Pan actin

Cells were harvested and lysed in radioimmunoprecipitation (RIPA) buffer containing protease and phosphatase inhibitors. An amount of 20 µg of protein for each sample was denatured in LDS sample buffer (Invitrogen Ltd., Paisley, UK) at 95 °C for 5 min and resolved by SDS-PAGE on 10% Bis-Tris gels (Invitrogen Ltd., Paisley, UK). The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane. Immunoblotting was carried out using antibodies against GAPDH (Abcam, Cambridge, UK), TRAF6, Total/p-IKKα/β, Pan-Actin, Total/p- TRAF6, Total/p-IKα, Total/p-p65 (Cell Signaling Technology, Hertfordshire, UK) and secondary antibodies anti-mouse and anti-rabbit (DAKO, Cambridgeshire, UK). Blots were probed with WesternBright™ ECL horseradish peroxidase (HRP) substrate (Advansta, Labtech, East Sussex, UK) for visualisation of protein.

Antibodies were used for both Western blots and immunofluorescence. Primary antibodies for Arp2, Arp3, α-tubulin, β-actin, E-cadherin, KinomeView Profiling kit, N-WASP, pan-actin, phospho-PLK1 (Thr210), PLK1, phospho-Rac1/Cdc42(Ser71), Rac1/Cdc42, vimentin, and WAVE-2 were purchased from Cell Signaling Technology (#3128, #4738, #2144, #4970, #3195, #9812, #4848, #8456, #9062, #4513, #2461, #4651, #5741, and #3659). PLK1 primary antibody used for immunofluorescence was purchased from Thermo Fisher Scientific (#37-7100); γ-actin antibody was purchased from Abcam (#ab123034). HCV NS5A sheep polyclonal antibody and NS5A mouse antibody were gifts from Professor Mark Harris (University of Leeds, UK) and Associate Professor Michael Beard (University of Adelaide), respectively. Secondary antibodies were either conjugated with HRP; anti-rabbit Ig/HRP (#P0448), and anti-mouse Ig/HRP (#P0161) (DAKO) or labelled with fluorescence dyes: antirabbit IgG Alexa Fluor 488 (#A21206), anti-mouse IgG Alexa Fluor 488 (#A21202), anti-mouse IgG Alexa Fluor 594 (#A21203), and anti-rat IgG Alexa Fluor 647 (#A21472) (Thermo Fisher Scientific).

Quercetin (Sigma-Aldrich, Darmstadt, Germany) was added to the culture media, and heated at 37 °C with continuous stirring for at least 30 min in order to ensure that it was dissolved. It was used at the working concentration of 400 μM. PI3K/AKT pathway modulators Wortmannin (Abcam, 120148, Cambridge, UK) and SC79 (Abcam, 146428, Cambridge, UK) were used at working concentrations of 2 μM and 22 nM respectively.
Primary antibodies used for immunofluorescence and western blotting were: Cldn1 (Invitrogen, 374900, Carlsbad, CA, USA), Cldn2 (Invitrogen, 516100, Carlsbad, CA, USA), Cldn3 (Abcam, 15102, Cambridge, UK), Cldn4 (Invitrogen, 364800, Carlsbad, CA, USA), Cldn7 (Spring Bioscience, E10594, Pleasanton, CA, USA), ZO1 (Invitrogen, 339100, Carlsbad, CA, USA), pPI3K (Cell signaling, 4228, Danvers, MA, USA), pAKT (Cell signaling, 9271, Danvers, MA, USA), AKT (Cell signaling, 9272, Danvers, MA, USA), and pan-actin (Cell signaling, 4968, Danvers, MA, USA,). In addition, secondary goat anti-rabbit (Alexa Fluor 595 and 488, Invitrogen, Carlsbad, CA, USA), goat anti mouse (Alexa Fluor 595 and 488, Invitrogen, Carlsbad, CA, USA), goat anti-rabbit-HRT conjugated (Cell Signaling, 70748, Danvers, MA, USA), and goat anti mouse peroxidase conjugated (Jackson ImmunoResearch, 115-035-146, West Grove, PA, USA) were used.