Rhodamine red x

Embryo fixation was performed according to standard methods. Embryos were fixed by a solution containing heptane (Sigma, St Louis, MO) and methanol^{57 (link),58 (link)}. Wing discs were fixed in PLP fixative (2% paraformaldehyde, 75 mM lysine, and 35 mM phosphate buffer, pH7.4) for 15–30 min at room temperature.
Antibodies used for immunohistochemistry were as follows: mouse anti-Cut (1:200, 2B10, Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa), guinea pig anti-Centrosomin (1:1000, from Jordan Raff), Rat anti-α-Tubulin (1:200, MAB1864, Millipore, Burlington, Massachusetts), rabbit anti-PH3 (1:200, 06–570, Millipore), Rabbit anti-Vtd (1:500, this study), rabbit anti-Myc (1:100, ab9106, Abcam, Cambridge, UK). Secondary antibodies conjugated with Rhodamine Red™-X (RRX), Alexa Fluor® 647 or fluorescein isothiocyanate (1:200, 715-095-151, 715-295-151, 715-605-151, 711-095-152, 711-295-152, 711-605-152, 706-095-148, 706-295-148, 706-605-148, 712-095-153, 712-295-153, 712-605-153) were from Jackson ImmunoResearch Inc. Vectashield with 4′, 6-diamidino-2-phenylindole (H-1200, Vector Laboratories, Burlingame, CA) was used for mounting. Fluorescent images were acquired using Carl Zeiss LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany).

JAWS II and GM-BM cells were seeded on coverslips placed in a 24-well plate at a density of 1.5 × 10⁵ cells per well. Cells were left uninfected or were treated with ECTV for 60 min. at 37 °C. After 4, 12 and 24 h, cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich, St Louis, MO, USA) and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in PBS. Next, JAWS II and GM-BM cells were blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich) in 0.1% Triton X-100 and incubated for 45 min. with anti-cathepsin B, anti-cathepsin L (both from Abcam, Cambridge, MA, USA) and anti-cystatin B (Thermo Fisher Scientific) primary antibodies. After washing with 0.1% Triton X-100 in PBS, cells were incubated with secondary anti-mouse or anti-rabbit antibodies conjugated with rhodamine Red-X (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) diluted in blocking solution for 1 h. ECTV antigens were stained with FITC-conjugated polyclonal antibodies for 1 h. Viral and nuclear DNA was stained with Hoechst 33342 (Sigma-Aldrich) solution for 10 min. in the dark. Slides were mounted in ProLong Gold Antifade Reagent (Life Technologies). Images were captured using Olympus BX60 fluorescence microscope and analyzed with Cell^F software (Soft Imaging System, Olympus, Tokyo, Japan) and ImageJ (NIH, Bathesda, MD, USA).

Antibodies against reticulon 4B (AB-163; Kinasource Ltd, Dundee, UK), reticulon 4A (ab62024; Abcam, Cambridge, UK), HA (MMS-101R-50; Covance, Princeton, NJ), FLAG (F7425; Sigma-Aldrich), calreticulin (2679S; Cell Signalling Technologies, MA) and β-actin (ab8227-50; Abcam) were used as primary antibodies. When indicated, rabbit anti-sheep bridging antibody (313-001-003; Jackson ImmunoResearch Labs Inc., West Grove, PA) was used. Secondary antibodies were Rhodamine Red-X (016-290-084; Jackson ImmunoResearch), Alexa 647 (A31571; Life technologies), Alexa 488 (A-11008; Life Technologies) and 1.4 nm nanogold-conjugated anti-rabbit antibody (Nanoprobes, Stony Brook, NY).

Samples of lesional skin from CLE patients were H&E stained to confirm the clinical diagnosis in every single case by an experienced dermatopathologist (JW). Immunohistochemistry was performed using the REAL Detection Systems with Fast Red as chromogen (Agilent, Santa Clara, CA, United States) with specific antibodies for pJAK (ABIN196869, antibodies-online), CXCL10 (ab9807, Cambridge, United Kingdom), and CD45 (550539, BD, New Jersey). The expression was scored semiquantitatively from 0 =̂ weak to 3 =̂ strong (18 (link)). Immunofluorescence analyses of JAK1-phosphorylation detected by anti-rabbit Rhodamine Red-X (711-295-152; Jackson ImmunoResearch, Baltimore, MD, United States) and DAPI (D9542, Sigma-Aldrich) were performed using a high-resolution microscope (Axio Observer Z1, Zeiss, Germany).

Polyclonal rabbit anti-PY⁷⁰⁵-STAT3 (#9145), anti-STAT3 (#4904) and anti-α/β-tubulin (#2148) were purchased from Cell Signaling Technology. For western blotting, the antibodies were probed using secondary donkey anti-rabbit antibodies, conjugated with horseradish peroxidase (Jackson Immunoresearch, #711-035-152). For immunofluorescence, the antibodies were probed using secondary donkey anti-rabbit antibodies conjugated to Rhodamine Red X (Jackson Immunoresearch, #711-295-152). Cell nuclei were fluorescently labelled using DAPI (Sigma-Aldrich, #D9564) or Syto 60 (Life Technologies, #S11342). Purified human IL-10 (#130-093-947), IL-6 (#130-095-365) and pure grade neutralizing anti-IL-6 (#130-096-093) and anti-IL-10 (#130-096-041) antibodies were purchased from Miltenyi Biotech. Nitric oxide synthase inhibitor L-NMMA (#0771, Tocris) was suspended in desionized water at the concentration of 50 mM.