Immunofluorescence analysis of PDK1 was performed using 70% confluent log phase HeLa 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 1 hour at room temperature. The cells were labeled with PDK1 Polyclonal Antibody(Product # PA5-79797) at 1µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 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). Panel d represents the merged image showing mitochondria like 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-Pyruvate dehydrogenase kinase isozyme 1 protein Polyclonal Antibody (Product # PA5-79797) and 51 kDa band corresponding to Pyruvate dehydrogenase kinase isozyme 1 protein was observed in all the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), Hep G2 (Lane 3), Mouse heart (Lane 4) and Rat heart (Lane 5) were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0342BOX). 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, 0.25 µg/mL, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).