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Fig. 7 | BMC Neuroscience

Fig. 7

From: Histological characterization of orphan transporter MCT14 (SLC16A14) shows abundant expression in mouse CNS and kidney

Fig. 7

Validation of antibody specificity. The specificity of the antibody: a representation of the human SLC16A14 and mouse Slc16a14 and the epitopes for the MCT14 antibody in human and mouse. The epitope for the commercially available MCT14 antibody is found in the second exon (E2) in both the human and mouse MCT14 proteins. The bottom most box shows the peptide sequences encoded by E2 in human and mouse, respectively. The underlined sequence encoded by the human exon constitutes the epitope of the antibody (31 amino acids) and the underlined sequence encoded by the mouse E2 is the corresponding epitope in mouse (ten amino acids). Graphics were generated using the Exon–Intron Graphic Maker [58] and the scale bars (upper right corners of each gene) represent 100 bp. b A Western Blot on wild-type mouse brain homogenate was performed to analyze the specificity of the polyclonal anti-MCT14 antibody. It generated a strong band at ~56 kDa corresponding to the mouse MCT14 protein (NP_082197) and a weaker band at ~45 kDa corresponding to the 48 kDa mouse isoform (EDL02188.1). c A peptide competition with anti-MCT14 and peptide corresponding to the epitope recognized by the antibody. The right lane shows a Western Blot of anti-MCT14 on wild-type mouse brain homogenate, resulting in a band at ~56 kDa. The left lane shows anti-MCT14 neutralized with an excess of peptide, resulting in a very faint band with the same size. Bottom blot shows the loading control, β-actin, with expected bands around 42 kDa. Densitometric analysis of MCT14 bands normalized against the β-actin control showed a 40 % reduction of band intensity when blocked antibody was blotted. d Double IHC on immortalized PC12 cells transfected with FLAG-tagged plasmids containing the full Slc16a14 mRNA sequence with the MCT14 antibody and FLAG antibody revealed colocalization of MCT14 and the FLAG. MCT14 staining is red, the FLAG marker is green and cell nuclei are stained with DAPI. Negative control for the immunocytochemistry (no primary antibodies) and for the transfection (no cDNA during transfection) can be seen in the upper and lower right corners, respectively. All images were acquired with ×40 magnification

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