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. 2011 Mar 31;6(3):e18334. doi: 10.1371/journal.pone.0018334

Figure 4. COMMD1 regulates CFTR ubiquitination.

Figure 4

(A) Representative gels for the same CFTR IP experiment with MAB25031 from HeLa cells stably expressing wt-CFTR and separated on 8% SDS-PAGE transferred to PVDF membrane. Half of the membrane was probed with anti-CFTR mAb and the other half with anti-ubiquitin mAb. Lysates were loaded onto an 11% SDS-PAGE and sequential probing of the membrane was performed (COMMD1, α-tubulin and lastly ubiquitin). Filled and empty arrowheads indicate the fully- (170 kDa) and core-glycosylated (140 kDa) CFTR, respectively. (B) Quantification of ubiquitinated CFTR. Ratio of ubiquitinated CFTR to total CFTR in each condition is shown, endogenous COMMD1 expression is referred as 100%. The means ± S.D. were obtained from five independent experiments.* P<0.05 was determined by t-test. (C) Stability of the mature wt-CFTR was determined upon inhibition of protein biosynthesis with cycloheximide (CHX). Cells were incubated in the presence of cycloheximide for the indicated time intervals. (D) Quantification of mature CFTR was normalized to α-tubulin level.