Extended Data Fig. 7: Controls and quantification for pulldown experiments.
From: DNA nanodevice for analysis of force-activated protein extension and interactions
a) Vinculin knockdown efficiency. Normalizing the vinculin band intensities by the GAPDH band intensities on a western blot of wild-type (WT) and vinculin-depleted (Vin-/-) cell lysates shows a knockdown efficiency of ~87%. b) FlnA-GFP pulldown by an elastic peptide (GPGGA)8, dubbed F40, shows no apparent differences on western blot before (-F) and after (+F) force application to F40. The gel on the right contains 8 times as much sample as the left. c) Western blot showing force-activated R1-R2 binding to FlnA-GFP, but not GFP, in lysates of NIH3T3 cells overexpressing the corresponding protein. Ctrl: DNA device without proteins, -F/+F: Origami-R1-R2 before/after force application. d) Purified FlnA-GFP examined by SDS–PAGE. e) SDS–PAGE of purified S-R1-R2-H (unconjugated, 87.3 kDa) and VinD1-FLAG (expressed in E. coli, 33.5 kDa) loaded with a range of known concentrations, as well as the mixture of a pulldown experiment loaded in two lanes with 2-fold concentration difference. f) Determining concentrations of S-R1-R2-H (top) and VinD1-FLAG (bottom) in the pulldown sample. Band intensities of the purified proteins (black dots) measured from e were used to construct calibration lines by linear regression. The intensities of the corresponding protein bands in the pulldown sample (crosses, measured from the 1× pulldown lane) were then used to determine the concentrations of S-R1-R2-H released from DNA devices and the pulled-down VinD1-FLAG (expressed in 293 T cells, 30.9 kDa). The resulting VinD1:R1R2 molar ratio is 163 nM/412 nM≈0.40. For experiments in b, c and e, DNA spring F1/2 ≈ 12.8 pN, gap=6 nm.
