Bolt mini gel tank

The bacterial outer membrane extracts (OMEs) of AaD7S and its mutants AaΔ29, AaΔ29P, and AaΔ29Δ29P were obtained as previously described [28 (link)], and outer membrane proteins (OMPs) were resolved by standard SDS-PAGE (10% acrylamide) [29 (link)]. Purified Aa OMP29^His was also detected after SDS-PAGE using the Bolt Bis-Tris Plus gel (Life Technologies, Carlsbad, CA, United States) and the Bolt Mini Gel Tank (Life Technologies). The gels were stained with Colloidal blue (Life Technologies). OMP29 protein has the approximate size of 29 kDa, however, appears as a 34 kDa band when denatured [14 (link)].

N9 cells were lysed in RIPA buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Sigma-Aldrich). Protein samples (30 μg) were electrophoresed on a 4–12 % gradient polyacrylamide pre-cast gel (Life Technologies), using Bolt^® Mini Gel Tank (Life Technologies) and transferred to nitrocellulose membrane (BioRad) using XCell II™ Blot Module (Life Technologies). Membranes blocked with 5 % non-fat milk in PBS/0.1 % Tween 20 were incubated with primary antibodies overnight, washed and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:4000, Merck-Millipore, AP307P) for 1 h, and visualized using ECL Plus (Thermo Fisher Scientific). Quantification of relative protein amounts was performed by densitometric analysis using ImageJ software (NIH), normalized to a loading control protein, growth factor receptor-bound protein 2 (GRB2). Primary rabbit antibodies used were: anti-phospho-5′ adenosine monophosphate-activated protein kinase (AMPK) Thr172 (1:1000, Cell Signaling, 2535), anti-AMPK (1:3000, Cell Signaling, 2603), anti-acetyl-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) Lys310 (1:1000, Cell Signaling, 3045), anti-NF-κB (1:3000, Cell Signaling, 4764), and anti-GRB2 (1:2000, Abcam, ab32111) antibodies.

Proteins were analyzed by Bolt^® Bis-Tris Plus 4–12% gradient gels (Life Technologies) with the Bolt^® MES as running buffer (B0002, Life Technologies). Samples were prepared by mixing 10 μl of SDS loading buffer (161–0747, Bio-Rad) with 40 μl of protein samples and boiling for 5 min at 95°C. Samples of 5 μg of the protein were loaded on the gel (Fig 2). Electrophoresis was run using the Bolt Mini Gel tank (Life Technologies) at 165 volts for 35 min.

Protein isolated from cell lines was separated on Bolt NuPAGE 4–12% Bis-Tris Plus gels and a Bolt Mini Gel Tank (all from www.lifetechnologies.com). Immunoblotting for hCFTR was performed by standard procedures according to the manufacturer’s instructions using the XCell II Mini-Cell and blot modules (www.lifetechnologies.com). After blocking for 1 h in 5% dry milk at room temperature, primary antibody against hCFTR clone 596 (1:500, kindly provided by the cystic fibrosis foundation therapeutics Inc.) or anti-GAPDH (1:1000) (www.scbt.com) was incubated overnight, horseradish peroxidase–conjugated secondary antibodies (anti-mouse from www.dianova.com) were incubated for 1 h at room temperature. All blots were processed by using ECL Prime Western Blot Detection Reagents for 30 min exposure time (www.gelifesciences.com). Semiquantitative analysis was performed using the ImageJ software and overexposure has been avoided as per as digital image and integrity policies.

At the indicated time points, soluble protein fractions were obtained as described above. NuPAGE LDS sample buffer (Invitrogen) and NuPAGE sample reducing agent (Invitrogen) were added, and samples were loaded on Novex 8% Tris-glycine gels (Invitrogen) in a Novex Bolt mini gel tank with MOPS (morpholinepropanesulfonic acid) buffer. Proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane using preassembled Trans-Blot Turbo transfer packs and a Trans-Blot Turbo transfer system (Bio-Rad). The voltage was set to 15 V, and samples were run for 7 min. The membrane was washed with Tris-buffered saline (TBS) supplemented with 0.1% Tween20 and blocked by incubation in blocking buffer (10% skimmed milk powder in TBS-Tween 20) for 2 h. Afterward, the membrane was washed with TBS-Tween 20, and GyrA was detected with polyclonal anti-GyrA (1/200). After overnight incubation with the primary antibody, membranes were washed with TBS-Tween 20. The secondary antibody, anti-rabbit IgG fused to horseradish peroxidase (1/50,000), was added, and the membrane was incubated for 55 min. After washing with TBS-Tween 20, detection was done by adding Clarity Western ECL substrate (Bio-Rad) and visualization with the Fusion FX imaging system (Vilber Lourmat). Pictures were taken, and band intensities were quantified using ImageJ.

The soluble byssal extract from two extraction rounds (20 TP total) was desalted and concentrated via three rounds of ultra-centrifugal filtration using 10% acetonitrile in water with Amicon filters with a cutoff particle size of 3 kDa (EMD Millipore, Billerica, MA, USA). Gel electrophoresis was performed using Life Technologies Bolt 12% Bis-Tris pre-cast 10 well gels, Bolt MES-SDS running buffer (Bis-Tris SDS-PAGE), and Bolt LDS sample buffer. Gels were run in a Bolt Mini Gel Tank, with a constant 165 V voltage setting, for 35–40 minutes and stained with SimplyBlue Safestain (Invitrogen). High-intensity stained gel bands at ~6, ~7, ~14, and ~28 kDa were clipped, de-stained, and stored in 1% acetic acid at 4 °C until use.