Ez ecl reagent

Cells were washed twice with cold PBS and lysed by adding 75 μl of buffer containing PBS, 100 mM PMSF, 1 mM sodium orthovanadate, 10 mM β-glycerophosphate, 1 μg/ml leupeptin, 1% Triton X-100, 10 mM NaF, and 1 μg/ml pepstatin, for 1 hour at 4 °C. The lysates (30 μg of protein) were subjected to SDS-PAGE and transferred to membranes, which were incubated with the following primary antibodies: pAKT (Ser 473) (Cell Signaling) and α-Tubulin (T6199) (Sigma). Horseradish peroxidase-conjugated secondary antibodies were utilized, followed by EZ-ECL reagent incubation (Biological Industries). A chemoluminescent image was captured by a Fujifilm LAS 3000 device.

Protein extracts were separated by SDS-PAGE (10% gels) at room temperature at 80 mV and then transferred to 0.2 µm-pore nitrocellulose membranes at 4 °C at a total of 600 mA using a Mini-PROTEAN Tetra Cell and a PowerPac Basic, both from Bio-Rad. Membranes were blocked with 5% non-fat milk 0.05% Tween 20 TBS for 1 h at room temperature, then incubated with primary antibodies overnight at 4 °C. Antibody dilutions were: anti-MTCO1 (Abcam ab90668) 1:1 000; anti-SDHA (Abcam ab137040) 1:6 000; anti-β-tubulin (Sigma-Aldrich T0198) 1:5 000. After washing in 0.05% Tween TBS, blots were incubated for 2 h with anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies (Calbiochem, San Diego, CA, USA) at dilution of 1:5 000. Protein bands were detected using EZ-ECL reagents (Biological Industries) and scanned with Dyversity 4 (Syngene, India). UN-SCAN-IT (Silk Scientific, Inc., USA) was used for densitometry analysis.

Samples were subjected to SDS-PAGE and transferred to nitrocellulose (pore size: 0.45 μm; Amersham Protran) or PVDF (pore size: 0.45 μm; Amersham Hybond) membranes. The blots were then blocked for 1 h with 5% (w/v) skim milk-PBST (0.1% Tween in phosphate-buffered saline), incubated with the primary antibody (diluted in 5% skim milk-PBST, for 1 h, at room temperature, unless otherwise indicated), washed, and then incubated with the secondary antibody (diluted in 5% skim milk-PBST, for 1 h, at room temperature). Chemiluminescence was detected with EZ-ECL reagents (Biological Industries). The following primary antibodies were used: mouse anti-HA (Abcam Inc.), diluted 1:1,000; mouse anti-HA.11 (Covance), diluted 1:1,000; mouse anti-V5 (Invitrogen), diluted 1:1,000; mouse anti His (Pierce), diluted 1:2000; mouse anti-JNK (BD Pharmingen), diluted 1:1,000 in Tris-buffered saline (TBS); and mouse anti-actin (MPBio), diluted 1:10,000. Antibodies directed against T3SS components included mouse anti-EspA, mouse anti-EspB and mouse anti-Tir, all a generous gift from Prof. B. Brett Finlay (University of British Columbia, Canada). Horseradish peroxidase-conjugated (HRP)-goat anti-mouse (Abcam Inc.), diluted 1:10,000, was used as the secondary antibody. Representative western blots of at least three independent experiments are presented in the results section.

Samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose (pore size, 0.45 μm; Bio-Rad) or polyvinylidene difluoride (PVDF; Mercury; Millipore) membranes. The blots were blocked with 5% (w/vol) skim milk–PBST (0.1% Tween in phosphate-buffered saline) for 1 h; then incubated with the primary antibody for 1 h at room temperature, or overnight at 4°C; washed with PBST three times; and then incubated with the secondary antibody (diluted in 5% skim milk–PBST, for 1 h, at room temperature). Chemiluminescence was detected with the EZ-ECL reagents (Biological Industries). A dilution of 1:1,000 was used for mouse anti-HA (Abcam), anti-V5 (Invitrogen), anti-His (Thermo Fisher Scientific), anti-HSV (Novagen), anti-actin (MPBio), anti-JNK (BD Pharmingen), anti-DnaK (Abcam), and rabbit anti-MBP (Thermo Fisher Scientific). Mouse anti-intimin (a gift from B. Brett Finlay, UBC) was diluted 1:2,000 (Gauthier et al., 2003 (link)), and horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Abcam) and HRP-conjugated goat anti-rabbit antibody (Abcam) were diluted 1:10,000.

Protein extracts were separated by SDS–polyacrylamide gel electrophoresis (10% gels) at room temperature at 100 mV and then transferred to 0.2-µm-pore nitrocellulose membranes (Macherey-Nagel, Düren, Germany) at 4 °C at 400 mA using a Mini-PROTEAN Tetra Cell and a PowerPac Basic, both from Bio-Rad. Membranes were blocked with 5% non-fat milk 0.05% Tween 20 TBS for 1 h at room temperature, then incubated with primary antibodies overnight at 4 °C. Antibody dilutions were: anti-CAV1 #610060 (BD Transduction Laboratories, San Jose, CA, USA) 1:3000; anti-ACTB #A5316 (Sigma-Aldrich) 1:5000; anti-pDRP1 #4867 (Cell Signaling Technology, Danvers, MA, USA) 1:500; anti-DRP1 #611113 (BD Transduction Laboratories) 1:1000; and anti-PKA RIIa, #MA3-517 (Thermo Fisher Scientific) 1:1000. After washing blots in 0.05% Tween TBS, blots were incubated for 2 h with anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies (Calbiochem) at dilution 1:5000. Protein bands were detected using EZ-ECL reagents (Biological Industries) and either scanned with a G-BOX (Syngene, Bangalore, India) or developed to X-ray films (Agfa-Gevaert, Mortsel, Belgium). ImageJ software (National Institute of Health, Rockville, MD, USA) was used for densitometric analysis.