Ab32036

The pGEX-4T vector expressing the PKR 1–265 fragment in fusion with GST at its N-terminus (GST-PKR Nter) and the pET15b vector expressing the PACT protein have been previously described^{2 (link),15 (link)}. The pENTR™-SD⁄D-TOPO® vector and the pDEST24 were purchased from Invitrogen. Normal mouse or rabbit IgGs were from Santa Cruz Biotechnology. Mouse monoclonal anti-PKR 71/10 antibody was produced by Agrobio Laboratories (FR) as reported^{49 (link)}. Mouse monoclonal Anti-ß-actin antibody was from Sigma (A1978). Rabbit polyclonal antibodies were used to detect human PKR phosphorylated at T446 (ab32036; Abcam). Anti-PACT (sc-377103) and anti-NFκB p65 (sc-109) polyclonal antibodies were purchased from Santa Cruz Biotechnology. Goat anti-Rabbit IgG (H&L) Secondary Antibody, DyLight 800 4X PEG (#SA5-35571), Goat anti-Mouse IgG (H + L) Secondary Antibody, DyLight 680 (#35518) and Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (A11034) were from Invitrogen.

Mouse monoclonal anti-NS5A antibody (9E10) was kindly provided by Dr. Joe Grove (UCL) and has previously been described (Lindenbach et al., 2005 (link)). Antibodies against β-actin (Abcam; ab8226 or ab8227), CypA (Enzo; BML-SA296-0100), CypB (Abcam; ab16045), CypD (Abcam; ab110324), RIG-I (Cell Signaling Technology; #3743) MAVS (Santa Cruz Biotechnology; sc166583), PKR (Abcam; ab32052) and phospho-PKR T446 (Abcam; ab32036) were also used. Secondary IRDye 680- or 800-labelled antibodies and AlexaFluor-conjugated antibodies were obtained from LI-COR Biosciences or Thermo Scientific, respectively. Anti-human interferon alpha/beta receptor chain two antibody (IFNAR2) (Pbl Assay Science, 21385–1) and an IgG2A control antibody (R and D Systems, 4460 MG-100) were used at 2 μg/mL.

Twelve microliters of supernatant or cell lysate were separated on a 4–12% Mini-Protean TGX gel (Bio-Rad) at 100 V for 1 h. Proteins were transferred onto a 0.2 μm nitrocellulose membranes (Li-Cor Biotechnology) at 100 V for 1 h. The membranes were blocked in 5% non-fat dry milk in PBS containing 0.2% Tween-20 for 1 h followed by incubation in PBS containing 5% BSA, 0.2% Tween-20, and either 1:1,000 rabbit polyclonal anti-Caspase-1 p20 (47 (link)), anti-IL-1β (70 (link)), anti-HMGB1 (abcam; ab18256), anti-PKR (phospho T446) (abcam; ab32036), anti-PKR (abcam; ab226819), anti-beta Actin antibody (abcam; ab8229), Anti-GFP antibody (abcam; ab6556), or 1:500 anti-Asc (Tyr-144) phospho-specific antibody (ECM Bioscience; AP5631) with rotation overnight at 4°C. The following day, donkey anti-rabbit IRDye 800CW and donkey anti-goat IRDye 680RD secondary antibodies (Li-Cor Biotechnology) were applied at a dilution of 1:15,000 with rocking for 1 h at room temperature. Membranes were imaged on a Li-Cor Odyssey CLx machine with auto exposure and high-quality setting.

Immunofluorescence was used to identify cells expressing ISG15 and PKR as described [49 (link), 50 (link)]. Briefly, after citrate buffer heat-mediated antigen retrieval and incubation with PBS containing 20 % horse serum or 20 % goat serum to block non-specific binding, paraffin sections were co-incubated with antibodies directed against ISG15 (sc-50366; 1:100) or PKR (ab-32036; 1:200) and antibodies directed against GFAP (ab-3554, Abcam; 1:200), Olig2 (ab-85900, Abcam; 1:500), or CD107b (MCA2293, AbD Serotec, Puchheim, Germany; 1:200) overnight at 4 °C. Negative controls included sections incubated with rabbit serum (R4505, Sigma-Aldrich; 1:3000/1:6000), goat serum (I9140, Sigma-Aldrich; 1:4500), sheep serum (1:5000), and rat serum (R9759, Sigma-Aldrich; 1:16000), respectively. After washing, sections were incubated Cy2- and Cy3-conjugated secondary antibodies (goat anti-rabbit, A-11034, Invitrogen; 1:200; goat anti-rat, 112-165-003; donkey anti-rabbit, 711-545-152; donkey anti-sheep, 713-765-147; donkey anti-goat, 705-765-147, Jackson ImmunoResearch, Suffolk, UK; 1:200) in the dark for 1 h at room temperature. Nuclei were counterstained with 0.01 % bisbenzimide (H33258, Sigma-Aldrich), and sections were mounted in Dako Fluorescence Mounting Medium (S3023, DakoCytomation GmbH, Hamburg, Germany).

PKR activation assays were performed as described (Zheng and Bevilacqua, 2004 (link)). RNAs from WT and mutant LCLs were tested for the ability to directly activate PKR protein in vitro, which was determined by levels of PKR phosphorylation in western blots. Briefly, 0.1 ng recombinant PKR (Life Technologies, PV4821) was dephosphorylated by treating with λ-PPase (NEB, P0753S) for 30 min at 37°C. λ-PPase was inactivated by treatment with freshly prepared sodium orthovanadate. The dephosphorylated PKR was then incubated with 10 ng RNA from WT and NIPBL-MS, NIPBL-NS, NIPBL-FS, and HDAC8 mutant cells in the activation buffer (20 mM HEPES [pH 7.5], 4 mM MgCl₂, 100 mM KCl, and 1 mM ATP) for 3 hr at 30°C. Reactions were stopped by adding SDS loading buffer and PKR was resolved on a NuPAGE 4%–12% Bis-Tris protein gel. The phosphorylation of PKR was determined with p-PKR antibodies (Abcam, ab32036).

Sixteen teeth from four dogs were extracted. Premolars were divided into mesial and distal roots to obtain two samples from one tooth. The extracted tooth sample was fixed in 4% paraformaldehyde (PFA) at 4°C for 24 h, decalcified with Kalkitox™ at 4°C for 48 h, neutralized in 5% sodium sulfate solution for 24 h (all purchased from FUJIFILM Wako Pure Chemical), and embedded in paraffin. Standard hematoxylin and eosin (H&E) staining was performed as previously described [16 (link)]. For immunohistochemical staining, the specimens were probed with specific primary antibodies against myeloperoxidase (MPO) (1 : 1000, ab208670; Abcam), CD68 (1 : 150, E3O7V; Cell Signaling Technology), and pPKR (phospho T446) (1 : 200, ab32036; Abcam) and counterstained with Mayer's hematoxylin solution (FUJIFILM Wako Pure Chemical).