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. 2021 Apr 23;153(7):e202012811. doi: 10.1085/jgp.202012811

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© 2021 Rasicci et al.

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Figure S2. — Quantitative Western blot for hRLC exchange efficiency. Representative Western blot used to quantitate human RLC (hRLC) exchange in M2β-S1. Image is a representative Western blot (primary antibody: α-HIS; secondary antintintibody: α-mouse 680 nm) used to calculate the efficiency of the RLC exchange. WT and K104E M2β-S1 were loaded in decreasing amounts and compared with a RLC standard curve. Lanes of the SDS-PAGE gel were loaded with the following samples: (1) mol wt marker (BioRad Precision Plus Protein Standard: 250, 150, 100, 75, 37, 25, 20, 15, and 10 kD, respectively), (2–6) decreasing amounts of His-tagged hRLC standard protein (564, 423, 282, 141, and 70.5 ng WT RLC, respectively), (7–9) decreasing amounts of M2β-S1 WT RLC (20, 10, and 5 µl, respectively), and (10–12) decreasing amounts of M2β-S1 K104E RLC (20, 10, and 5 µl, respectively). Concentration was calculated by gel densitometry and compared with Bradford assay of total M2β-S1 concentration as a measure of exchange efficiency. Blots from three protein preparations were averaged to calculate heavy chain:light chain ratio (± SD): 0.97 ± 0.11 M2β-S1:WT RLC and 0.96 ± 0.11 for M2β-S1 K104E RLC.