Fig 1: Kindlin-3 expression in human tumorsA. Downregulation in Kindlin-3 mRNA observed in human tumors from several origins compared to normal corresponding tissues Relative expression of Kindlin-3 mRNA to the reference gene PPIA assessed by qRT-PCR (a) Melanoma tissues compared to benign melanocytic naevi and normal skin tissues (n=71, n=11 and n= 17 respectively); (b) Primary breast tumors compared to normal breast tissues (n=129 and n= 23 respectively); (c) Kidney cancers compared to normal kidney (n=15 and n= 11 respectively); (d) 18 matched pairs of lung cancer versus normal lung tissues from the same patients. P values were calculated using two-sided Student t test. Means and 95% confidence intervals are shown;*, P <0.05. B. Immunohistochemical staining of Kindlin-3 in sections of human cancer and normal tissues (a) Expression of Kindlin-3 in endothelial cells within blood vessels, as positive control; (b and c) naevi and melanoma; (d and e) normal and breast cancer (f and g) normal and kidney cancer respectively. Higher magnification images are shown below each image for breast and kidney tissues. The illustrated immunostaining shown was performed with the Genosphere anti-Kindlin-3 Ab. Images are representative of 6 tumor specimens for each cancer type. Similar staining was obtained using the Abcam anti-Kindlin-3 Ab. Note that staining is always lower in the corresponding cancer tissues. Scale bar = 20μm. C. Specificity of the anti-Kindlin-3 antibodies used Western blot analyses of N-terminal GST-tagged recombinant Kindlin-1 (300ng), Kindlin-2 (300ng) or Kindlin-3 (150ng) proteins (H00055612-P01, H00010979-P01, H00083706-P01; Novus Biologicals Cambridge, UK). Blots were probed with 2 different kindlin-3 antibodies (upper panels) or GST antibody; Millipore) (lower panels). A single band corresponding to GST-tagged Kindlin-3 was obtained with both antibodies used, ab68040 (Abcam, Cambridge MA, USA) and the custom made anti-Kindlin-3 antibody produced against a human Kindlin-3 specific peptide corresponding to amino acids 19 –31 (RVFVGEEDPEAES) and affinity purified on a sepharose matrix (Genosphere, France). Importantly, neither of the two antibodies recognized GST tagged Kindlin-1 or Kindlin-2 even when 2x quantity of these recombinant proteins was loaded.