Anti pkcδ

Cells were lysed with RIPA buffer (25 mM Tris-Cl pH 7.6, 150 mM NaCl, 1% Triton-X-100, 0.1% SDS, 1% sodium deoxycholate, 1% PMSF, and 1% proteinase inhibitor cocktail; Roche) and samples were quantified using a protein assay (Micro BCA; ThermoFisher Scientific). The same amount of protein was separated using SDS-PAGE (10%) and the gel scanned with a Typhoon 9410 Gel and Blot Imager (GE healthcare life sciences).
For immunoblots, proteins were transferred to PVDF membranes (Roche, Mannheim, Germany), and the membrane was blocked for 1 h at RT in 5% non-fat milk in TBS-T (137 mM NaCl, 2.7 mM KCl, 25 mM Tris, 0.1% Tween-20), and probed overnight at 4 °C with the following primary antibodies: rabbit polyclonal anti-Kv1.1 (Alomone Labs, # APC-161 and APC-009), rabbit polyclonal anti-PKCδ (Santa Cruz, # sc-937), rabbit polyclonal anti-phospho-PKCδ (Tyr311) (Cell Signaling, # 2055), rat monoclonal anti-ARTD10 (Merck, #5H11), or mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich, # T7451). Blots were visualized using secondary HRP-conjugated anti-rabbit or anti-mouse antibodies and SuperSignal West Pico or Femto PLUS Chemiluminescent Substrate (ThermoFisher Scientific). The data was analyzed with ImageJ.

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).

Western blotting was performed by modifying a previously described procedure [26 (link)]. Briefly, platelets were lysed in buffer A and B and centrifuged at 20,000× g for 30 min, and the supernatant was collected to obtain the cell membrane lysate. Then, proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and reacted with anti-PKC-α antibody (1:500, Santa Cruz), anti-PKC-βI (1:500, Santa Cruz), anti-PKC-βII (1:500, Santa Cruz), anti-PKC-γ (1:500, Santa Cruz), anti-PKC-δ (1:500, Santa Cruz), or anti-actin (1:3000, Cell Signaling) overnight. All samples were analyzed using a LAS 4000 mini (Fuji Photo Film, Tokyo, Japan). This experiment was repeated a total of three times.

Western blot was performed as described in a previous publication [37 (link)]. Primary antibodies used were anti-PKCδ (1:500, Santa Cruz), anti-Smac (1:500, Santa Cruz), anti-Actin (1:2000, MP Biomedicals), anti-HSV (1:1000, Novagen), anti-GST (1:2000, GE Healthcare) and anti-GFP (1:1000, Invitrogen). Secondary horseradish peroxidase-labeled antibodies used were from GE Healthcare and Dako. For the chemiluminescent reaction, Supersignal Substrate (Thermo Scientific) was used according to manufacturer’s instructions. Chemiluminescence was detected with a LAS-1000 charge-coupled device camera (Fujifilm) and Image Reader LAS-1000 Pro v2.6 software (Fujifilm). Image quantifications were performed using ImageJ 1.48v and by normalizing band intensities to input fractions and control.

After fixing the slices, antibody treatments were performed as described previously6 (link)25 (link)47 (link) with minor changes. Slices were rinsed twice with 1 ml of 1X PBS and fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. Before antibody treatments, slices were washed twice with PBS for 15 min each at RT to remove residual PFA and incubated with blocking buffer (0.05% TritonX-100 and 3% goat serum in 1X PBS) for 1 h at RT. Primary antibodies were diluted (1:1000 unless otherwise stated) in blocking buffer and incubated for 2–3 days at 4 °C. The following antibodies were used: anti-NeuN (clone A60, Alexa Fluor^®488 conjugated Millipore, Cat No. MAB377X) and anti-PKCδ (Santa Cruz). After incubation with primary antibody, membranes were washed 4 times with PBS for 15 min each and incubated 1 h in the dark with the following secondary antibodies diluted 1:1500 in blocking buffer: Alexa Fluor 555-conjugated anti-mouse secondary antibody or Alexa Fluor 488-conjugated anti-rabbit secondary antibody. The cell-permeable dye Hoechst 44432 (1:5000 for 3 min in PBS) was used to stain all nuclei. The culture membranes were removed from the inserts and mounted directly on microscope slides, with membranes facing the slide, using Fluoromount mounting medium (Sigma) and imaged with a SPOT color digital camera attached to a Nikon TE2000-U microscope.