Figure 4
From: RILP interacts with HOPS complex via VPS41 subunit to regulate endocytic trafficking
VPS41 subunit plays a crucial role in RILP-HOPS interaction.
(A). Hela cells expressing GFP-RILP were immuno-labeled with anti-VPS41 antibody to demonstrate the recruitment of endogenous VPS41 by RILP. (B). Hela cells were co-transfected with pSuper.GFP-shRNA-VPS41, myc-tagged HOPS subunits and HA-RILP, then processed for immuno-staining with myc and HA tag antibodies. Knockdown cells were revealed by co-expresed GFP signal. Myc-tagged proteins were revealed by Texas-red conjugated secondary antibodies and HA-tagged proteins were revealed by Cy5-conjugated secondary antibodies. Immuofluorescence microscopy showed that depletion of VPS41 disrupts the co-localization of other HOPS subunits with RILP. (C). GST-pulldown experiments demonstrated that depletion of VPS41 decreases the amount of HOPS subunits bound to RILP. (D). Quantitative analysis of the results from triplicate experiments revealed that the amount of HOPS subunits bound to RILP is significantly decreased when VPS41 was knocked-down. Un-cropped blots/gels are presented in Supplementary Figure s9. bar = 20 μm.
