SUPPLEMENTAL DATA:
LEGENDS:
TABLE-S1:Purification table of inhibitory protein from AMI subject plasma.
The table summarizes the protein concentration and the nitric oxide production of the AMI subject plasma sample, the pooled NO inhibitory fractions of the DEAE-cellulose separation and the pooled NO inhibitory fractions of Sephadex G-50 separation. The specific activity (units of inhibitory activity per mg of inhibitor protein) was calculated at every step and respective fold purification was calculated.
FIGURE-S1:
Figure-S1: DEAE-Cellulose separation of inhibitory protein from plasma of Normal subject.
Plasma of Normal subject was loaded in the prepared DEAE cellulose column and eluted with 50mM Tris-HCl buffer pH 7.4 containing 1.0 mM EDTA, 2.0 mM PMSF and 0.3 M sucrose with increasing gradient of NaCl i.e., 0M, 0.1M, 0.2 M, 0.3 M, 0.4M and 0.5 M. Each gradient constitute 50 fractions and each fraction of 1.5ml and to sum up 300 fractions were collected. Protein concentration and insulin activated nitric oxide synthesis (IANOS) of each fraction were quantified. The Solid circle (---●---) and Solid Square (---■---) represented protein concentration and insulin activated nitric oxide synthesis respectively.
FIGURE-S2: Immunoblot for IgG-Fc.
Lane 1: Cell culture medium (DMEM) 30µl as negative control, Lane-2: IgG 5µg(Papain cleaved whole IgG) unreduced, Lane-3: AIHD inhibitory protein 5µg wererun on a SDS-PAGE and transferred to PVDF membrane and immunoblotted for IgG-Fc using Anti-human IgG-Fc specific antibody (Sigma: I2136).
Table-S1: Purification of IANOS inhibitory protein from AMI subject PFP.
Step / Total protein(mg) / Specific activity (nmol NO inhibited/mg protein/h). Units of inhibitory activity per one mg of inhibitor protein. / Total protein yield (%) / Fold purification
Plasma / 127.95 / 0.5111 / 100 / 0
DEAE Cellulose separation / 13.50 / 31,001 / 10.55 / 60,668
Sephadex G-50 separation / 3.00 / 69,741 / 2.34 / 136,480