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. 2008 Aug 19;105(34):12319–12324. doi: 10.1073/pnas.0800340105

Fig. 3.

Fig. 3.

V-ATPase accessory subunit Ac45 is abundant in islets of Langerhans and is ex vivo processed by furin. (A) Relative Ac45 mRNA expression in different tissues determined by quantitative reverse transcription PCR: 1, islets of Langerhans; 2, pancreatic acini; 3, pituitary gland; 4, adrenal gland; 5, lung; 6, brain; 7, bone marrow; 8, small intestine; 9, liver; 10, heart. The Ac45 expression of every sample was normalized to GAPDH and relative to the Ac45 expression in βTC3 cells, which was arbitrarily set at 1. Data represent mean ± SEM, n = 3. *, P < 0.05. (B) RPE.40 cells were transfected as indicated. (Co)transfection with empty pcDNA3 vector (−) was used as a negative control. Transfected RPE.40 cells were radiolabeled, and FLAG-tagged proteins were immunoprecipitated with M2 anti-FLAG antibody. Because of heterogeneity of glycosylation, the separation of glycosylated proAc45 and Ac45 by SDS/PAGE resulted in a smear (indicated with {). After deglycosylation with N-glycosidase F (N-glyc F; right two lanes), distinct protein bands were detected. The indicated positions of proAc45 and Ac45 correspond to the predicted Mw of the unglycosylated peptide backbone (46 and 24 kDa, respectively). Molecular masses are indicated (kDa).