Anti α β tubulin

WBs were performed as we previously described^{17 (link),53 (link)} using anti-p21 Waf1/Cip1 (12D1) (#2947, diluted 1:1000), anti-Myosin Light Chain 2 (MLC2) (#3672, diluted 1:500), anti-Phospho (Ser19) Myosin light chain 2 (MLC2S19*) (#3671, diluted 1:500), anti-αTubulin (#3873, diluted 1:1000) and anti-α/βTubulin (#2148, diluted 1:1000) antibodies from Cell Signaling Technology, anti-IL8 (#ab106350, diluted 1:500), anti-IRF5 (#ab21689, diluted 1:1000), anti-IL1β (#ab2105, diluted 1:1000) and anti-iNOS (#ab3523, diluted 1:500) antibodies from Abcam; an anti-IL6 antibody (RD, #AB-206-NA, diluted 1:500); an anti-SIRPα antibody (Invitrogen, #PA1-30537, diluted 1:2000); an anti-p53 (DO-1) antibody (Santa Cruz, #sc-126, diluted 1:1000) and an anti-GAPDH antibody (EMD Millipore, #MAB374, diluted 1:1000). Blot scan images were analyzed using GeneSys software version v1.3.9.0 (Genesys). The uncropped and unprocessed scans of all the blots are provided in the Source Data file.

The primary antibodies for the following proteins were used for Western analysis and immunofluorescence (IF): anti-Nup62 (BD Biosciences #610497, used at 1:2000 for Western), anti-Nup98 (Abcam #45584, used at 1:1000 for Western), anti-Nup153 (Abcam #96462, used at 1:1000 for Western), anti-eIF4G (BD Biosciences #610536, used at 1:1000 for Western), anti-PABP (Cell Signaling Technology #4992, used at 1:1000 for Western), anti-nucleolin (Abcam #22758, used at 1:3000 for Western), anti-hnRNP-C1/C2 (Santa Cruz #32308, used at 1:500 for IF), anti-SC35 (Sigma #4045, used at 1:500 for IF), anti-Sam68 (Santa Cruz #sc333, used at 1:500 for IF), and anti-α/β-tubulin (Cell Signaling Technology #2148, used at 1:1000 for Western). Antibodies to 3C^pro were kindly provided by S. Amineva (Madison, WI, USA; Amineva et al., 2004 (link)) and antibodies to dsRNA were kindly provided by S. Bowden (VIDRL, Melbourne, VIC, Australia).

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.

Oligo primers were synthesized by Operon. The anti-KIAA1199 (CEMIP) antibody was purchased from Abcam. The anti-HIF-1α antibody was purchased from BD Transduction Laboratories and the anti-HIF-2α antibody was purchased from Novus Biologicals. The anti-α/β-tubulin, -E-cadherin, and -Jarid1A antibodies were purchased from Cell Signaling Technology. HIF-1α P402A/P564A (plasmid 18955), HIF-2α P405A/P531A (plasmid 18956), and Jarid1A plasmids (plasmid 14800) were purchased from Addgene. Dual Glo-Luciferase assay was purchased from Promega. aiRNA against CEMIP was purchased from Boston Biomedical, Inc. [27 ].

Exponentially growing cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing 1 mM 4-(2-aminoethyl)-benzensulfonyl fluoride hydrochloride. The cell lysate was cleared by centrifugation at 15,000 rpm for 10 min at 4 °C, and then supernatant was used as the total cellular protein. Total protein concentration was determined by the BCA protein assay (Pierce, Rockford, IL). Protein samples (8 or 16 µg) were electrophoresed on SDS-polyacrylamide gel and were electrophoretically transferred to a polyvinyl difluoride membrane in a transfer buffer (100 mM Tris, 192 mM glycine). After overnight incubation with blocking solution (10% skim milk), the membrane was incubated with the primary antibodies, a biotinylated anti-mouse or anti-rabbit IgG antibodies, and streptavidine-alkaline phosphatase. The bands were visualized after addition of nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as a substrate. The primary antibodies used in this study are anti-adiponectin (clone 19F1, Abcam Co. Ltd., Tokyo, Japan), anti-FABP4 (Abcam Co. Ltd., Tokyo, Japan), anti-PPARγ (clone 81B8, Cell Signaling technology Japan, Tokyo, Japan), and anti-α/β-tubulin (Cell Signaling technology Japan).