Anti gm130 antibody

Western blot analysis was performed as previously described [21 (link)]. Macrophages or cementoblasts were washed and solubilized with RIPA lysis buffer. Protein concentrations were measured and adjusted to be the same, followed by electrophoresis on a precast gel and then transferred to a polyvinylidene difluoride membrane. After being blocked with 5% BSA, the transferred membranes were incubated at 4°C overnight with anti-iNOS antibody, anti-ALIX antibody, anti-GM130 antibody (Proteintech, Chicago, IL, USA), anti-CD63 antibody, anti-Arg-1 antibody, anti-BSP antibody, anti-COL-1 antibody (Cell Signaling Technology, Danvers, MA, USA), anti-RUNX2 antibody, anti-OSX antibody, or anti-β-actin antibody (Abcam, Cambridge, MA, USA) diluted at 1 : 1000. Subsequently, the membranes were incubated with anti-rabbit or anti-mouse secondary antibodies (ZB-2301 and ZB-2305, Zhongshan Golden Bridge Biotechnology, Beijing, China), which were diluted at 1 : 5000 at room temperature for 1 h. The expression of associated proteins was visualized with a chemiluminescence reagent and analyzed using ImageJ software.

Cells grown on coverslips were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, followed by incubation with primary antibodies overnight at 4 °C. The following primary antibodies were used: anti-GM130 antibody (1:1000, Proteintech), anti-GSH antibody (1:1000, Thermo Fisher), anti-paxillin antibody (1:500, Proteintech) and anti-ezrin antibody (1:250, NOVUS Bilogicals). The cells were then incubated with secondary antibodies (Alexa Fluor 488 donkey anti-mouse IgG/ Alexa Fluor 555 donkey anti-mouse IgG, 1:5000, Thermo Fisher) for 1 h at room temperature. DAPI (1:1000, Thermo Fisher) was used for nuclear staining. For F-actin staining, cells were fixed in 4% paraformaldehyde, blocked for 1 h and subjected to FITC-conjugated/rhodamine-conjugated phalloidin (1:1000, Thermo Fisher) staining at room temperature. The percentage of cells containing thick actin stress fiber with a diameter >0.5 μm was analyzed and used as an index for coarse quantification of the stress fiber. Immunofluorescence signal was detected using the Olympus FV1200 confocal system (FLUOVIEW Ver.4.2 software).