JCS02917 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Glycosylation of LOX1. Effects of tunicamycin and PNGase F on LOX1 glycosylation. (A) LOX1-Flag transfected HEK-293T cells were incubated in the presence or absence of 10 µg/ml tunicamycin for 16 hours before cells were lysed. Equal amounts of total cell lysate were western blotted with sheep anti-LOX1 antibodies. (B) LOX1-Flag transfected HEK-293T cells were lysed and equal amounts of total cell lysate treated with or without PNGase F for 16 hours as described by supplier (Invitrogen, Holland). Affinity-purified sheep anti-LOX1 antibodies were used for western blotting. Mature, deglycosylated and immature LOX1 forms indicated in B are similar to those in A.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Acid stability of LOX1, OxLDL and antibody complexes. Wild-type (WT) LOX1 was expressed in HeLa cells and analysed by immunofluorescence microscopy. (A) LOX1-expressing HeLa cells were fixed and incubated with mouse anti-Flag (see Materials and Methods), washed and incubated with DMEM at different acidic pH (shown under each panel) before rinses, fixation and labelling with secondary anti-mouse fluorescent conjugate and visualisation using microscopy. (B) This experiment is similar to the experiment shown in A except that the mouse anti-Flag was incubated with LOX1-expressing HeLa cells in DMEM buffers at different acidic pH (shown under each panel) before processing and visualisation using microscopy. (C) HeLa cells expressing tagged LOX1 were incubated with mouse anti-Flag antibodies on ice and then warmed up to 37°C for the indicated times prior to fixation. Cells were permeabilised (apart from the 0 time point) and then LOX1 detected using an affinity-purified sheep anti-LOX1 antibody. Primary antibodies were visualised using AlexaFluor 594-conjugated anti-mouse IgG (red) and AlexaFluor 488-conjugated anti-sheep (green). Nuclei were stained with DAPI (blue). Boxes show enlarged areas of colocalisation (yellow) between the internalised anti-Flag and the post-fixation labelled LOX1. Scale bars: 10 µm.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. LOX1 and OxLDL codistribution with clathrin and caveolin-1. (A) HeLa cells expressing tagged LOX1 were pre-incubated with labelled OxLDL (red) on ice and then warmed up to 37°C followed by fixation, permeabilisation and labelling with rabbit anti-clathrin light chain or rabbit anti-caveolin-1 followed by Alexa Fluor 488-conjugated anti-rabbit IgG antibodies (green). Percentage calculated for colocalisation of labelled OxLDL with clathrin or caveolin-1 at different time points after addition of ligand (n=3 separate experiments in each of which five cells were quantified, ± s.e.m.). Comparison with t=0 minutes to calculate P values, *P<0.05. HeLa cells were also incubated with saturating rhodamine-conjugated Tf on ice prior to warming up followed by fixation, labelling with anti-caveolin-1 antibodies and secondary Alexa Fluor 488-labelled antibodies and calculation of percentage colocalisation. (B) Serum-starved HeLa cells expressing tagged LOX1 were incubated with or without saturating amounts of labelled OxLDL on ice, warmed up for different periods and fixed, permeabilised and labelled with mouse anti-Flag and either rabbit anti-clathrin light chain or rabbit anti-caveolin-1, followed by Alexa Fluor 594-conjugated anti-mouse IgG (red) and Alexa Fluor 488-conjugated anti-rabbit IgG antibodies (green). Percentage colocalisation between the different markers was calculated as described in A. Boxes display enlarged areas with colocalisation appearing yellow. Scale bars: 5 µm.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Further LOX1 mutational analyses. Wild-type (WT) and mutant tagged LOX1 constructs were expressed in HeLa cells and analysed by western blotting (A) and immunofluorescence microscopy (B). Blots were probed with affinity-purified sheep anti-LOX1 or sheep anti-TGN46 (control). Fixed cells were labelled with mouse anti-Flag and FITC-conjugated anti-mouse IgG antibodies. Arrow indicates DD(4,5)AA LOX1 clustered on the surface of a transfected cell. (C) HeLa cells transfected with the tagged LOX1 WT or mutant constructs were pulsed with labelled OxLDL and chased at 37°C before fixation and labelling with anti-Flag antibodies to detect LOX1. The percentage of transfected cells with internalised OxLDL was counted (n=3 separate experiments ±s.e.m.). Comparison with control LOX1 WT was used to calculate P values, *P<0.01. (D) Transfected cells dual-labelled for OxLDL (red) and WT LOX1 (green) or LOX1-DD(4,5)AA (green) are shown. Nuclei were stained with DAPI (blue). (E) Cell control or LOX1-DD(4,5)AA-transfected HeLa cells were labelled with FITC-conjugated Con A (green) immediately before fixation. Fixed cells were then incubated with sheep anti-LOX1 and Alexa Fluor 594-conjugated anti-sheep IgG antibodies (red). Areas of colocalisation appear yellow and arrow indicates clustered LOX1. Scale bars: 10 µm.