Irdye cw secondary antibodies

The specificity of the CXCL16 and CXCR6 antibodies was investigated by Western blotting. Cell lysates were incubated with NuPAGE LDS Sample Buffer (Life Technologies, USA) for 5 minutes at 85 °C, sonicated briefly, and run on a NuPAGE® 4-12 % Bis Tris Gel (cat#NP0322, Life Technologies, USA). Blotting was performed on a Hybond nitrocellulose membrane (cat# RPN2020D, GE Healthcare) using the NuPAGE blotting system (Life Technologies, USA). The membrane was incubated with Odyssey blocking buffer (cat# 927–40000, LI-COR Biosciences, Germany) for 1 hour at room temperature. Primary and secondary antibodies were diluted in the blocking buffer. Anti-CXCL16 antibody (cat# 101404) was used in the dilution of 1:500, anti-CXCR6 in the dilution 1:500 (cat# 125115) and anti-actin (cat#A2066, Sigma) 1:2000. IRDye CW secondary antibodies (cat# 926–32213 and 926–68073, LI-COR, Germany) were used in dilution 1:10000. Molecular weight markers used were SeeBlue Plus 2 (cat# LC5925, Life Technologies, USA), and Magic Mark XP (cat# LC5602, Life Technologies, USA). Images were acquired on the ODYSSEY Sa Infrared Imaging System (LI-COR, Germany).

Cell lysates were incubated with NuPAGE LDS Sample Buffer (Life Technologies, USA) for 5 minutes at 85°C, sonicated briefly and run on a NuPAGE 4–12% Bis Tris Gel (Life Technologies, USA). Blotting was performed onto a Hybond nitrocellulose membrane (GE Healthcare) using the NuPAGE blotting system (Life Technologies, USA). The membrane was incubated with Odyssey blocking buffer (LI-COR Biosciences, Germany) for 1 hour at room temperature. Primary and secondary antibodies were diluted in the blocking buffer. Anti-MCT1 antibody (Millipore, USA, cat#AB3538p) was used in the dilution of 1∶500, anti-MCT4 in the dilution 1∶500 (Santa Cruz, USA, cat#SC-50329) and anti-actin (Sigma, cat#A2066) 1∶2000. IRDye CW secondary antibodies (LI-COR, Germany) were used in dilution 1∶10000. Molecular weight markers used were SeeBlue Plus 2 (Life Technologies, USA, cat#LC5925) and Magic Mark XP (Life Technologies, USA, cat#LC5602). Images were acquired on the ODYSSEY Sa Infrared Imaging System (LI-COR, Germany).