Anti mouse cy3

BMMs and murine cardiac fibroblasts were cultivated in 8-chamber slides (Sigma-Aldrich, Germany). Cells were fixed with 4% Histofix (Roth, Germany) and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich). BMMs were stained against F4/80 (1:100, Abcam, UK) or CD11b (1:100, Abcam, UK) (Figures 1B–E). Fibroblasts were stained with antibodies against vimentin (1:100), CD31 (negative control for endothelial cells) (1:100), and desmin (negative control for smooth muscle cells) (1:100) (Figures 1F–K). The secondary antibodies anti-mouse FITC (1:100) (Dianova, Germany) or anti-mouse Cy3 (Dianova, Germany) were applied according to the manufacturer’s protocol. Nuclei were stained using DAPI (1:50000) (Sigma, Germany) and cells were mounted with Fluoromount G (Southern Biotech). Negative controls were performed by omitting the primary antibodies. Images were acquired using a BZ-9000E fluorescence microscope (Keyence, Germany). All evaluations were performed in a blinded manner.

All reagents were obtained from Sigma-Aldrich, Roth or Merck unless otherwise stated. The following commercial antibodies were used: Mouse anti-GM130 (BD Transductions Laboratories, 610822, final conc. 2 ng/ml). Rabbit anti-VPS13B (442) was described earlier^{6 (link)}. Secondary antibodies for Western blotting were anti-rabbit IgG-HRP and anti-mouse IgG-HRP (both DAKO GmbH) and for immunofluorescence donkey anti-mouse-Cy3, anti-rabbit-Cy3, and anti-rabbit-Cy2 (all Dianova GmbH). 6-Diamidino-2-phenylindole (DAPI) (Invitrogen) was applied for nuclear DNA-staining.

Two days post‐transfection, secretion of pC4 reporter construct was induced with incubation of 3.3 µM rapamycin (cayman chemicals, Michigan, USA) for 0 or 10 min. Cells were washed twice with ice‐cold PBS and fixed in 4% PFA in PBS. After permeabilization with 0.1% Triton X‐100 in PBS for 3 min, cells were blocked with 1% goat serum for 1h at RT. Primary antibody (anti‐GM130, mouse, cell signaling, 1:400 in 1% goat serum) was incubated over night at 4°C. Secondary antibody incubation with anti‐mouse Cy3 (Dianova) was performed in a 1:400 dilution in 1% goat serum for 1h at RT. Nuclear staining was performed with Hoechst 33342 (Hoechst, Frankfurt/Main, Germany) followed by mounting with Mowiol. Confocal microscopy images were taken with a LSM700 (Carl Zeiss, Oberkochen, Germany).