Alexa 546 conjugated anti mouse antibody

To detect HB-EGF molecules on the surface of intact cells, cells were incubated with 1 μg/mL of each primary mAb at 4°C for 1 hour and then fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at room temperature for 30 minutes. Cells were then stained with the Alexa546-conjugated anti-mouse antibody (Life Technologies, Thermo Fisher Scientific) at 4°C for 1 hour. Images were captured by a conventional fluorescent microscope (Olympus BX50, Tokyo, Japan). For CRM197 competition, cells were incubated with 50 μg/mL of CRM197 at 4°C for 1 hour before incubation with the primary mAb.

HeLa cells that were incubated with SirReal2 (20 and 50 μM), SirReal6 (50 μM), AGK2 (Sigma-Aldrich, 20 μM) or DMSO as a control in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, antibiotics and DMSO (1% (v/v)) for 4 h, were fixed with ice-cold methanol (10 min), washed with PBS and blocked with PBS supplemented with 0.1% (v/v) Triton-X-100 and 5% (v/v) FCS (30 min). Cells were then stained with an anti-acetyl-α-tubulin antibody (Sigma-Aldrich, T6793) and then probed with a secondary Alexa 546 conjugated anti-mouse-antibody (Invitrogen). Nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Coverslips were mounted with FluoroMount (Sigma-Aldrich) and sealed with DPX Mountant (Sigma-Aldrich). Images of the mounted samples were acquired on a Leica DM500 microscope equipped with a Leica DFC 395 FX camera and HBO 100 W lamp40 (link). The microscope was run with the Leica Application Suite 4.4.0 software. Chroma UV filter set (No. C40888) and Leica N2.1 filter set (No. 513832) were used for DAPI and Alexa 546 signal acquisition, respectively, with a HCX FL Fluotar 40x/0.75 (dry) objective. Further details about the equipment and the settings that were used to acquire the images are found in the Supplementary Methods section. Unprocessed images are found in Supplementary Fig. 9.