P stat6

Mφ after activation and CM induction were harvested and lysed in lysis buffer containing protease and phosphatase inhibitors. The lysates were centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected, and protein concentrations were determined by BCA protein assay. Proteins were subjected to SDS-PAGE analysis, transferred to nitrocellulose membranes, and incubated with the primary antibodies against STAT3, p-STAT3 (Cell Signaling Technology, MA, USA), IL10, IL27 Rα (Abcam, Cambridge, UK), IL-12b p40, p-STAT6 (Santa Cruz Biotechnology, Santa Cruz, CA), GAPDH, IRF-4, and SOCS3 (Bioworld Technology, Nanjing, China). Protein bands were detected using an enhanced chemiluminescence reagent (Sigma-Aldrich, St. Louis, MO, USA) following incubation with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit secondary antibodies (Bioworld Technology, Nanjing, China). The expression levels of the proteins were compared to the control based on the relative intensities of the bands.

Appropriate RIPA lysate containing protease and phosphatase inhibitor cocktails (CWBio Biosciences) were added to lysate the cells. Standard curves were drawn with BSA for protein quantitation. Then, protein denaturation was performed. After isolation in SDS–PAGE, the denatured protein was transferred into the immobilon‐polyvinylidene fluoride (PVDF) membrane (Millipore). PVDF membranes were incubated with the corresponding primary antibody at 4°C overnight. Primary antibody includes β‐actin (CWBio Biosciences), CD31, vascular endothelial growth factor (VEGF), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 2 (MMP2), ZO‐1, E‐cadherin, vimentin, Snail, Slug, Twist, CD44, Oct4, Sox2, Bmi‐1, Nanog, p‐STAT3, STAT3, p‐STAT6, STAT6 (Santa Cruz Biotechnology). Then, membranes were incubated with secondary antibody at RT for 1 h. The gel imaging system (Bio‐Rad) was used to collect the image.

The granulation tissues together with underlying bowel loops used for histology were fixed with 10% neutral formaldehyde. The samples were removed from the formaldehyde, followed by dehydration and then embedded in paraffin. Serially section was performed using a microtome according to standard protocols. The sections, 5 μm in thickness, were processed for hematoxylin and eosin staining (H&E), Masson’s trichrome staining and immunohistochemical evaluation. Sections for immunohistochemistry were stained with p-STAT1 (Santa Cruz, sc-135648) and p-STAT6 (Santa Cruz, sc-11762). For neovascularization evaluation, sections were reacted with primary antibodies CD31 (KEYGEN, KGYM0118–7) and α-smooth muscle actin (α-SMA) (KEYGEN, KGYT5053-6) overnight at 4 °C. Sections were washed and incubated with TRITC- (KEYGEN, KGAA98) and FITC- (KEYGEN, KGAA26) conjugated secondary antibodies. Sections were rinsed and mounted with 4, 6-diamidino-2-phenylindole (DAPI) mounting medium to label Nuclei.

In all, 1 × 10⁶ uninfected or KSHV-infected cells were lysed, subjected to electrophoresis and transferred to nitrocellulose membranes, as previously described.^{29 (link)} Membranes were blocked in PBS-0.1% Tween 20 solution containing 3% BSA, probed with specific antibodies and developed using ECL Blotting Substrate (Advansta). The following antibodies were used: mouse monoclonal antibody against Kb-ZIP (Santa Cruz Biotechnology, sc-69797), pSTAT6 (1:100; Santa Cruz Biotechnology Inc., sc-136019), STAT6 (1:100; Santa Cruz Biotechnology Inc., sc-1689), mouse monoclonal anti-STAT3 (1:1000; BD Transduction Laboratories, 610189), mouse monoclonal anti-phospho-STAT3 (p-Tyr705, 1:100; Santa Cruz Biotechnology Inc., sc-8059), pSTAT1 (1:100; Santa Cruz Biotechnology Inc., sc-136229), STAT1 (1:100; Santa Cruz Biotechnology Inc., sc-464), mouse monoclonal anti-Ire1α (1:100; Santa Cruz Biotechnology, sc-390960), XBP1s (NovusBio NBP1-77681SS), ATF4 (R&D system, MAB7218), rabbit polyclonal anti-BIP (1:100; Cell Signaling, 3177), mouse monoclonal anti-CHOP (1:100; Santa Cruz Biotechnology, sc-7351), and anti-β-actin (1:10000; Sigma Aldrich, A5441). Goat anti-mouse IgG-HRP and anti-rabbit IgG-HRP (1:10.000 Santa Cruz Biotechnology Inc) were used as secondary antibodies.

Monosialoganglioside GM1 and N-Hexanoyl-biotin-Monosialoganglioside GM1 (GM1-biotin) were purchased from Matreya LLC (State College, PA, USA). GM1-Pentasaccharide was obtained from Carbosynth (Compton, UK). An antagonist for chemokine receptor CCR2 (RS 102895) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). D-(+)-Mannose, N-Acetyl-D-galactosamine, D-(+)-Galactose and D-(+)-Glucose were obtained from Sigma-Aldrich (St. Louis, MO, USA). Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Heparin was obtained from JW Pharmaceutical (Seoul, Korea). Antibodies were purchased as follows: Anti-Arginase (Arg) 1, mannose receptor (CD206), YM1, MCP-1, IL-10, interferon gamma (IFN-γ), IL-2 receptor gamma (γC), and integrin αV were purchased from Abcam (Cambridge, UK). Anti-p-STAT3, p-STAT5, TNF-α, and IL-1β were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-p-STAT1, p-STAT6, STAT, STAT3, STAT5, STAT6, F4/80, VEGF, and GAPDH were purchased from Santa Cruz Biotechnology Inc. Anti-β-actin was obtained from Sigma-Aldrich. Anti- nitric oxide synthase (iNOS) was purchased from Millipore (CA, USA).

Samples containing equal amounts of protein were fractionated on a 10% SDS-PAGE gel and transferred onto a Hybond TM-P membrane (GE Healthcare, Little Chalfont, UK) by using Trans-Blot cell (Bio-Rad Laboratories, Hercules, CA, USA). After blocking solution (8% skim milk in TBS-T, according to vendor's suggestion) at room temperature for 1 h, the membranes were incubated with specific antibodies against MFHAS1, p-STAT6, STAT6, PPAR-γ (Santa Cruz Biotechnology, Dallas, TX, USA), p-JAK1, JAK1, Cyclin D1, N-cadherin, E-cadherin, β-actin (Cell Signalling Technology, Danvers, MA, USA) and KLF4 (Abcam, Cambridge, MA, USA), respectively, at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Protein bands were detected by enhanced chemoluminescence.

Isolated enterocytes were lysed with sodium dodecyl sulfate sample buffer (50 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, and 5% mercaptoethanol). The lysate was then heated for 5 min at 100 °C, and the protein concentration was determined using the RC-DC kit (Bio-Rad, Hercules CA). Proteins were loaded in equal amounts for Western blotting. Antibodies used in this study were Raptor (#2280), p-S6K (#9234), pS6 (S240/244) (#5364), DCLK1 (#62257), Stat 6 (#5397), GAPDH (#5174) (all from Cell Signaling Technology, Danvers MA) and p-Stat 6 (sc-11762, Santa Cruz Biotechnology, Dallas Texas). The proteins were detected using Bio-Rad ChemiDocTM XRS + system with image Lab TM software (Bio-Rad, Hercules CA).