Knockdown of LOXL2 was achieved by transfecting U87MG cells with LOXL2 specific validated siRNA.

Western blot analysis was performed using modified whole cell extract (1% SDS) from the LOXL2 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and nontransfected cells (lane 1).

The blots were probed with ab277807 at 1-3 μg/mL and a Goat anti-Rabbit IgG (H+L)-HRP secondary antibody (0.4 μg/mL, 1/2500 dilution).

Loss of signal upon siRNA mediated knock down confirms that antibody is specific to LOXL2.

For immunofluorescence analysis, HeLa cells were fixed and permeabilized for detection of endogenous LOXL2 using ab277807 at 5 μg/mL and labeled with Goat anti-Rabbit IgG (H+L) secondary antibody, Alexa Fluor^® 488 conjugate (1/2000). Nuclei (blue) is stained with DAPI and cytoskeletal F-actin (red) staining using Rhodamine Phalloidin (1/300).

Panel a-d) Shows representative un-treated cells that were stained for detection and localization of LOXL2 protein (green) with less signal.

Panel e-h) Demonstrate enhanced perinuclear localisation of LOXL2 in cells treated with Cobalt chloride (100M 16h).

The images were captured at 60X magnification.

Western blot analysis was performed on whole cell extracts (30 μg lysate) of HeLa (human epithelial cell line from cervix adenocarcinoma) (Lane 1), HeLa treated with cobalt chloride (100 M for 16 hrs) (Lane 2), U-87 MG (human glioblastoma-astrocytoma epithelial cell line) (Lane 3), U-87 MG treated with cobalt chloride (100 M for 16 hrs) (Lane 4), MDA-MB-231 (human breast adenocarcinoma cell line) (Lane 5), MDA-MB-231 treated with cobalt chloride (100 M for 16 hrs) (Lane 6), A549 (human lung carcinoma cell line) (Lane 7) and A549 treated with cobalt chloride (100 M for 16 hrs) (Lane 8).

The blots were probed with Anti-LOXL2 Recombinant Rabbit Multiclonal Antibody (ab277807, 2.5 μg/mL) and detected by chemiluminescence using a Goat anti-Rabbit IgG (H+L)-HRP secondary antibody, (0.4 μg/mL, 1/4000 dilution). A 87 kDa band corresponding to LOXL2 was observed across the cell lines tested.

4-12% Bis-Tris gel.

The membrane was probed with the relevant primary and secondary antibody following blocking with 5% skimmed milk.

Chemiluminescent detection was ECL.