Rabbit anti halotag

HUVECs were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X/PBS. Rabbit anti-phospho-PKA substrate (dilution, 1:200; #9624; Cell Signaling Technology), mouse anti-Myc-Tag (dilution, 1:200; #9B11; Cell Signaling Technology), rabbit anti-HA-tag (dilution, 1:200; #C29F4; Cell Signaling Technology), mouse anti-VE-cadherin (dilution, 1:200; #sc-9989; Santa Cruz), and rabbit anti-Halo-tag (dilution, 1:200; #G9281; Promega Corporation) were used as primary antibodies. Alexa 488 and 546 dye-labeled antibodies (dilution, 1:200; Molecular Probes) were used as the secondary antibodies. The actin cytoskeleton was detected using Acti-stain™ 555 phalloidin (dilution, 1:1000; #PHDH1-A; Cytoskeleton Inc.). The samples were mounted with ProLong™ Glass Antifade Mountant with NucBlue™ (P36981; Invitrogen Corporation). Immunofluorescence images were obtained using a Leica SP-8 confocal microscope. The intensity of stress fibers was quantified using ImageJ software (https://imagej.nih.gov/ij/).

Cells were fixed and immunolabeled similar to COS-7 ER samples described by Huang et al. (2016) (link). Briefly, cells were fixed with 3% paraformaldehyde (15710; Electron Microscopy Sciences) + 0.1% glutaraldehyde (16019; Electron Microscopy Sciences), permeabilized with 0.3% IGEPAL CA-630 (18896; Sigma-Aldrich) + 0.05% Triton X-100 (T8787; Sigma-Aldrich), and blocked with goat or donkey normal serum (Jackson ImmunoResearch). For Fig. 1 F, goat anti-Rtn4 (Nogo N-18; sc-11027; Santa Cruz Biotechnology) was labeled with custom-labeled secondary antibody: unlabeled donkey anti-goat antibodies (Jackson ImmunoResearch) were labeled per manufacturer’s directions with Atto594 NHS ester (Sigma-Aldrich). Goat anti-Rtn4 was imaged in cells that transiently expressed SNAP-Sec61β, which was labeled with SiR-BG in living cells (see Preparation for live-cell imaging section). For 4Pi-SMS (Fig. 2, E–I) and STED imaging of overexpressed GFP-Sec61β (Fig. S1, B–D), GFP was immunolabeled with rabbit anti-GFP (A-11122; Invitrogen) and a secondary goat anti-rabbit Alexa Fluor 647 (A-21245; Invitrogen) for 4Pi-SMS or Atto594 antibody (77671-1ML-F; Sigma-Aldrich) for STED imaging. Immunoblots were probed with rabbit anti-HaloTag (G9281; Promega) or goat anti-Rtn4 (sc-11027; Santa Cruz Biotechnology).