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Rab GTPases are essentially molecular switches. They serve as master regulators in intracellular membrane trafficking from the formation and transport of vesicles at the originating organelle to its fusion to the membrane at the target organelle. Their functions are diversified and each has their specific subcellular location. Their expression may vary significantly in the same cell when the level of protein production is significantly different in different physiologic status. One of the best examples is the transition from fetal to mature status of cells. Expression and localization of Rab GTPases in mature and developing brains have not been well studied. Immunohistochemistry is an efficient way in the detection, semiquantitation, and localization of Rab GTPases in tissue sections. It is inexpensive and fast which allow efficient mass screening of many sections. In this chapter, we describe the immunohistochemical assay protocol for analyzing several Rab protein expressions of the Rab5 subfamily, including Rab5, Rab17, Rab22, and Rab31, in developmental (fetal) and mature human brains.

// Moshe Shashar 1 , Jamaica Siwak 1 , Umit Tapan 2 , Shin Yin Lee 2 , Rosana D. Meyer 3 , Paige Parrack 1 , Josenia Tan 3 , Fatemeh Khatami 1 , Jean Francis 1 , Qing Zhao 3 , Kevan Hartshorn 2 , Vijaya B. Kolachalama 4,5 , Nader Rahimi 3 and Vipul Chitalia 1,4 1 Department of Medicine, Boston University School of Medicine, Boston, MA, USA 2 Department of Medicine, Hematology-Oncology Section, Boston University School of Medicine, Boston, MA, USA 3 Department of Pathology, Boston University School of Medicine, Boston, MA, USA 4 Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA 5 Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA, USA Correspondence to: Vipul Chitalia, email: // Keywords : colorectal cancer, Cbl, Wnt Received : June 16, 2016 Accepted : August 21, 2016 Published : September 20, 2016 Abstract Despite the loss of Adenomatous Polyposis Coli ( APC ) in a majority of colorectal cancers (CRC), not all CRCs bear hallmarks of Wnt activation, such as nuclear β-catenin. This underscores the presence of other Wnt regulators that are important to define, given the pathogenic and prognostic roles of nuclear β-catenin in human CRC. Herein, we investigated the effect of Casitas B-lineage lymphoma (c-Cbl) on nuclear β-catenin, which is an oncoprotein upregulated in CRC due to loss-of-function APC or gain-of-function CTNNB1 mutations. Despite mechanistic rationale and recent discoveries of c-Cbl’s mutations in solid tumors, little is known about its functional importance in CRC. Our study in a cohort of human CRC patients demonstrated an inverse correlation between nuclear β-catenin and c-Cbl. Further investigation showed that the loss of c-Cbl activity significantly enhanced nuclear β-catenin and CRC tumor growth in cell culture and a mouse xenograft model. c-Cbl interacted with and downregulated β-catenin in a manner that was independent of CTNNB1 or APC mutation status. This study demonstrates a previously unrecognized function of c-Cbl as a negative regulator of CRC.