Fig. 4. The transport of cell surface localized PDIs is influenced by KDELR1. (A) The reduced expression of the KDELR1 in two HEK293T cell lines, created by shRNA, was analyzed by flow cytometry with permeabilized cells. The bar diagram shows average mean values and standard deviations of at least three independent experiments. Following Student's test, the calculated p values were **p < 0.01 and therefore considered as significant. (B) KDELR1 can be detected on the cell surface of non permeabilized HEK293T cells. The cell surface association of KDELR1 was comparably reduced at the cell surface of the KDELR1 knock-down cell lines. Following Student's test, the calculated **p < 0.01 was considered as significant. (C) Typical graphs of the flow cytometric analysis summarized in Fig. 4A (upper panel) and 4B (lower panel. (D) Down regulation of KDELR1 expression reduces cell association of PDIA6 and PDIA6wo. Wild-type and KDELR1 knock-down clone 3 and clone 7 cell lines were either incubated with PBS or with fluorescently labeled proteins: PDIA6, PDIA6wo, or IL-6. After 30 minutes, cells where washed and analyzed by flow cytometry. Binding of PDIA6 was significantly reduced in the knock-down cell line as indicated by the asterisks. *p< 0.05, **p< 0.01, and ***p< 0.001 were considered as significant. Additionally, the interaction of PDIA6wo was reduced in KDELR1 knock-down cell lines. (E) Representative graphs of the flow cytometric analysis shown in Fig. 4D. (F) Bar diagram summarizing the flow cytometric analysis of the cell surface expression of three different PDIs (PDIA1, PDIA3, and PDIA6) well known as being located at the cell surface. The analysis was performed at least three times independently, with wild-type HEK293T cells and with two KDELR knock-down cell lines. Significance is indicated by **p < 0.01 and ***p < 0.001. The control samples were only treated with secondary antibody and not primary antibodies. (G) Examples of flow cytometric analysis summarized in Fig. 4F.