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. 2015 Jul 21;1:15018. doi: 10.1038/celldisc.2015.18

Figure 5.

Figure 5

Activation of ALIX by ALG-2 has a rate-limiting role in MVB sorting of activated EGFR. (a) HEK293 cells were first transfected with si-NC or si-ALG-2(1) and cultured for 48 h. These cells were then transfected with the expression plasmids for WT FLAG-ALG-2* or Mut FLAG-ALG-2* (E47A/E114A mutation in ALG-2) and cultured for another 12 h. After serum starvation for 12 h, these cells were stimulated with EGF for 30 min and assayed for MVB sorting of activated EGFR by the proteinase K protection assay (left and middle panels). The average percentages of protected EGFR and s.d. were determined from three independent experiments and plotted (right panel). (b) HEK293 cells were transfected with indicated siRNAs and expression plasmids for GFP-ALIX* and processed as described in a. (c, d) HEK293 cells transfected with indicated siRNAs were stimulated with EGF for the indicated minutes (c) or hours (d), and cell lysates were immunoblotted with indicated antibodies to visualize phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, and actin (top panel). The relative levels of p-ERK at different time points were determined, normalized against the level at 60 min in c and 1 h in d. The average levels of p-ERK and s.d. were determined from three independent experiments and plotted (bottom panel). ALG-2, apoptosis-linked gene-2 product; EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; MVB, multivesicular body; NS, not significant; si-NC, negative-control small interfering RNA; siRNA, small interfering RNA.