Researchers uncover immunesystem signaling complex NatureComms
. HEK293 cells were commercially obtained . ‘High’ panel reactive antibody sera were obtained as pooled, de-identified sera from the tissue typing laboratory at Yale New Haven Hospital. ‘High’ PRA sera were taken from renal transplant candidates showing allo-sensitization of ≥80% and negative testing for numerous infectious agents. Prior to use, PRA sera was supplemented with human complement .
For immunostaining, HUVEC were grown on glass coverslips, fixed and permeabilized with ice cold methanol for 15 min, blocked with PBS containing 0.1% Tween and 5% FBS for 1 h at room temperature . Primary antibodies were then incubated overnight at 4 °C at 1:200 dilution using the following antibodies: Ulex , P65 , ZFYVE21 , Rubicon , and RNF34 .
For co-immunoprecipitations, cells were lysed in RIPA buffer without SDS containing protease inhibitor tablets in 1.5 mL Eppendorf tubes with gentle agitation for 1 h at 4C. Following this incubation, lysates were pre-cleared with 15 μL protein A/G agarose beads and incubated with 10 μL of antibody against Rab5 , Rab7 , FliI or caspase-1 antibody at 4 °C overnight.
Following the above, 4X Laemli’s buffer and 1 mM DTT were added to 32.5 μL sample, and this mixture was heated for 95 °C for 13 min. Subsequently, samples were loaded onto pre-cast polyacrylamide gels , and proteins were electrophoretically separated and transferred to methanol-activated PVDF membranes at 4 °C for 90 min. Membranes were washed for 15 min three times using Tris-buffered saline containing 0.1% Tween-20 pH 7.
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