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. 2012 May 25;7(5):e37628. doi: 10.1371/journal.pone.0037628

Figure 4. ADAM12 p.L792F does not affect cell proliferation or EGF shedding.

Figure 4

(A) Comparable proliferation of ADAM12 WT and ADAM12 p.L792F expressing cells. 293VnR cells transiently transfected with ADAM12-GFP or ADAM12-GFP p.L792F were plated on FBS-coated coverslips in triplicates. Proliferating cells were stained with Edu. Four images were taken from each triplicate and EdU- and GFP-positive cells were calculated as percentage of total cells (DAPI staining). The data are shown as +/− standard error of the mean (SEM) (n = 2 independent experiments performed in triplicates); “ns.” indicates no statistical difference (p>0.05, two-tailed unpaired t-test). (B) ADAM12 p.L792F does not affect ADAM12-mediated proEGF shedding. 293VnR cells were transiently transfected with alkaline phosphatase (AP)-tagged proEGF together with ADAM12-GFP WT, CI ADAM12-GFP, or ADAM12 p.L792F (p.L792F). EGF-shedding was calculated from the ratio of AP activity in the cell media to total AP activity in the media and corresponding cell lysate, corrected for background (sample without AP substrate) (n = 2 independent experiments performed in triplicate). * indicates a statistically significant difference (p<0.0001, two-tailed unpaired t-test) and “ns.” indicates no statistical difference (p>0.05). (C) Representative western blot of protein expression control from experiment in (B). Transfected 293VnR cells were lysed in RIPA buffer and analyzed by western blot with the appropriate antibodies. Note the reduction in the upper band of AP-EGF, indicating shedding of proEGF-AP from the cell surface. Actin was used as a loading control.