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Protein Solubilisation and Preparation

For in vitro experiments, hPASMCs, at the end time point of an experiment, were placed on ice. The media was aspirated off, and the monolayer of cells was then washed twice with DPBS. 120μL of 1% (v/v) lauryl maltoside (LM, Abcam, UK) buffer in DPBS was added to each well, when protein was extracted from 6-well plates. When protein was extracted from unstimulated hPASMCs growing in a T75cm² culture flask, 600μL of 1% LM, supplemented with protease inhibitors (0.1mmol/L PMSF, 1μg/mL soybean trypsin inhibitor and 1μg/mL benzamidine), was used. The cells were then collected using a sterile plastic scraper, collected in pre-chilled 1.5mL Eppendorf tubes and left on ice for 30 minutes to allow for improved disassociation of protein complexes and protein solubilisation. Cell lysates were then sonicated in iced water, before centrifuging at 10,000rpm (4°C) to remove any debris.

Alternatively, when extracting protein from tissue, a piece of tissue was placed in a 2mL Eppendorf tube, containing 300µL of 1% LM buffer. A 5mm stainless-steel ball (Qiagen, UK) was added, and the tissue homogenised using the Tissue Lyser II (Qiagen, UK).

Homogenised tissue was then left on ice for 30 minutes to promote disassociation of protein complexes and improve protein solubilisation. Tissue samples were then centrifuged at 10,000rpm for 10 minutes (4°C).

Protein supernatant was collected into a new pre-chilled Eppendorf tube, and stored at -80°C until further.

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Bicinchoninic Protein Assay

Protein concentration in supernatants was determined using the bicinchoninic acid (BCA) assay (Pierce, Thermo Fisher, USA). To quantify protein concentration in collected samples, a standard curve was constructed within the 0.0–2.0mg/mL concentration range, using the albumin standard ampules consisting the BCA Kit, diluted in 1% LM. Briefly, suitable volume of the albumin standards and unknown samples was pipetted into a 96-well plate.

BCA assay working solution was prepared by combining 9.8mL of reagent A with 200µL of reagent B. 200µL of the working solution was quickly added to each well and incubated at room temperature with gentle agitation on micro-plate shaker for 30 minutes. Protein concentration was then evaluated against the standard curve, by measuring the absorbance at 652nm with POLARstar OPTIMA microplate reader (BMG Labtech, Germany).

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis

Samples, containing equal amounts of protein, were prepared by adding NuPAGE® LDS (lithium dodecyl sulphate) Sample Buffer (4X) and NuPAGE® Reducing Agent (10X) (10mmolL/L dithiothreitol), and heated at 70°C for 10 minutes. Samples were then separated per their molecular weight on Novex® Tris-Glycine gels (Invitrogen, Life Technologies, UK) using Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis, under constant voltage at 110–150V in NuPAGE MOPS SDS running buffer (20x), containing 1% (v/v) NuPAGE® Antioxidant.

Protein Transfer

Once adequate separation of protein according to their weight was achieved, the protein was blotted onto hydrophobic Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore, USA) using a wet blotting system by Life Technologies. Due to its hydrophobic character, the membrane was immersed into 100% methanol prior to use in order to activate it and expose membrane’s full protein binding capacity. The buffer for wet blotting contained 5% (v/v) NuPAGE Transfer Buffer (20x) and 20% (v/v) methanol. Proteins were transferred onto PVDF membranes at constant voltage (35V) for 2 hours and 15 minutes.

Transfer efficiency was assessed by a short incubation of membranes in 0.1% (w/v) solution

91 of Ponceau S stain, prepared in 5% (v/v) acetic acid solution. The stain was washed off the membranes in 10% (v/v) TBS buffer containing 0.1% (v/v) Tween-20 (Fisher Scientific, UK) (TBST) washing buffer, before blocking in 5% (w/v) milk prepared in TBST, for 1 hour at room temperature in order to minimise non-specific binding of the antibody. The residual blocking solution was washed off the membrane by numerous short washes in TBST.

Immunoblotting

The detection of a specific protein was achieved by an overnight incubation of the membrane in primary antibody (the dilutions and concentrations are provided in Table 2-1 on a shaker at 4°C. The membranes were then washed for 3x10 minutes in TBST washing buffer to remove any unbound primary antibody, before incubating in secondary antibody conjugated with horseradish peroxidase (HRP) (Table 2-1) prepared in 5% (w/v) BSA (Sigma, UK) in TBST for 1 hour at room temperature. The membrane was again washed in TBST buffer for 3x15 minutes. The secondary antibodies used were coupled with HPR, catalysing the oxidation reaction of the luminal, which is present in the substrate Immobilon western chemiluminescence HRP substrate (Merck Millipore, USA) or Pierce ECL Western Blotting Substrate (Thermo Scientific, USA). The light emitted during the reaction catalysed by HPR, was captured on X-ray film (Kodak, UK), allowing protein visualisation after film processing using MIN-R Mammography Processor. When samples were probed for several proteins, membranes were washed in TBST for 5 minutes, following a 15-minute incubation in the Restore western blot stripping buffer (Thermo Scientific, UK) on a shaker, during which the bound antibodies were stripped. Equally, to reduce the number of times the PVDF membranes were stripped, the membranes were cut alongside suitable molecular-weight size markers, labelled and probed as described previously.

The membranes were re-probed for another protein of interest, or for loading control (β-actin, GAPDH). Protein expression was quantified by densitometry analysis using TotalLab TL100 v2006 programme (TotalLab, United Kingdom). In the following chapters the representative immunoblotting images are shown. Where images have been altered from full blots to remove redundant samples, it is stated in the legend.

92 Table 2-1: List of antibodies used for immunoblotting.

Primary

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Id3 1:1000 M100 Cal

Bioreagents

17 Rb 1:5000

pSmad1/5/9 1:1000 9511 Cell Signalling

55–60 Rb 1:5000

GAPDH 1:1000 ab9484 Abcam 40 Ms 1:10000

AhRR= AhR repressor, PHD2= Prolyl hydroxylase 2, HIF1α= Hypoxia-inducible factor 1α, ARNT (HI1β)= Aryl hydrocarbon receptor nuclear translocator, vHL= von Hippel-Lindau tumour suppressor , VEGFR2= Vascular endothelial growth factor receptor 2, p27/Kip1= Cyclin-dependent kinase inhibitor 1B (p27/Kip1), CYP1A1=, Cytochrome P450 1A1, CYP1B1=, Cytochrome P450 1B1, 17βHSD2= 17β-hydroxysteroid dehydrogenase type 2, AhR= Aryl hydrocarbon receptor, BMPRII= Bone morphogenetic protein receptor II, Id1= Inhibitor of DNA binding 1, Id3= Inhibitor of DNA binding 3, pSmad1/5/9= phosphorylated Smad 1/5/9

Table 2-2: List of positive controls.

Protein Positive Control # Supplier CYP1A1 HeLa whole cell

lysate

ab29545 Abcam

CYP1B1 Human liver tissue lysate, total protein

ab29889 Abcam

CYP1B1 Rat liver lysate N/A

AhR Rat kidney lysate N/A

HIF1α CoCl2-treated hPASMC lysate

N/A Caspase 3 Resveratrol-treated

EC lysate

N/A

HIF1α= Hypoxia-inducible factor 1α, ), CYP1A1=, Cytochrome P450 1A1, CYP1B1=, Cytochrome P450 1B1, AhR= Aryl hydrocarbon receptor

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