Iblot device

Cells treated as indicated were washed with ice-cold PBS and lysed using RIPA buffer with protease and phosphatase inhibitor cocktails (Sigma). Protein concentrations were quantified using a BCA assay (Pierce). Proteins were separated on 4–12% or 10% polyacrylamide gels (Invitrogen) and transferred onto nitrocellulose membranes using an iBlot device (Invitrogen). Membranes were incubated in TBS +0.1% Tween (TBST) and blocked using 5% BSA (wt/vol) or 5% dried nonfat milk (wt/vol) in TBST to inhibit non-specific antibody binding. Membranes were incubated overnight in 1% blocking solution in TBST and primary antibodies. Membranes were washed in TBST and incubated with secondary antibodies at room temperature for 1 h. Immunoblots were visualized by ECL using a GE Amersham imaging system. Antibodies used were mouse monoclonal anti-HCMV immediate-early (IE), pp65, pp28 (All at 1:200) (Kind gift from Dr. Bill Britt), pp52 (1:250) (Santa Cruz Biotechnology) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:500) (Sigma-Aldrich).

2 mg cell pellets were re-suspended with 200 μl lysis buffer (50 mM HEPEs) and were sonicated for 12 s at 75% power (Sonics VCX-130 Vibra-Cell Ultrasonic Liquid Processor). Both the supernatant and pellet of the cell lysate were loaded onto Bis-Tris gels with 2X SDS sample buffer and electrophoresis was run at 200 V for 35 min. SDS gels were then transferred to nitrocellulose membranes using the iBlot device from Invitrogen. Membranes were incubated with Odyssey Blocking Buffer (PBS) for an hour before being blotted with 2D2-AF680 antibody (SCBT, sc-20067 AF680) at room temperature for 2 h. Membranes were then washed with 1× PBS buffer with 0.1% tween-20 three times, and then was scanned at 700 nm channel using Odyssey infrared scanner (Li-Cor) where the intensity of each band was determined using the Odyssey software. The concentration of Bax for each sample was determined by comparing the band intensity of the sample to a sample with known Bax concentration.

Samples were mixed with SDS-PAGE sample buffer (0.5 M Tris-HCl pH 6.8, 10% SDS, 3 M glycerol, 0.05% bromophenol blue) and separated with 15% tris-glycine SDS-PAGE according to the Laemmli procedure (Laemmli 1970 (link)). SDS-PAGE gels were run with or without reducing agent. If added reducing agent (10X, ThermoFisher, Waltham, MA), it was diluted to 0.25% and added to the buffer in the inner chamber of gel tank to maintain the protein samples in a reduced state during electrophoresis. Reducing agent (1%) added to the protein samples prior to loading. The gels were stained with Coomassie Brilliant Blue G-250 or silver nitrate. Separated proteins on the SDS gel were transferred to a PVDF membrane using the Invitrogen iBlot device. The membranes were blocked with 5% skim milk in PBS-T (0.1% Tween 20) buffer for 1 hour, incubated overnight at 4°C with anti-PFN1 primary antibody (Sigma-Aldrich #P7749) diluted in PBS-T 5% milk (1:1000), and rinsed 3 times with PBS-T buffer. Then, proteins were visualized with an HRP-conjugated secondary antibody (Amersham Corp., USA) diluted in PBS-T 5% milk (1:5000) and detected with the ECL system (Amersham).