Immobilon p pvdf membrane

Pre-fusion osteoclasts were washed with PBS then lysed in ice cold Modified RIPA Buffer supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Proteins were resolved on 8% SDS-PAGE and transferred to Immobilon-P PVDF Membrane (Thermo Fisher). Blots were blocked and incubated with primary antibodies diluted at 1:1000 in TBST, 3% BSA at 4 °C overnight. They were washed three times for 5 minutes with TBST, then incubated for 2 hours with HRP-conjugated secondary antibodies at 1:6000 dilution in TBST, 5% nonfat milk, and visualized using WesternBright Sirius chemiluminescent substrate (Advansta, San Jose, CA, USA). Antibodies used were the same PKD antibodies as for immunofluorescence or α-tubulin (Cell Signaling Technology #2144). Band quantitation was performed using Image Lab 6.0 Software (Bio-Rad, Hercules, CA, USA). P-PKD intensities were normalized to α-tubulin from the same lane, and then graphed as fold change relative to the indicated comparison group. Each of the experiments shown in Figure 3 was performed and quantitated four independent times with comparable results. Representative Western blot images from a single experiment are shown, while the accompanying graphs show the aggregate data from all four of the experiments performed. Please refer to Section 4.12 for details on quantitative data analysis.

Control and tested cells were lysed to create whole cell extracts as described in our previous research [34 (link)]. A total of 80 μg of proteins was electroblotted onto an Immobilon-P PVDF membrane (Thermo Scientific, Rockford, IL, USA), after being separated by 10% SDS-PAGE. After blocking the membranes with 5% low-fat milk for 30 min, they were incubated with the primary-mice monoclonal antibodies against Beclin 1, and procaspase 3 (0.5 μg/mL; Santa Cruz Biotechnology, Dallas, TX, USA) within an entire night at 4 °C. Next day, after washing them 3 x in PBS buffer with 0.05% Triton X-100 (Sigma), membranes were incubated with secondary, alkaline phosphatase (AP)-conjugated, antibodies. The NBT/BCIP Solution (ABCAM, Waltham, MA, USA) was used to detect proteins. The acquired results were subjected to a qualitative analysis based on the color depth, band width, and band thickness. ImageJ Software version 1.8.0 was used to analyze protein bands quantitatively. The information was adjusted in relation to β-actin (0.5 μg/mL; Santa Cruz Biotechnology). There were three separate experiments carried out. As Supplementary Materials, whole blot membranes are attached (Figures S3 and S4).