Immunofluorescent analysis of Calreticulin (green) U2OS cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA in PBS 0.1% triton-X (Product # 37525) for 30 minutes at room temperature. Cells were probed with a rabbit polyclonal antibody recognizing Calreticulin (Product # PA3-900) at a dilution of 1:50 for at least 1 hour at room temperature. Cells were washed with PBS and incubated with DyLight 488 goat-anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:250 for 30 minutes at room temperature. Actin filaments (red) were stained with DyLight 554-Phalloidin (Product # 21834) at a dilution of 1:300 (i.e. 1unit/mL final concentration) in PBS and incubated for 30 min including. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249) at a dilution of 1 µg/mL. Images were taken on a Thermo Scientific ArrayScan VTI at 20X magnification.

Western blot was performed using Calreticulin Polyclonal Antibody (Product # PA3-900) and a ~55kDa band corresponding to Calreticulin was observed across all the cell lines tested with a uncharacterized bands (*). Whole cell extracts (30 µg lysate) of HL-60 (Lane 1), LNCaP (Lane 2), HeLa (Lane 3), MCF-7 (Lane 4), Mouse Liver (Lane 5) and Rat Liver (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 (1:1000 dilution) 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).