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. 2014 Jun 2;9(6):e98606. doi: 10.1371/journal.pone.0098606

Figure 1. VPS35 localizes with MHCII and Vps35 is required in MHCII cell surface retention.

Figure 1

(A) BMDCs were transduced with shRNA constructs targeting VPS35 (shVPS35) or luciferase (shLuc) as a control. Cells were treated with the Golgi transport inhibitor Brefeldin-A for a 5-hour chase, and MHCII cell surface expression was determined by flow cytometry (see Materials and Methods section for details). The percent of initial MHCII remaining on the plasma membrane after chase was calculated for three independent samples and plotted as mean ±s.d. To confirm efficient knockdown, VPS35 expression relative to β-actin was determined by quantitative PCR. (B) BMDCs were fixed and labeled with antibodies against MHCII and VPS35 for microscopic analysis. (C) Following the MHCII endocytosis assay, BMDCs were fixed and labeled with anti-VPS35. Zoomed images are demarcated by the white box and dashed lines in the adjacent image. For each condition, >20 individual cells were imaged. Images were collected with 100× oil objective. Scale bars, 10 µm and 1 µm.