Anti rabbit 568

Anti-E-cadherin (HECD-1) antibody was from Invitrogen. Anti-CAR (H300), anti-TNFR1 (H5), anti-ICAM1 and anti-PKCδ antibodies were from Santa Cruz (Germany). Anti-phospho-PKCδ was from Cell Signalling Technology (USA). p-CAR thr290/ser293 polyclonal antibody was previously described in Morton et al.15 (link), and was developed by Perbioscience (Thermofisher) using the peptide Ac- RTS(pT)AR(pS)YIGSNH-C and was affinity purified before use. Anti-TNFR1 blocking antibody (Mab225) was obtained from R&D Systems (USA). Anti-GFP antibody was from Roche (UK). Anti-mouse HRP and anti-rabbit-HRP were from DAKO, anti-mouse-568, anti-rabbit-568 and phalloidin-633 were all obtained from Invitrogen (UK). CalyculinA, sodium orthovanadate and PI-3-kinase inhibitor (LY294002) were obtained from Calbiochem. TNFα, IL-5, IL-1β, IL-13 and IL-17 were obtained from Sigma-Aldrich (UK). FK was produced and purified as previously described35 (link). PKCδ targeted siRNA and non-targeting controls were obtained from Ambion (USA).

To investigate co-localisation of hnRNP E2 with ubiquitin and TDP-43, double immunofluorescence was carried out on a subset of the FTLD-TDP cases. Seven micrometer sections cut from formalin-fixed paraffin-embedded blocks were deparaffinised in xylene and dehydrated in 99% industrial methylated spirit. Sections were pre-treated with microwave heating in citrate buffer and normal serum blocking was performed using normal goat serum (1:10 for 45 min). Sections were incubated overnight at 4°C with hnRNP E2 (hnRNP E2-23G: sc-101136, Santa Cruz) at 1:200 with either TDP-43 (Proteintech, 10782-2-AP) at 1:250 or anti-ubiquitin (Dako, Z045801) at 1:100.
After washes, sections were incubated with AlexaFluor secondary antibodies (goat anti-mouse 488 and goat anti-rabbit 568, Invitrogen, Paisley, United Kingdom). Sections were treated with Sudan Black for 10 min to quench autofluorescence. Following numerous washes in phosphate buffered saline, sections were mounted with Vectashield hard set media containing DAPI. Sections were visualized using a fluorescent microscope (Zeiss Axiovert S 100, Gottingen, Germany) and images captured using ImagePro Express (v6) (Meyer Instruments, Houston, TX, United States). Sections were also examined using confocal microscopy (Leica confocal SP system) and captured images analyzed using ImageJ 1.47v software.

Anti-B7-H3 (ThermoFisher, Loughborough, UK (IP), AF1027 BD Bioscience, Wokingham, UK (WB), and MAB1027 Santa Cruz, Heidelberg, Germany (IF)), anti-EEA1 (Cell Signaling, Leiden, The Netherlands), anti-HSC70 (Santa Cruz), anti-IMPDH2 (Proteintech), anti-Rab10 (Abcam, Cambridge, UK), anti-Rab11 (Cell Signaling), and HTII-280 AT2 (terrace Biotech, San Francisco, USA) were used. IgG negative control was used for IP and obtained from DAKO. DAPI (Sigma-Aldrich, Gillingham, UK) was used as nuclear stain for immunofluorescence. LysoTracker™ Deep Red was used to stain lysosomes (ThermoFisher). Anti-mouse-HRP, anti-rabbit-HRP, and anti-goat-HRP were from DAKO; anti-mouse-568, anti-rabbit-568, anti-rabbbit-488, and phalloidin-488 and -647 were all obtained from Invitrogen. Other reagents used were CellROX™ Deep Red Reagent (for oxidative stress detection) (ThermoFisher). CellEvent™ Caspase-3/7 Green Detection Reagent (ThermoFisher), Cisplatin (Cambridge Bioscience, Cambridge, UK), and Mycophenolic acid (MPA; IMPDH inhibitor) (Sigma-Aldrich).

Anti-integrin β1 (12G10, Santa Cruz), anti-active β1 integrin (9EG7, Merck Millipore), anti β-catenin [Santa Cruz (IF) and BD Bioscience (WB)], anti-CAR antibody (Santa-Cruz), anti E-Cadherin (Abcam), anti-FAK (Cell signalling), anti-green fluorescent protein (GFP) (26 (link)), anti- Heat Shock Cognate 70kDa (HSC70) (Sigma-Aldrich), anti-paxillin (BD Bioscience), anti phospho-FAK (Y397, Cell signalling), anti-phospho-paxillin (Y118, Cell signalling), anti-phospho-src (Y418, Millipore), anti-rap1 A/B (R&D Systems) and anti-src (Millipore) antibodies were used for western blot. Adenovirus Type5 fiberknob (Ad5FK) was produced and purified as previously described (27 (link)). CAR thr290/ser293 polyclonal antibody was developed by Perbioscience (Thermofisher) using the peptide Ac-RTS (28 (link))AR(pS)YIGSNH-C and was affinity purified before use. DAPI (Sigma Aldrich) was used as nuclear stain for immunofluorescence. Anti-mouse-HRP, anti-rabbit-HRP, anti-goat-HRP were from DAKO, anti-mouse-568, anti-rabbit-568, anti-goat-568 and phalloidin-647 were all obtained from Invitrogen. Inhibitors to FAK (PF228), Rap1A (GGTI298) and src (PP2) were all obtained from Tocris.

Immunocytochemistry was performed as previously described.^{35 (link)} Briefly, HMVEC were fixed with 4%PFA (paraformaldehyde, Thermo Fisher Scientific) for 1 h at room temperature, permeabilized for 15 min with PBS supplemented with 0.3% Triton-X (Sigma) and 0.5% BSA (Sigma), blocking was performed with PBS supplemented with 5% BSA and 0.1% Triton-X. Cells were subsequently stained for vWF overnight at 4C (NB600–586, Novus Biologicals diluted 1:300 in PBS with 0.5% BSA). Cells were washed with PBS and incubated with anti-Rabbit 568 (Invitrogen, diluted 1:500) and DAPI (4′,6-diamidino-2-phenylindole, Sigma, 1 µg/mL) in PBS with 0.5% BSA for 2 h. Cells were then washed with PBS prior to imaging. Images were acquired on a Leica SP5 confocal microscope and analysed using FIJI ImageJ software.