Proteins were separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose. Membranes were blocked with 3% nonfat dry milk (NFDM) in PBST (phosphate-buffered saline supplemented with Tween 20) for 1 hour at RT and probed with primary antibody diluted in 0.5% NFDM in PBST overnight at 4°C. After incubation with horseradish peroxidase–coupled secondary antibody, signals were visualized by enhanced chemiluminesence (GE Healthcare) and exposure of x-ray films. X-ray films of two to three exposures were scanned using a CanoScan LiDE220 scanner (Canon), and ImageJ was used for quantification of Western blot signals. For lysate quantifications, a 3× twofold dilution series of the strongest signal was fit to a linear or logarithmic trend line by Excel. The equation of the scan with the best R2 value was used to calculate the values of the Western blot signals (fig. S1B).

All experiments were performed at least three times (n), and the values were presented as means ± SD; n in the figure panels and/or figure legend indicates the number of independent experiments. Statistical significance of immunoblotting and ELISA data were assessed using Student’s t test for pairwise comparisons and one way analysis of variance (ANOVA) and a Tukey’s honestly significantly different (HSD) post hoc test for threefold comparisons. In all cases, P values of <0.05 were considered to be statistically significant and are indicated with *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. Statistical analysis was performed in Excel using the data analysis and real statistics add-ins.

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