Anti c ebpβ

Anti-COL11A1 was obtained from Abcam. Anti-c/EBPβ, goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP antibodies were purchased from Santa Cruz Biotechnology. Anti-Akt, anti-p-Akt, anti-PDK1, and anti-ubiquitin antibodies were obtained from Cell Signaling. Anti-β-actin antibody was obtained from Sigma. Cisplatin (Fresenius Kabi Oncology Ltd.), paclitaxel (Corden Pharma Latina S.P.A.), gemcitabine (Eli Lilly and Company), and pegylated liposomal doxorubicin (TTY Biopharm) were provided by the Cancer Center at National Cheng Kung University Hospital. ERK inhibitor (PD98059), p38/MAPK inhibitor (SB203580), JNK inhibitor (SP600125), and Akt inhibitor (LY294002) were obtained from InvivoGen. MG132 and cycloheximide (CHX) were obtained from Sigma-Aldrich.

Hepatic and pancreatic tissue lysates from control and STZ-induced male and female rats containing equal amounts of protein were separated by SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes, blocked for 1h with 5% skim milk at room temperature, and incubated with the indicated primary antibodies for 2 h at 1:1000 dilution (anti-β-actin, anti-SPARC, anti-C/EBPβ, anti-ATP5B, anti-CD95, anti-MCP1, anti-iNOS, anti-Cyt C, anti-SOD2, anti-INS, anti-CA3, anti-RARhoGAP, anti-CPS1, anti-BHMT, anti-PA, anti-APC2 [Santa Cruz Biotechnology, Santa Cruz, CA, USA], anti-TNFα, anti-PARP-1, anti-NFκB, anti-HSP90 [Cell Signaling Technology, Beverly, MA, USA], and anti-CRP [AbFrontier, Seoul, Korea]). After washing with Tris-buffered saline containing Tween 20, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Then, immune complexes were detected using the ECL method, and immunoreactive bands were quantified by densitometric analysis using ImageMaster 2D software version 4.95 (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Relative intensity (%) values of proteins were normalized to β-actin levels.

After specific treatments, proteins (20 µg) were separated using 12% SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and transferred to nitrocellulose membrane by an electroblotting Bradford assay, according to the manufacturer’s instructions (Sigma-Aldrich). The anti-Id4 monoclonal antibody (sc-365656, 1:100) and anti-C/EBPβ (CCAAT/enhancer-binding protein β) polyclonal antibody (sc-150, 1:400) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the β-actin antibody (A3854, 1:10,000) was purchased from Sigma-Aldrich.

The cells were collected for immunoblotting analysis as previously described^{38 (link)}. The primary antibodies were as follows: anti-HIF1α (Novus Biologicals, Littleton, CO); anti-HIF2α (Novus Biologicals, Littleton, CO); anti-GLUT1 (Abcam plc, Cambridge, UK); anti-C/EBPβ (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-α-tubulin (Cell Signaling Technology, Danvers, MA). HRP-conjugated anti-rabbit IgG (Bio-Rad Laboratories, Hercules CA) or anti-mouse IgG (Bio-Rad Laboratories) antibodies were used as secondary antibodies. The signals were detected with the ECL Plus reagent (Thermo Fischer Scientific, Waltham, MA) using the chemiluminescence protocol. For detecting the signals of GLUT1 and HIF-2α, SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fischer Scientific) was used.

Primary cultures of mouse astrocytes were treated with LPS (10 μg/ml) and 24 hr later chromatin immunoprecipitation (ChIP) assay was performed, following the manufacturer’s recommendations (Santa Cruz Biotechnology). For immunoprecipitation, the following antibodies were used: 5 μg of rabbit polyclonal anti-C/EBPβ (Santa Cruz Biotechnology) and 2 μg of normal rabbit IgG (Santa Cruz Biotechnology, Mountain View, CA, USA). The precipitated DNA was analyzed by PCR using the following primers: C/EBPβ, sense: 5’-TGC CCT CAC CCC TTA TCC TCC TC-3’; antisense: 5’-GGC AGG TTC AGG AAG GTG GAG AC-3’ (which amplified the C3 promoter from −669 to −525) and GAPDH, sense: 5’-GAG GTC CAC CAC CCT GTT GCT GTA GC -3’; antisense 5’-GCT GAA CGG GAA GCT ACA TGG CAT GG -3’ (which amplified a fragment from +208 to +712). Thermal cycling conditions: one cycle at 95°C for 1 min; 38 cicles at 95°C for 30 s, 60°C for 30 s and 72°C for 30 s; and one cycle at 72°C for 7 min.

Cells and exosomes were lysed using CHAPS lysis buffer (30 mM Tris-Cl, pH 7.5; 150 mM NaCl; 1% CHAPS) mixed with 25X protease inhibitor (Roche), and sonication. Proteins were quantified by Bradford method (Bio-Rad). Electrophoresis was performed using 10% acrylamide gel, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and probed with the following antibodies: anti-TSG101 (Abcam), anti-HSC70, anti-GAPDH, anti-Calnexin, anti-C/EBPβ, anti-CDK2 (all from Santa Cruz Biotechnology), anti-FOXO-1 (Cell Signaling), and appropriate species-specific HRP-conjugated secondary antibodies (Santa Cruz), and detected using ECL reagent (Roche).

Mature 3T3-L1 adipocytes were stimulated by MacCM for 5, 10, 15 minutes. After stimulation, 3T3-L1 cells were washed with ice-cold PBS and scraped with RIPA buffer. Cells lysates were centrifuged at 12,000 rpm, 4 °C, 10 min for remove cell debris and supernatant were collected new tubes. Protein concentration of the supernatant was determined using the Bio-Rad protein assay kit (Bio-Rad) with BSA as a standard. 20 μg (protein equivalents) of the supernatant were resolved by SDS-PAGE, transferred onto a polyvinylidene difluoride (PVDF) membranes and immunoblotted with anti-CEBP/β (Santa Cruz Biotechnology), anti-p-CEBP/β (abcam) antibodies. Uncropped scans of the blots were supplied in Supplementary Fig. 7.