Goat anti rabbit igg secondary antibody

The cells were first lysed with radioimmunoprecipitation assay lysis buffer (Solarbio, Beijing, China). The protein levels were then quantified using a Bradford assay kit (Beyotime, Haimen, China). After the protein samples were separated by 12% SDS-polyacrylamide gel electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were blocked and incubated with primary antibodies, followed by washing with Tris-buffered saline and Tween 20 (TBST). Secondary antibodies were then incubated with the membranes followed by washing with TBST. Finally, the proteins were detected using WesternBright ECL HRP (Advansta, Menlo Park, CA, USA) and the signals were detected with a Clinx GenoSens 1600 integrated gel imaging analysis system (Clinx, Shanghai, China). The primary antibodies that were directed against phosphates and tensin homolog deleted on chromosome ten gene (PTEN), phosphatidylinositide 3-kinase (PI3K), and protein kinase B (AKT), as well as the goat anti-mouse IgG and goat anti-rabbit IgG secondary antibodies, were all purchased from Cell Signaling Technology Company (Danvers, MA, USA).

Cells (2 × 10⁶) were harvested and lysed by RIPA buffer. In brief, cell extracts were collected and stored at −80 °C. Proteins were separated by homogeneous SDS-PAGE and transferred to a nitrocellulose membrane (pore size 0.45 μm; Merck Millipore). Primary antibodies which were used in this study: phospho-p38 (D3F9, Cell Signaling Technology, 4511 S, 1:1000); p38 (D13E1, Cell Signaling Technology, 8690 S, 1:1000); phospho-ERK (D13.14.4E, Cell Signaling Technology, 4370 S, 1:1000); ERK (137F5, Cell Signaling Technology, 4695 S, 1:1000); GAPDH (D16H11, Cell Signaling Technology, 5174 S, 1:1000); CLEC2D monoclonal antibody was generated by our lab (Clone 2260CT10.1.3.2), western blot validation of extracts from primary bone marrow cells. Primary antibody binding was visualized by chemiluminescence using HRP-conjugated goat anti-mouse (1:5000) or goat anti-rabbit IgG secondary antibodies (1:5000; both Cell Signaling Technology).

Antibodies against collagen type II (COL2A1), aggrecan (ACAN), a disintegrin-like and metalloprotease with thrombospondin type-1 motifs 5 (ADAMTS5), matrix metalloproteinase 13 (MMP13), Melatonin receptor 1A (MTNR1A), Melatonin receptor 1B (MTNR1B), tumor necrosis factor-alpha (TNF-α), and GNAI2 (Gαi2) were purchased from Abcam (Cambridge, UK). Antibodies against Hippo signaling proteins (YAP, phospho-YAP [p-YAP], LATS1 and LATS2), and NF-κB signaling proteins (p65, p-p65, IκBα and p-IκBα), and goat anti-mouse IgG and goat anti-rabbit IgG secondary antibodies were purchased from Cell signaling Technology Inc (Boston, MA, USA). Antibodies against p-LATS1/2 was obtained from Affinity Biosciences LTD (OH, USA). Anti-GADPH and anti-α-tubulin was obtained from Proteintech Group Inc (Rosemont, IL, USA). Melatonin, luzindole, 4P-PODT and cycloheximide (CHX) was obtained from Sigma-Aldrich (St. Louis, MO, USA), and recombinant human TNF-α was purchased from R&D Systems (Minneapolis, MN, USA). S26131 was purchased from Med Chem Express (MCE, New Jersey, USA). Verteporfin (VP) was obtained from Selleck Chemicals (Houston, TX, USA).