Bca 1 kit

Standard methods were used throughout (Coligan, 2003 ; Ausubel et al., 2005 ). Protein concentrations were determined using the BCA-1 kit (Sigma-Aldrich). SDS-PAGE was performed as in Laemmli (1970) (link) using a Low Molecular Weight Calibration Kit for SDS electrophoresis (Cytiva).

Standard methods were used throughout (Ausubel et al., 2005 ). All chemicals were purchased in analytical grade unless otherwise indicated. Competent E. coli were prepared as described in Hanahan et al. (1991) (link). Plasmid and PCR product purification used the PureLink Plasmid DNA Miniprep and PCR Purification Kits (Life Technologies). Genomic DNA was isolated using the Genomic DNA Mini Kit (Life Technologies). Restriction enzymes were from Thermo Fisher (Waltham, MA, United States); T4 Ligase and RNAse inhibitor were from Promega. General PCR used GoTaq Master Mix Green (Promega), and high-fidelity amplification used Phusion Master Mix (Finnzymes/Thermo Fisher). Protein concentrations were determined using a bicinchoninic acid-based assay (BCA-1 kit, Sigma-Aldrich, St. Louis, MO, United States). Small-scale cell extracts of H. influenzae were prepared using BugBuster Master Mix (Novagen) as per the manufacturer’s instructions.

Standard methods were used throughout [25 ]. Genomic DNA, plasmid and PCR products were isolated using the Genomic DNA mini kit (Life Technologies, Carlsbad, CA, USA), the Purelink Plasmid DNA miniprep kit and the Purelink Quick PCR Purification Kit (both Life Technologies, Carlsbad, CA, USA), respectively, accordingly to the manufacturer’s protocols. Restriction enzymes were from Life Technologies or NEB, T4 ligase from Promega (Madison, WI, USA) or Life Technologies. SDS-PAGE used the method of [26 (link)], desalting of protein samples used PD-10 columns (Cytiva, Marlborough, MA, USA), and BCA protein determinations (BCA-1 kit, Sigma Aldrich, St. Louis, MO, USA) were performed according to the manufacturer’s protocol.

NTHI strains were grown under anaerobic or microaerophilic conditions in 150 mL of sRPMI. Bacteria were harvested at 2300 × g for 10 min at 4°C and cell pellets stored at –20°C before being used. Cells were disrupted using BugBuster® Master Mix (Novagen) and insoluble components were removed by centrifugation at 13000 × g (15 min, room temperature). The resulting supernatant (crude extract) was collected and used for enzyme assays.
Sulfoxide and nitrate reductase activities were assayed at 37°C by monitoring the oxidation of reduced benzyl viologen at 600 nm (ε₆₀₀ = 7.4 mM⁻¹ cm⁻¹; Dickie and Weiner, 1979 (link)). Enzymes assays were performed anaerobically in 20 mM sodium phosphate buffer (pH 6.8) containing 0.2 mM benzyl viologen, 0.3 mM sodium dithionite, and one of the following terminal electron acceptors: 17 mM DMSO, 5 mM DL-methionine sulfoxide (MetSO), or 10 mM potassium nitrate.
Formate dehydrogenase activity was determined anaerobically at 600 nm in assays containing 75 μM of dichlorophenolindophenol (DCPIP; ε₆₀₀ = 21 mM⁻¹ cm⁻¹) in 20 mM sodium phosphate buffer (pH 6.8), 0.25 mM phenazine metasulfate, and 20 mM sodium formate as electron acceptor (Enoch and Lester, 1982 (link)). Specific activities are given as μmol of substrate reduced or oxidized per min (U) and mg of protein present. Protein concentrations were determined using the BCA-1 kit (Sigma-Aldrich).

Nuclear fractions were prepared from 450 ml of synchronized cultures with ∼2 × 10⁶ cells ml⁻¹ that had been incubated with or without 2 μM RB under light for 40 min. The cells were collected and treated with autolysin for 40 min and examined for the removal of cell walls by addition of 1 volume of 0.1% Triton-X. Nuclear extract was prepared as described previously (Winck et al., 2011 (link)) using CelLytic PN kit (Sigma-Aldrich, St. Louis, MO). Because there were bands detected in the nuclear extract close to the size of SAK1, nuclear extract was prepared from WT (4A+) and sak1 rather than a cell wall-deficient strain (cw15). Chloroplasts were isolated from cell wall-less strain cw15 as described previously (Klein et al., 1983 ). Mitochondria were isolated as described (Eriksson et al., 1995 (link)). After unbroken cells, chloroplasts, and mitochondria were collected, the ER fraction was collected by centrifugation at 100,000×g for 90 min at 4°C. The remaining supernatant was enriched for cytosol. Protein was extracted and prepared for SDS-PAGE as described (Calderon et al., 2013 (link)) with minor modifications. Protein was quantified by using BCA1 kit (Sigma-Aldrich, St. Louis, MO) after extraction with the methanol-chloroform method (Wessel and Flügge, 1984 ).

Standard methods were used throughout (Ausubel et al., 2005 ). All chemicals were purchased in analytical grade or equivalent. Genomic DNA was isolated using DNAzol reagent (Thermo Fisher Scientific). GoTaq Green MasterMix (Promega) was used for general PCR (primer sequences, Supplementary Table S2), the GeneJET Plasmid Miniprep Kit and GeneJET PCR Purification kit (both Thermo Fisher Scientific) for plasmid and PCR product purification. Restriction enzymes were from Thermo Fisher Scientific, T4 ligase (NEB) was used for ligations. Cell-free extracts of NTHi were generated using BugBuster Mastermix (Novagen) according to the manufacturer’s instructions (750 μL were used for pellets from 50 mL culture). Protein concentrations were determined using the BCA-1 kit (Sigma Aldrich).

Cells were seeded in 24-well plates and incubated for 3, 6, and 24 h with NPs loaded with 0.1, 0.5, 1.25, 2.5, or 5 μg/μL C12DOXO. At the end of the incubation period, the culture medium was aspirated; then, the cells were washed twice with PBS, detached with 0.50 μL of trypsin, and suspended in 250 μL of PBS. An aliquot of 50 μL was sonicated and used for protein quantification with the BCA-1 kit (Sigma-Merck). The remaining cell suspension was transferred to a 96-well plate and used to read the intracellular fluorescence of C12DOXO as an index of drug uptake using a Synergy HTX multiplate reader. The excitation and emission wavelengths were 596 and 615 nm, respectively. Fluorescence units were converted to nmol of C12DOXO on the basis of a calibration curve generated using free C12DOXO solutions at the following concentrations: 1, 10, 100, 250, and 500 nmol/mL. The results were expressed as nmol/mg cellular proteins.