Immunofluorescence analysis of Reticulon-4 was performed using 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Nogo-A Polyclonal Antibody (Product # PA5-20366) at 2.5 µg/mL in 0.1% BSA, incubated at 4-degree Celsius overnight, and labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Western Blot was performed using Anti-Nogo-A Polyclonal Antibody (Product # PA5-20366) and a 200 kDa band corresponding to Reticulon-4 was observed across all the cell lines and tissue lysates tested except for MCF7 as reported across protein expression databases. Bands at ~29 kDa and ~50 kDa corresponds to Nogo- B. Membrane enriched extracts (30 µg lysate) of U-87 MG (Lane 1), SH-SY5Y (Lane 2), IMR-90 (Lane 3), Daudi (Lane 4), MCF7 (Lane 5), Mouse Brain (Lane 6), Rat Brain (Lane 7), Mouse Cerebellum (Lane 8), Rat Cerebellum (Lane 9), Mouse Heart (Lane 10), Rat Heart (Lane 11) were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).