The Human Phospho Kinase array is a nitrocellulose membrane where antibodies against 46 kinase phosphor ylation sites ref 3 have been spotted in duplicate. Cell lysates from untreated, 5 minute, 20 minute, and 1 hour MSU activated cells were prepared in lysis buffer provided with the proteome profiler. In total, 250 ug of protein was used for each array and incubated with the nitrocellu lose membrane Inhibitors,Modulators,Libraries array Inhibitors,Modulators,Libraries overnight at 4 C. The array was washed and then incubated with a cocktail of phospho site specific biotinylated antibodies for 2 hours at room temperature, and washed before adding Streptavidin HRP for 30 minutes. Signals were developed with an Inhibitors,Modulators,Libraries enhanced chemiluminescence Western blotting detection system and recorded on x ray film.
Densities of individual dots corresponding to a phosphorylated kinase were measured Inhibitors,Modulators,Libraries by Image J software, and a comparison between untreated and MSU activated samples was performed. Immunoblot analysis After incubation, around 5. 105 confluent adhering OBs were washed with PBS and then directly lysed in Laemmli buffer. Cells were boiled for 10 minutes. Samples were subjected to 15% SDS polyacrylamide gel electrophoresis and transferred to Immobilon membranes. Equal pro tein loading and transfer efficiency were visualized with B actin evaluation. Membranes were saturated for 30 mi nutes at room temperature in Tris buffered saline with 0. 5% Tween 20, containing 5% dried milk, and subsequently exposed overnight at 4 C to the LC3 B rabbit polyclonal antibody, NLRP 3b mouse monoclonal antibody, P IB or IB mouse antibodies, or 1 hour at room temperature to the actin mouse monoclonal antibody.
Membranes were washed twice in TBS Tween and incubated Inhibitors,Modulators,Libraries with second ary antibodies. Bounded antibodies were revealed with the enhanced chemiluminescence Western blotting detection system after TBS Tween washes, as specified by the man ufacturers protocol. LC3 GFP transfection OBs were transfected with LC3 GFP plasmid for 24 hours by using lipofectamine, according to the manufacturers protocol. After 4 hours of MSU stimulation, cells were then observed with confocal mi croscopy laser, 400 magnification. Small interfering RNA knockdown of NLRP3 expression Knockdown of NLRP3 expression was achieved by trans fecting OBs with a combination of two small interfering RNAs against NLRP3 or AllStars Negative Control siRNA.
Predesigned siRNAs against NLRP3 target sequences were SI02634009 and SI02634030. OBs were transfected with these siRNAs in the presence of HiPerFect Trans fection Reagent by following the manufacturers protocol. After 24 hours of transfection, knockdown of NLRP3 protein expression was confirmed with immuno blot, and these cells were stimulated or not with 0. 5 mg MSU for selleck chemicals Tofacitinib 8 hours. Densitometric analyses Immunoblots were analyzed by using ImageJ software to quantify band intensity assessed with densitometry.