using 5% (w/v) milk (Campina) in TBST (150 mM NaCl, 10 mM Tris, 0.1% Tween 20, pH 8.0) for 30 min. Immunoblotting was performed with the indicated antibodies by incubation with primary antibodies o/n at 4ºC and with secondary antibodies for 1 h at RT. Membranes were washed 3 × with TBST after each step. Proteins were visualized using ECL chemiluminescence reagent (Thermo Fisher Scientific), light sensitive films (Fuji Film) and a film processor (Konica Minolta, SRX-101A). Quantification of bands was performed by densitometry using ImageJ. Band intensity of the protein of interest was corrected to that of α-tubulin.

The resolved proteins were transferred to 0.45-μm nitrocellulose membranes (Serva Electrophoresis, Germany). Membranes were subsequently blocked with 5% skimmed milk in Tris-buffer saline with Tween^® 20 (TBST) buffer consisting of Tris, 125 mM NaCl, and 0.1% Tween 20 for 1 h at room temperature followed by incubation with primary antibodies against T. vitulorum (control positive serum sample) diluted at 1:100, overnight at 4°C. Excess antibodies were removed by extensive washing in TBST, and blots were then reprobed with horseradish peroxidase-conjugated anti-bovine IgG (1/1000 dilution, Abcam). Membranes were then extensively washed with TBST and treated with enhanced chemiluminescence reagent (Optiblot™, Abcam, UK). Light-sensitive films (Fuji, Japan) were used for X-ray film exposure [24 (link)].