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. Author manuscript; available in PMC: 2015 Oct 1.
Published in final edited form as: J Surg Res. 2014 Jun 4;191(2):268–279. doi: 10.1016/j.jss.2014.05.084

FIGURE 2. Degradation of internalized α5 integrin is MG-132 sensitive and in the absence of fibronectin, α5 integrin cytoplasmic tail is ubiquitinated and CBL associates with α5 integrin.

FIGURE 2

(A): CHO-α5 cells were cultured overnight without FN and then pretreated for one hour with either lysosomal inhibitors (Bafilomycin or Chloroquine) or the proteosomal inhibitor, MG-132. Cells were labeled with NHS-SSBiotin at 4°C and either stripped of surface biotin (0H) or transferred to 37°C. After one hour, the remaining surface label was stripped from the cells followed by cell lysis and analysis of whole cell lysates for labeled α5 to allow monitoring of internalized labeled α5 integrin as described in Fig. 1. (Untreated control lanes: Cu = unstripped cells at 0H; Cs = stripped cells at 0H). Pretreatment with MG-132 leads to accumulation of internalized α5 integrin which was not seen in cells pretreated with lysosomal inhibitors. (B): CHO cells stably expressing an α5 integrin with a C-terminal GFP-tag were transiently transfected with an HA-tagged ubiquitin cDNA (HA-Ub) or a control vector. Cells were then cultured overnight in the presence or absence of FN followed by pretreatment for one hour with MG-132. Whole cell lysates were analyzed by immunoprecipitation using an anti-HA monoclonal antibody (IP HA) followed by immunoblotting under reduced conditions with an anti-GFP antibody to detect the GFP-labelled α5 integrin cytoplasmic domain (~50 kDa). α5 integrin is found to be ubiquitinated only in the absence of FN. (whole cell lysate controls: CG = α5-GFP cells; CW = wild-type α5 cells) (C): Cells transiently transfected to express the E3 ubiquitin ligase CBL (Cbl) or with a control vector were cultured overnight in the presence or absence of FN and pretreated with MG-132 for one hour. Whole cell lysates underwent immunoprecipitation with anti-α5 antibody (IP alpha 5) followed by immunoblotting with an anti-CBL antibody. α5 integrin association with CBL is detected only in the absence of exogenous FN. (whole cell lysate controls: CFN+ = FN present; CFN- = FN absent)