Saxena SK , Singh M , Kaur S , George C .

DK-57717 NIDDK NIH HHS , R01 DK057717 NIDDK NIH HHS

	Fig 1. Syntaxin1A augments currents - HT-29 cells grown on cell inserts were transfected with wild-type syntaxin1A (Syn1A) and its truncated constructs. Inset- Structure of syntaxin1A. TMD represents the transmembrane domain. 48 hours later the amiloride-sensitive currents were recorded as described in the text. The data represents a mean of five individual experiments. Experimental conditions that resulted in a significant change (p < 0.05) from the relevant control values (Bar one) are denoted by multiple asterisks. Three asterisks denote higher statistical significance.
	Fig 2. Syntaxin1A up-regulates amiloride-sensitive currents in a dose-dependent manner - HT-29 cells grown on cell inserts were transfected with wild-type syntaxin1A (Syn1A) at different concentrations. 48 hours later amiloride-sensitive currents were recorded (A) as described in the text. (B) The protein extracts from the transfected cells were solubilized and analyzed by Western blot analysis. The blots were developed with syntaxin1A antibody. The data represents a mean of three individual experiments. Experimental conditions that resulted in a significant change (p < 0.05) from the relevant control values (Bar one) are denoted by multiple asterisks. Three asterisks denote higher statistical significance.
	Fig 3. Syntaxin1A antibody inhibits amiloride-sensitive currents - The HT-29 cells were targeted with anti-syntaxin, or anti-ENaC antibodies or irrelevant IgG using the Chariot® protein delivery system. Antibodies were complexed with the Chariot® reagent at a ratio of 1 ng IgG: 2 μL Chariot® in 100 μL PBS for 30 min. Then the IgG: Chariot® complexes were overlaid onto cultured cells in the presence of fresh serum free culture medium for 3 hr and amiloride-sensitive currents were measured. Data represent a mean of three individual experiments. Experimental conditions that resulted in a significant change (p < 0.05) from the relevant control values (Bar one) are denoted by multiple asterisks.
	Fig 4. ENaC interacts with different syntaxin1A domains - Endogenous ENaC interacts with syntaxin in HT-29 cells - GST-syntaxin proteins were used to pull down proteins bound to natively expressed ENaC in HT-29 cell lysates. The immune complex was adsorbed on glutathione beads and then separated by SDS-Polyacrylamide gel electrophoresis and transferred to PVDF membrane. The blots were probed with affinity purified subunit specific γENaC antibody. The data shows interaction of multiple syntaxin1A domains with ENaC and points to its involvement and physiological significance in the regulation of the amiloride-sensitive epithelial sodium channel in native cells.
	Fig 5. Syntaxin1A domains modulate ENaC expression at the cell surface - HT-29 cells were transfected with wild-type syntaxin1A and its truncated constructs. Cell surface proteins were biotinylated with Sulpho-NHS-SS-biotin, pulled down with streptavidin-agarose separated by SDS-PAGE and transferred to PVDF membrane. The blots were probed with γENaC antibody (A). The protein was analyzed by densitometry (B). The data reflect increased expression of ENaC in HT-29 cells transfected with syntaxin1A, which is reflected in enhanced amiloride-sensitive currents reported in the text. Data represent three individual experiments each performed with different batches of HT-29 cells.
	Fig 7. Syntaxin1A stimulates amiloride-sensitive channel (ENaC) activity by multiple signaling modules - HT-29 cells (60-70% confluent) on cell inserts were transfected with wild-type syntaxin1A (Syn1A) and incubated in the presence of indicated inhibitors; PD98059 (PD; 50 μM) or LY294002 (LY; 50 μM), RpCPTcAMP (RpcAMP; 30 μM) and PMA (200 nM) 48 hours later. The inhibition or activation in intact cells was measured with the indicated concentration of reagents or dimethyl sulfoxide carrier (0.1%, v/v) for 45 min at 37°C. The amiloride-sensitive currents were recorded as described in the text. The data represents a mean of three individual experiments. Experimental conditions that resulted in a significant change (p < 0.05) from the relevant control values (Bar one) are denoted by multiple asterisks. Three asterisks denote higher statistical significance.

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