Angular distributions of ER proteins around tubule cross-sections. (A) Possible models of ER protein distributions in tubule cross-sections. Model (i) shows the vectors that angle φ is being calculated between. (B–D) Representative regions of interest taken from datasets of each protein. Skeletons, Rtn4, Sec61β, and KDEL are displayed in white, green, magenta, and yellow, respectively. (E–G) Polar histograms of φ. The Rtn4, Sec61β, and KDEL observed data is shown by the green, magenta, and yellow histograms, respectively. The gray histograms are from simulated datasets of points randomly distributed in a circular ring around the skeleton that was created based on each set of observed data (P <<< 0.001 for each protein’s distribution compared to their simulated random distributions; Wilcoxon rank-sum statistic; n = 3 cells from unique sample preparations). The Rtn4, Sec61β, and KDEL data represents 2,589,190; 1,881,961; and 2,222,140 unique angle calculations total across the n = 3 biological replicates, respectively. Error bars represent the standard deviation of the frequency of each histogram bin between biological replicates. (H) Overview of a U-2 OS cell overexpressing mCherry-Sec61β (magenta) labeled with an anti-RFP nanobody and endogenous Rtn4 (green) labeled with primary and secondary antibodies. (I–N) Cross-sections of ER tubules with 3D views orthogonal to the tubule axis. Data was recorded with a 4Pi-SMS microscope. Scale bar: 1 μm (B–D and H); 50 nm (I–N).