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. 2010 Mar 5;6(3):e1000792. doi: 10.1371/journal.ppat.1000792

Figure 2. Endocytosis of PA is β-arrestin and AP-1 dependent.

Figure 2

A-B: Hela cells were transfected 72 hrs with control siRNAs or siRNAs against Clathrin Heavy Chain (CHC), the ubiquitin ligase E3 Cbl (Cbl), Grb2, AP-1, AP-2, β-arrestin-1 (β-Arr-1), β-arrestin-2 (β-Arr-2) or Dab2. The efficiency of siRNAs was analyzed on cell extracts (40 µg of proteins) by SDS-PAGE and western blotting (Fig. S1). Cells were incubated with 500 ng/ml PA63 and 100 ng/ml LF for 1 hour at 4°C and different times at 37°C and cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal PA SDS-resistant heptamer (pPA7mer) and N-terminus of MEK1 (MEK1 (N)). B: Levels of pPA7mer and full length MEK1 were quantified using the Typhoon scanner and normalized to 1 and 100% respectively at time 0 (1 hour at 4°C). The plot represents the means of 4 independent experiments. Errors represent standard deviations. C: Hela cells were transfected 72 hours with CMG2-V5 and control siRNAs or siRNAs against AP-1, AP-2 or β-arrestin-2 (β-Arr-2). Cell extracts were subsequently analyzed by SDS-PAGE and western blotting to reveal the different forms of PA, CMG2-V5 and N-terminus of MEK1 (MEK1 (N)).