Author response image 1. Ire1α concentration in HEK 293 microsomes.
Ire1α concentration in either HEK 293 microsomes or canine microsomes (CRM) was quantified relative to purified His-tagged Ire1α. We estimate 1μl of HEK microsomes (OD280 = 50) contain ∼150 pg or 1.4 nM of Ire1α. Tyedmers et al. (2000, PNAS) estimated that 1ul of CRM (OD280 = 50) contains ∼ 2.12 uM. Thus, 1 μl of HEK microsomes contain ∼ 0.4 uM since they contain 5X less concentration of Sec61. We therefore estimate that Ire1α concentration is ∼ 300X less than the Sec61 in HEK microsomes. Note that the five times less concentration of Sec61 in HEK relative to CRM is not likely biased by our Sec61α antibodies since both human and canine Sec61α peptide sequence are nearly identical.