Fig 1: KDELR3 regulates expression and processing of the metastasis suppressor KAI1.a Screen of known melanoma metastasis suppressor expression following KDELR3 knockdown (3 days post knockdown). P, parental; C, siControl; K3, siKDELR3. b qPCR of KAI1 RNA expression (CD82 gene) in siRNA-knockdown cells (indicated), 3 days post knockdown. c–e KAI1 protein (c) and RNA (d, e) expression in 1205Lu cells transfected with CD82/KAI1 overexpression (KAI1) or PCMV6-AC control vector (Vec.), KDELR3 transcript 1 with DDK tag (K3_1), KDELR3 transcript 2 with DDK tag (K3_2), or PCMV6 control vector (Vec.1). They were harvested 3 days post transfection. Equal protein amounts subjected to immunoblot analysis with an anti-KAI1 and anti-DDK antibody and anti-vinculin loading control (c). f 1205Lu cells parental (P), and 1205Lu cells transiently transfected with control siRNA (C), and KDELR3 siRNA (K3), harvested 3 days post transfection and equal protein amounts subjected to immunoblot analysis with an anti-KAI1 and anti-gp78 antibody. Red arrow indicates high-molecular-weight KAI1. g KAI1 protein expression in siRNA-knockdown (indicated) 1205Lu cells harvested 3 days post transfection and treated with deglycosylation enzymes (De-G). h qPCR of gp78 RNA expression (AMFR gene) in siRNA-knockdown cells (indicated), 4 days post knockdown. f, g Anti-vinculin antibody used to control for protein loading. i qPCR of KDELR3 RNA expression in siRNA-knockdown cells (indicated), 4 days post knockdown. j Co-immunoprecipitation of endogenous gp78 and mCherry-tagged gp78 (gp78-mCh) with FLAG-tagged KDELR3 (K3-DDK) in stably transduced 1205Lu cells. k pol2 > KDELR3-GFP (green) co-localizes with pol2 > gp78-mCherry (red) in 1205Lu metastatic melanoma cells. Scale bars, 50 µm. l Schematic of the KDELR3–KAI1 axis in melanoma metastasis. a Representative of four independent experiments. b–f, h, j–k Representative of three independent experiments. g, i Representative of two independent experiments. b, d, e, h, i Bars and error bars represent mean ± s.e.m. N = 3 (representative of three independent experiments). a, c, f, g Square brackets depict KAI1 molecular weights. j Square bracket depicts all forms of gp78, including endogenous and mCherry-tagged gp78.