Same protein, different shape explains protein's effectiveness against bacteria nature
In vitro ubiquitination reactions were performed in buffer A . Components were mixed as indicated at concentrations of 0.4 µM E1 , 2 µM E2 , 10 µM E3 , 200 µM ubiquitin and 10 µM GSDM . Reactions were incubated at 37 °C for 2 h and stopped by the addition of SDS–PAGE loading dye, followed by boiling for 5 min before electrophoresis. Ubiquitination was evaluated by Coomassie blue staining of SDS–PAGE..
Beads were washed three times with PBS buffer then eluted with 50 μl of PBS buffer containing 100 μg mlFLAG peptide . Eluted samples were boiled with an equal volume of 2× SDS Loading buffer then processed for immunoblotting with one of the following antibodies: anti-FLAG , anti-actin , anti-HA or anti-ubiquitin .One each of plasmids pcDNA-FLAG-GSDMB and pcDNA-FLAG-GSDMD was cotransfected with 500 ng of the pCMV-HA-IpaH7.8 plasmid into HEK293T cells seeded in a 12-well plate at 1.
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