Ecl plus reagent

RPMI-1640 and McCoy's 5A medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Lonza Milano SRL (Milan, Italy). ZSTK474, BYL719, TGX221, AS605240, CAL101, IPI145, Imatinib, Nilotinib and GZD824 were obtained from Selleck Chemicals (Houston, TX, USA). For cell viability determination, CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega (Milan, Italy). Annexin V/7-ADD detection kit was from Merck-Millipore (Darmstadt, Germany). Western blot antibodies for total Akt-1, Ser473 p-Akt-1 and Thr308 p-Akt-1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while all the other antibodies were from Cell Signaling Technology (Danvers, MA, USA), including the rabbit secondary antibody. Chloroquine, the mouse secondary antibody and the monoclonal β-Actin antibody were purchased from Sigma Aldrich (Milan, Italy). Signals were detected using ECL Plus reagent from Perkin Elmer (Boston, MA, USA).

CHO-K1 and MEF cells stably expressing receptors of interest were starved for 12–18 h in serum-free medium prior to stimulation. Cells were stimulated with 300 nM chemerin for 2 minutes, then collected by centrifugation and heated to 100°C for 5 min in 2X Laemmli sample buffer. Whole cell lysates were resolved on 10% Tris/Tricine polyacrylamide gels and transferred to nitrocellulose membranes. Phosphorylated ERK1/2 and total ERK1/2 were detected by using rabbit polyclonal anti-phospho-ERK1/2 (Cell Signaling #4370, 1:1,000) and anti-ERK1/2 (Cell Signaling #4695S, 1:2,000) antibodies. Chemiluminescent detection was performed using ECL-Plus reagent (Perkin Elmer).

Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium, fetal bovin serum (FBS), nonessential amino acids (NEAA), penicillin and streptomycin were from Lonza (Lonza Milano SRL, Milan, Italy). For cell viability determination, Cell Proliferation Kit I (MTT) was purchased from Roche Applied Science (Basel, Switzerland). Annexin V/7-AAD and cell cycle kits were from Merck-Millipore (Darmstadt, Germany). NVP-BGT226 and Z-VAD-FMK were provided by Selleck Chemicals (Houston, TX, USA). Antibodies to total Akt-1, Ser473 p-Akt-1 and VEGF were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HIF-1α antibody was provided by BD Biosciences Pharmigen, while all the other antibodies were from Cell Signaling Technology (Danvers, MA, USA), including the rabbit secondary antibody, SQSTM1/p62 (#5114) and Cleaved Caspase-7 (Asp198, #9491) antibodies. The mouse secondary antibody, 3-Methyladenine (3-MA), Chloroquine, 1,4-Diazabicyclo[2.2.2.]octane (DABCO) and 4′,6 diamidino-2-pheny-lindole (DAPI) were from Sigma Aldrich (Milan, Italy). Signals were detected with the ECL Plus reagent purchased from Perkin Elmer (Boston, MA, USA).

The nuclear and cytoplasmic protein from the chondrocytes was isolated as recommended by the manufacturer using the extraction kits (Beyotime, Shanghai, China). The nuclear and cytoplasmic protein concentration was determined using an Enhanced BCA Protein Assay Kit. The total protein was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene difluoride (PVDF) membrane (BIO-RAD USA). After blocking with 5% nonfat milk for 2 h, the membrane was incubated with the Nrf2 primary antibody (1:1000) and HRP-conjugated secondary antibodies. ECL Plus reagent was used to expose the membrane in an enhanced chemiluminescence detection system (PerkinElmer, USA). Semi-quantitative analysis of band intensity was analyzed byAlphaEaseFC 4.0 software.

7WD10, 7WD10-NotchΔE, or 7WD10-Notch ∆E-St6gal1 cells were seeded in 24-well plates and cultured at 37 °C, in the presence or absence of the indicated concentrations of tetravalent or monomeric peptides. After 48 h incubation, culture medium and cells were recovered separately. Cells were lysed in lysis buffer, containing 2% sodium dodecyl sulfate (SDS), 80 mM Tris-HCl (pH 6.8), 7% glycerol, 250 mM dithiothreitol (DTT), and 0.025% phenol red. Proteins in culture medium and lysates were separated by tricine SDS-polyacrylamide gel (12% acrylamide) electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Wako Chemicals). To separate immature APP and mature APP, glycine SDS-PAGE (7% acrylamide) was used. After boiling in phosphate-buffered saline (PBS) for 5 min and then blocking with 5% skim milk, the membranes were immunoblotted with the indicated primary antibodies, followed by HRP-conjugated secondary antibodies. Protein bands were visualized using ECL Plus Reagent (PerkinElmer) and analyzed using an ImageQuant LAS 500 CCD imager (GE Healthcare Life Sciences). Total amounts of target proteins were measured using Image J software (National Institutes of Health; NIH), after normalization to β-actin as an internal control.