Figure 4.

TG2 interacts with autophagy-related cargo proteins. (a) Western blot analysis of TG2, p62 and VCP proteins in human 2fTGH cells subjected to immunoprecipitation for TG2. After 4 h of treatment as indicated, cells were lysed and proteins were immunoprecipitated using anti-TG2 antibody (see Materials and Methods). Immuno- and co-immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. WC, whole cell lysate, was used as protein control. (b) Western blot analysis of TG2, p62 and VCP proteins in HEK293^TG2 cells stably transfected with TG2 protein, subjected to immunoprecipitation for TG2. After 2 h of treatment as indicated, cells were lysed and proteins were immunoprecipitated using anti-TG2 antibody. Immuno- and co-immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. WC, whole cell lysate, was used as protein control. (c) Representative blot of the TG-catalyzed incorporation of 5-(biotinamido)pentylamine into proteins in 2fTGH cell line. Cells were labelled with 5-(biotinamido)pentylamine and treated as indicated for 4 h. After separation by SDS-PAGE, biotinylated proteins were revealed with HRP-conjugated streptavidin. p53 and p27 proteins were used as a control for monitoring proteasome inhibition, and actin was used as loading control