Alexa fluor 488 conjugated streptavidin

After a single wash with PBS, samples were fixed with 4% paraformaldehyde (153799, Aname) for 20 min at room temperature. Next, samples were washed twice with PBS and further blocked using Tris-buffered saline (TBS) with 6% donkey serum (S30, Millipore) and 1% Triton X-100 (T8787, Sigma) for 1 h at room temperature. Samples were then treated overnight at 4 °C with primary antibodies diluted in antibody dilution buffer consisting of TBS with 6% donkey serum and 0.5% Triton X-100. After three rinses with antibody dilution buffer, samples were treated for 4 h at room temperature with fluorescent-conjugated secondary antibodies (Alexa Fluor (A) 488-, Cy3- or A647-; 1:200). A previous blocking step with a streptavidin/biotin blocking kit (SP-2002, Vector Labs) was performed when samples were assayed for biotinylated LTL (B-1325, Vector Labs) and Alexa Fluor 488-conjugated streptavidin (SA5488, Vector Labs) was used to detect LTL⁺ cells. Nuclei were detected using 4,6-diamidino-2-phenylindole (DAPI; 1:5000, D1306, Life Technologies) for 30 min. For mounting, samples were immersed in Fluoromount-G (0100–01, Southern Biotech). Image acquisition was carried out using an SP5 Leica microscope or a Zeiss LSM780 confocal microscope. Primary antibodies and associated information are provided in Supplementary Table 5.

Dimethylsulfoxide (DMSO) and EDTA-free protease inhibitor cocktail tablets were purchased from Sigma (St. Louis, MO). Peanut agglutinin (PNA) and Alexa Fluor 488-conjugated streptavidin were purchased from Vector. 4969-Diamidino-2-phenyl-indole (DAPI) for the nuclear staining was purchased from Life Technologies (Grand Island, NY). Polyvinylidene difluoride (PVDF) membrane was obtained from Millipore (Burlington, MA). The GALR3 inhibitor SNAP-37889 was purchased from Alomone Labs (Jerusalem, Israel) (PubChem CID 1471834). The GALR3 activator galnon was purchased from Sigma (PubChem CID: 5311268). pCMV6-GALR3-(Myc-DDK-tagged) DNA was purchased from OriGene Technologies (Rockville, MD). Polyethylenimine was obtained from Sigma. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was also purchased from Sigma. All the antibodies used in this study are listed in Table 1.