Fig. 6

CTR1 localization regulated by extracellular stiffness and glycosylation. a Immunoblot analysis of CTR1 in m5-2D MM cell line, comparing mock and overexpression (OE) of flag-CTR1 group; g, glycosylation (30–50 kDa); CTR1 (25 kDa); t: truncate (15 kDa). b Schematic images of the culture system. High stiffness culture corresponds to direct plating whereas low stiffness culture corresponds to plating on the Matrigel layer (scale bar = 100 μm). c Immunoblot analysis of CTR1. Matrigel, Matrigel layer in the legend (b). MβCD (10 μM, 1 h); g, glycosylation (30–50 kDa); CTR1 (25 kDa); t: truncate (15 kDa). d Immunofluorescent images from m5 Flag-CTR1 stably expressed cell line. Left images; cells were cultured on glass slip as high stiffness culture. Right images; cells were cultured on Matrigel layer as a low stiffness culture. Flag-CTR1 (Alexa 488, green), F-actin (CF 568, magenta) and nuclear staining (Hoechst33342, blue; scale bar, 5 μm). e, f Immunoblot analysis of m5 Flag-CTR1 stably expressed cell after treatment of TM (tunicamycin) or BP (2-bromopalmitate) for 9 h. g, glycosylation (30–50 kDa); CTR1 (25 kDa); t, truncate (15 kDa). g Immunofluorescent images from m5 Flag-CTR1 stably expressed cells with after treatment of TM or BP for 9 h. Flag-CTR1 (Alexa 488, green), F-actin (CF 568, magenta) and nuclear staining (Hoechst33342, blue; scale bar: 20 μm). Refer to text for details