RabGEF1 mutants can be expressed in mast cells. (A) Schematic of the RabGEF1 mutants used in this study, with the ZnF, Vps9, and coiled coil domains indicated. Numbering corresponds to amino acid residues in the mouse RabGEF1 sequence. Arrows indicate point mutations. All constructs were HA-tagged at the N-terminus. * indicates α-RabGEF1 (Ac-KSER) Ab epitope; **, α-RabGEF1 (BD Biosciences) Ab epitope. (B) Total cell lysates from +/+ and −/− BMCMCs infected with the indicated constructs were analyzed for RabGEF1 expression by Western blot using α-HA and α-RabGEF1 Abs. The blot was reprobed with α-GAPDH Abs to show loading. Results are representative of those obtained in 4 or more separate experiments. (C) RabGEF1−/− BMCMCs cells transiently transfected (24 hours) with the indicated GFP constructs were induced to adhere to fibronectin-coated coverslips and imaged in real time by spinning-disc confocal microscopy. Bar (for scale of all images in C) represents 10 μm. Cells were imaged by spinning disc confocal Axiovert 200M microscope (Zeiss, Germany) with a 63× oil objective and CoolSNAP HQ CCD camera. Images were collected using slidebook software (v. 4.1.15; 3I, Denver, CO) and exported into Adobe Photoshop (v. 7.0), where signals were balanced and cropped.