Nupage reducing agent

Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (protease inhibitor P8340; phosphatase inhibitor cocktail 2 P5726, Sigma-Aldrich). Proteins were quantified with BCA protein assay (Pierce) and then reduced in NuPAGE LDS Sample buffer (Invitrogen) with NuPAGE Reducing Agent (Invitrogen) at 70°C for 10 min. Proteins were separated on SDS-PAGE 4-12% (Invitrogen) and transferred onto PolyVinylidene Fluoride (PVDF) membrane (Millipore). After saturation in PBS 0.2% of casein, membranes were incubated with primary antibodies overnight at 4°C. References of antibodies are listed in the supplemental Table S1. Membranes were washed with PBS 0.5% Tween for 30 min and incubated with secondary antibodies conjugated with Horse Radish Peroxidase (HRP) for 2 h at room temperature. Membranes were analysed with SuperSignal west Dura Chemiluminescence Substrate (Pierce).

Protein lysate from one million cells were extracted on ice using Golden Lysis Buffer (10 mM Tris pH 8.0, 400 mM NaCl, 1% Triton X-100, 10% Glycerol+Complete protease inhibitor cocktail (Roche), phosphatase inhibitor (Sigma)). Protein concentration was measured using Pierce’s BCA Protein Assay Kit and analyzed on the Versamax spectrophotometer at a wavelength of 560nm. Appropriate volumes containing 20ug of protein lysates combined with NuPage LDS Sample Buffer and NuPage Reducing Agent (Invitrogen) were run on 4–12% (or otherwise indicated) NuPage Bis-Tris gels in MOPS buffer. Proteins were transferred onto a PVDF membrane (Millipore), blocked in 5% milk (TBST + dry milk) for one hour and incubated in the primary antibody (in 5% milk) overnight at 4°C. Membranes were washed with 0.05% TBST (TBS + 5% Tween) and secondary antibody incubations were done at room temperature for one hour. Proteins were visualized using Amersham ECL Prime Western Blotting Detection System and Amersham Hyperfilm ECL (GE Healthcare).
The following primary antibodies were used: mouse anti-Actin (1:10,000; Abcam), mouse anti-myc-tag (1:1000; Cell Signaling). Secondary antibodies goat anti-rabbit (Santa Cruz) and goat-anti-mouse (GE Healthcare) were used at concentrations of 1:10,000.

Virus samples were subjected to SDS gel electrophoresis under denaturing and reducing conditions in 1× NuPAGE LDS Sample Buffer (Invitrogen) with 10% NuPAGE Reducing Agent (Invitrogen). NuPAGE 4–12% Bis-Tris precast gels (Invitrogen) were used with MES running buffers and Novex Sharp Pre-stained Protein Standard (Invitrogen) in an XCell Sure Lock system (Invitrogen) according to manufacturer’s instructions. Protein band detection was performed by using acidic Coomassie Brilliant Blue R250 solution or by silver staining as described previously [47 (link)].
Protein bands were excised from the gel, digested by trypsin (porcine, Roche), peptides were extracted from the gel, purified and prepared for MS analysis as described previously [48 (link)].

6-well plates were seeded with approximately 1.0 x 10⁶ HEp2 cells, mock-infected or infected next day at an MOI of 5, then proteins were extracted using MPER (Pierce) with Protease Arrest and scraping from triplicate wells at respective times post-infection. Cell lysates plus NuPAGE Reducing Agent (Invitrogen) were heated at 70°C for 10 minutes and equal amounts of protein were resolved on 4–12% Bis-Tris gels (Invitrogen) in triplicate, followed by transfer to nitrocellulose. Blots were blocked in Rockland IR blocking buffer overnight and then probed with Goat anti-Ap3Mu3A (1:200), Mouse anti-RSV Matrix (1:1000), Rabbit anti-delta adaptin (SA4) (1:1000), Rabbit anti-GAPDH (1:10000) for one hour. Washed extensively in Tween-20 containing wash buffer, then blotted with species-specific IR labeled antibodies: Donkey anti-Goat IR 800 (1:25000), Donkey anti-Rabbit IR 800 (1:25000), Donkey anti-Rabbit IR 700 (1:5000), Donkey anti-Mouse IR 700 (1:5000), Donkey anti-Mouse IR 800 (1:20000) for an hour and then washed extensively. Blots were imaged on Licor IR Imager, fluorescent intensity analysis was performed using Licor imaging program and subsequent data analysis was performed by comparing infected versus uninfected triplicate averages of the integral intensity ratio between GAPDH loading control and AP3 integral intensities via unpaired two tail t-test.

For immunoblot analysis, HEK293 T-REx drTPRM7 wild type or truncation mutant cells were induced for 16 h with 1 μg/mL tetracycline added to the media. Cells were harvested and dissolved in lysis buffer (10 mM Tris-HCl, 75 mM NaCl, 5% glycerol, 0.5% triton, 5 mM EDTA, 1 mM PMSF) containing protease inhibitors (104 mM AEBSF, 80 μM Aprotinin, 4 mM Bestatin, 1.4 mM E-64, 2 mM Leupeptin and 1.5 mM Pepstatin A) for 30 minutes at 4 °C. After incubation, the lysates were centrifuged at 12,000 rpm for 5 min. at 4 °C and the protein concentration of the lysates were measured using a protein assay reagent (Thermo Scientific, USA). Soluble lysates were boiled with NuPAGE LDS sample buffer and NuPAGE Reducing Agent (Invitrogen) at 95 °C for 8 minutes. Equal amounts of proteins (40 μg) were loaded and separated in a NuPAGE 4–12% gel (Invitrogen) then transferred to a PVDF membrane. Proteins were detected by immunoblotting with the antibodies anti-HA (3F10, Roche), anti-GAPDH (6C5, Abcam, UK) followed by treatment with horseradish peroxidase (HRP)-conjugated anti-rat or anti-mouse antibody (GE Healthcare, USA). GAPDH was evaluated as an internal control. The antibody-bound protein was visualized using ECL solution (Life Technologies, USA.)

Proteins were extracted from the synchronized cell pellets with a non-denaturing lysis buffer (1% Triton X-100, 50 mM Tris-HCl, 300 mM NaCl, 5 mM EDTA) containing phosphatase and protease inhibitor cocktail (Roche, Penzberg, Germany) for 30 min at room temperature (RT) in 1:3 ratio (volume-to-volume, v:v). Nuclear proteins were extracted with lysis buffer containing 1% (v:v) NP-40, 0.1% (weight-to-volume, w:v) Na-deoxycholate, 300 mM NaCl, 50 mM Tris-HCl, and phosphatase and protease inhibitors. The lysates were centrifuged at 18,000× g for 15 min at 4 °C, and the supernatant was recovered and stored at −80 °C. Protein concentration was determined with the BCA (bicinchoninic acid) assay calibrated with BSA (bovine serum albumin provided with the kit) according to the manufacturer’s recommendations. Protein samples (50 μg) were denatured by incubation at 70 °C for 10 min with the NuPAGE Reducing agent (1:10, v:v; Invitrogen, Carlsbad, CA, USA) and the NuPAGE LDS Sample buffer (1:4, v:v; Invitrogen, Carlsbad, CA, USA).