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. Author manuscript; available in PMC: 2017 Feb 8.
Published in final edited form as: Dev Cell. 2016 Feb 8;36(3):331–343. doi: 10.1016/j.devcel.2016.01.001

Figure 7. The S718-S721 phosphorylation does not affect the function of ALIX in MVB sorting of activated EGFR.

Figure 7

(A) HEK293 cells ectopically expressing indicated forms of GFP-ALIX were mock-treated or stimulated with EGF for 1 h, and the average percentage of each GFP-ALIX in the M fraction was determined by membrane flotation centrifugation of the PNS (n = 3 ± SD). (B-F) Left: HEK293 cells were processed as diagrammed, and cell lysates were immunoblotted with the indicated antibodies. Right: cells were stimulated with EGF for 30 min, and assayed for MVB sorting of activated EGFR by the proteinase K protection assay (n = 2 ± data range). (C&D) Cells were stimulated with EGF for indicated minutes, and cell lysates were immunoblotted with the indicated antibodies. The relative levels of p-ERK were determined and normalized against the level of si-NC cells at 60 min (n = 3 ± SD). (E&F) Cells were stimulated with EGF for indicated hours, and cell lysates were immunoblotted with the indicated antibodies. The percentages of remaining EGFR at different time points were determined. See also Figure S5.