Next, we measured the internalization rate of EGFR during 3 min of EGF treatment.
To investigate the involvement of endogenous PLD1 in EGFR endocytosis, we designed siRNA for human PLD1 (siPLD1), corresponding to the human PLD1a coding nucleotides 1455 1475, and measured the internalization of EGFR in HeLa cells.
To confirm the role of PLD1 and to check the involvement of the lipase activity of PLD1 in EGFR endocytosis, we transfected wild type or a lipase inactive mutant PLD1 (K898R) after depleting the endogenous PLD1 from HeLa cells by siPLD1, and measured the internalization of EGFR after 2 min of EGF treatment.
Because PU-KKmt and PU-KKmt-Bpy contain a His-tag at their N-termini, we measured the internalization of the conjugates by Western blot using an anti-His tag antibody.
Assays to measure the internalization of promastigotes into canine monocyte-derived macrophages and peritoneal macrophages and visualization of amastigotes were performed in a similar way to the binding assays after the Leishmania had interacted with the cells for 48 h.
To evaluate whether other important membrane-dependent events that occur early upon TLR activation were affected by SMPDL3B depletion, we measured the internalization rates of TLR4 and the phagocytic uptake of CpG.
C6-NBD-PS and C6-NBD-PC are the fluorescent analogs of PS and phosphatidylcholine; we measured the internalization of C6-NBD-PS C6-NBD-PS C6-NBD-PSippand and scramblase, respectively, by comparing the fluorescence intensity associated with the cells byflippase ander Bscramblasetrespectivelydescribyd in "Materials and Methods".
Since the method described above allowed us to discriminate Alexa488/InlA-beads that had become internalized from those that had not, we further used it to calculate the rate of internalization by measuring the fraction of beads internalized at various time points after initial bead binding for MDCK and Caco-2 cells (Figure 3A and 3B – gray line).
This was achieved by measuring the shift in NECA internalization concentration-response curves at both A3-YFP and A3 W243F-YFP in the presence of 25 nM CA200645 (Supporting Information Figure S2).
The results of several studies measuring the time-course of EGFR internalization following continuous EGF treatment correlate with our observations of the temporal response of PRL-array decondensation to EGF.
