Figure 2. Extracellular loop (L) 3 and C-terminal domains of CRACM3 are critical for binding the syntaxin4 N-peptide.

All plasmids were transfected into cells using RBL-2H3 Cell Avalanche^TM Transfection Reagent. At 72 h after transfection, the cells were stimulated with 0.5 μM TG for 15 min, and His-tag immunoprecipitation followed by western blotting using specific antibodies was performed. (a) CRACM3 protein, which was co-immunoprecipitated using the His-tag in rSx4-His-wild- (lane 1), rSx4-His-mTM- (lane 2), rSx4-His-mH3- (lane 3), rSx4-His-mIIS- (lane 4), and rSx4-His-mNp-transfected cells (lane 5), was detected using western blotting. Full-length blots are presented in Supplementary Fig. 1. Upper: CRACM3 expression in plasmid-transfected cells. Lower: CRACM3 expression normalized against actin levels in total cell lysates. (b) The protein levels of CRACM3 in His-tagged CRACM3 truncation-transfected cells after His-tag immunoprecipitation. (a.u., arbitrary area units; ***P < 0.001; n = 3). (c) Syntaxin4 protein, which was co-immunoprecipitated with His-tag in rM3-His wild- (lane 1), rM3-His-mNTD- (lane 2), rM3-His-mL1- (lane 3), rM-His-mL2- (lane 4), rM3-His-mL3- (lane 5), and rM3-His-mCTD-transfected cells (lane 6), was detected by western blotting. Full-length blots are presented in Supplementary Fig. 1.Upper: syntaxin4 expression in the plasmid-transfected cells. Lower: syntaxin4 expression normalized against actin levels in total cell lysates. (d) The protein levels of syntaxin4 in His-tagged CRACM3 truncation-transfected cells after His-tag immunoprecipitation. (a.u., arbitrary units; *** P < 0.001; n = 3).