Fig 2: Study 4: Inter- and intra-individual SMN signal variability.While PBMC SMN levels normalized by total protein levels or cell counts are similar within a group; measures of individuals across timepoints reveal large differences. The 6 h timepoint is 3 h post-prandial to a large carbohydrate rich meal. A: PBMC SMN by protein level are not different by group analysis from at T = 0 h through 30d after initial collection. B: PBMC SMN by cell counts is not different by group analysis. C,D: SMN protein levels in PBMCs normalized by protein or cell counts by individual vary up to 8x over multiple blood collection timepoints, and between individuals the SMN levels can diverge by >25x. In Figures 5A–B the bodies of the boxplots indicate the first and third quartiles, while the horizontal bar indicates the median. In Figures 5C–D error bars depict standard deviation.

Fig 3: SMN protein levels in tissues of C/C-allele and WT mice measured by SMN-ECL and SMN-ELISA.Protein levels were measured in the spinal cord of C/C-allele and WT mice using (A) SMN-ECL and (B) SMN-ELISA. Both assays showed a statistically significant difference in SMN levels between WT and C/C-allele mice (p < 0.0001). (C) SMN protein levels in the whole blood of C/C-allele, WT and heterozygous mice measured by SMN-ECL.

Fig 4: Study 1: Impact of short-term PBMC processing delays.PBMCs were collected via CPT tubes from 3 individuals and processed with delays of 0, 45 minutes, 2 h and 24 h prior PBMC isolation by centrifugation. A: Cell viability was similar at all timepoints for all subjects, ranging from 94–99%. B: Cell counts were consistent through 45 minutes, but were significantly reduced by 30–40% with delays of 2 h and 24 h compared to the 0 h timepoint. C: Total soluble protein was consistent with up to 2 h processing delays, however at 24 h there was a trend towards increased protein concentrations by up to 40%. D: SMN levels with 24 h processing delays were accordingly reduced when normalized by total protein. E: SMN levels by cell counts were similar with all delays examined, albeit with trends for higher variability than the SMN signal generated by protein normalization. In Figure 1 error bars indicate the minimum and maximum values while the horizontal bar indicates the median value.

Fig 5: SMN protein stability in whole blood: short term, long term, and freeze / thaw events.Whole blood of healthy subjects was used in the study. (A) SMN protein was measured in previously frozen, undiluted whole blood samples incubated at 4°C or at room temperature. (B) SMN protein was measured in undiluted whole blood samples of two subjects stored at -80°C or at -20°C. (C) SMN protein levels were measured in samples of two subjects that went through freeze-thaw cycles. *FDA acceptance criteria (below 85%).