Recombinant human SLC44A2 was produced in Sf9 insect cells transfected with full‐length SLC44A2 cDNA as described (Kommareddi et al, 2009 (link)). SLC44A2–NT (CTL2‐NT) rabbit antiserum against synthetic SLC44A2 peptides was coupled to CNBr beads as previously described and used to immunoprecipitate SLC44A2 from cell lysates as described (Nair et al, 2004 (link)). Whole cell lysates from lung tissues of Slc44a2 wild‐type and knockout FVB mice (Kommareddi et al, 2015 (link)) were prepared in lysis buffer with 1% NP‐40 and stored frozen until use. UM‐SCC‐47, a human squamous cell carcinoma cell line, naturally expresses wild‐type SLC44A2 and served as a source of wild‐type SLC44A2 protein. Cell lysates of Sf9 insect cells expressing full‐length rHuSLC44A2 and UM‐SCC 47 expressing wild‐type SLC44A2 protein were similarly prepared. The cell lysates (150 μg protein) were immunoprecipitated with anti CTL2‐NT beads at a concentration of 2 μg/ml, collected and washed by centrifugation and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) as described previously (Kommareddi et al, 2009 (link)). The primary antibodies, rabbit anti‐CTL2‐NT (1/500 or 2 μg/ml), human alloantibodies RIF, and VER were used at 1:10. Antibody binding on Western blots was detected with enhanced chemiluminescence using the appropriate affinity purified secondary antibody. Rabbit antihuman IgG‐IgM‐specific antiserum was diluted 1:2,000. Goat anti‐rabbit IgG heavy and light chain specific was used at 1:5,000.
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