Western blot was performed using Anti-HB9 Polyclonal Antibody (Product # PA5-67195) and a 50 kDa band corresponding to HB9 was observed across all cell lines tested except DU 145 and PC-3 which are known to be low expressing (DOI: 10.1158/0008-5472.CAN-16-0087). Nuclear enriched extracts (30 µg lysate) of Caco-2 (Lane 1), HEK-293 (Lane 2), BeWo (Lane 3), LNCaP (Lane 4), DU 145 (Lane 5) and PC-3 (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (0.2 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 Reagent Kit (Product # WP20005). Two uncharacterized bands were observed at ~15 kDa and 110 kDa.
Immunofluorescence analysis of HB9 was performed using 70% confluent log phase Caco-2 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 HB9 Polyclonal Antibody (Product # PA5-67195) at 0.2 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then 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). Panel d represents the merged image showing predominantly nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60x magnification.
Supplier Page from Thermo Fisher Scientific for HB9 Antibody