Figure 3. Forming styrene maleic acid lipid particles (SMALPs).

(A) Schematic representation of the SMALPs extraction and nAChRs pull-down for mass spectrometric analysis. (B) Negative staining of extracted SMALPs by transmission electron microscopy, n=3. Scale bar 100 nm. (C) Western blot for Dα6-mVenus nAChR with and without enrichment using α-Btx, n=2. Detected with anti-GFP antibody. The fusion protein was detected at approximately 83 kDa. Ponceau S staining was used as sample equal loading control. (D, E) Negative staining of extracted SMALPs after α-Btx pull-downs, n=3, ring-like protein structures are boxed (scale bar = 100 nm) with an example in the magnified image (scale bar = 20 nm). A top view of the nAChR structure from PDB entry 4HQP is shown for reference.

Figure 3—figure supplement 1. Coupling α-Btx and testing pull-down efficiency with affinity beads.

(A) Fluorescence signal of uncoupled α-Btx in solution before and after coupling to affinity beads (two-tailed t-test, ***p<0.001, n=4). (B) Biological replicate: same condition as in Figure 3C, samples were reduced with 1% DTT. To allow the detection of the fusion protein Dα6-mVenus an anti-GFP (Ab252881) antibody was used. The fusion protein was detected at approximatly 83 kDa. (C) Pull-down samples were not reduced with 1% DTT. A signal at about 180 kDa was also detected and mostly indicative of a dimer. (D) Biological replicate: same condition as in (C), samples were not reduced with 1% DTT. Ponceau S staining was used as sample an equal loading control.