LRIM1 can form protein complexes with all members of the APL1 family.

A. Structure of expression plasmids used for co-immunoprecipitation of proteins expressed in Trichoplusia cells. Fusion tags: purple, FLAG tag; pink, 6xHis tag. Small boxes represent LRR repeats; yellow, Cys-rich region; green, coiled-coil (CC) domain with helix-loop-helix region between CC domains. Additional structural details in [23]. B. FLAG-tagged LRIM1 was used to pull down 6xHis-tagged LRIM1, APL1A1, APL1B and APL1C. Western blots were performed with anti-6xHis/HRP (top panel) and anti-FLAG/HRP (bottom panel) to detect the 6xHis-tagged co-precipitated proteins and FLAG-tagged LRIM1, respectively. C. Expression plasmids used for pull-downs of proteins expressed in Drosophila S2 cells. Fusion tags: orange, V5 tag; light green, Strep tag; other protein features as in (A). D. S2 cells were transfected with Strep-tagged APL1A, V5-tagged LRIM1, or co-transfected with both plasmids. Cell extracts were purified by Strep-tag column affinity, and analyzed by Western blot using anti-Strep or anti-V5 antibodies. Lane labels: A, cell extract applied to Strep column; E, eluted fraction from Strep column. E. Coomassie-stained SDS-PAGE of extract from co-transfected cells (APL1A-Strep+LRIM1-V5) after elution from Strep column. Lane NR, sample non-reduced, arrow indicates APL1A/LRIM1 heterodimer; lane R, sample reduced, arrow 1 indicates APL1A-Strep monomer, arrow 2 LRIM1-V5 monomer.