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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: J Neurochem. 2013 Nov 25;129(1):48–59. doi: 10.1111/jnc.12527

Figure 1. Under-expression of CHGB by siRNA in chromaffin cells.

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1a. Left: Immunoblot of CHGB protein in siRNA-transfected PC12 cells. The rat CHGB siRNA siGENOME “smart pool” was transfected into PC12 cells and silencing of CHGB expression was evaluated by immunoblotting at the target protein level. ~95% knockdown of CHGB expression was achieved by 15 nM siRNA. Right: Quantification of CHGB in the blot. Band intensity was scanned and scored by ImageJ. 1b. Expression of catecholamine pathway (storage, synthesis) genes in CHGB siRNA-silenced PC12 cells. The rat CHGB siRNA siGENOME “smart pool” was transfected at 15 nM into PC12 cells and transcript expression was scored by real-time qPCR, and normalized to the signal for β-actin. Each condition was repeated four times. During knockdown, the CHGB transcript was under-expressed, while Chga and Dbh were over-expressed, but Scg2 and Th were unchanged. 1c. Visualization of dense-core granules in CHGB siRNA-silenced PC12 cells by transmission EM. PC12 cells were transfected with the rat CHGB siRNA siGENOME “smart pool” at 15 nM and visualized by Zeiss EM10B electron microscope. CHGB siRNA-silenced PC12 cells display fewer dense-core granules than control (mock-transfected) cells. Arrowheads: Dense core secretory granules. Bar: 2 µm. 1d. Quantification of dense-core chromaffin granule abundance after CHGB silencing by siRNA. The data come from figure 1C. Left, granule number, n=8 cells were counted for each condition. Right, granule diameter, n=20 granules were measured for each condition. 1e. Effect of CHGB silencing on exogenous catecholamine uptake and secretagogue-triggered release. PC12 cells were labeled with [3H]-L-norepinephrine and treated with nicotine at 60 µM.The supernant and cell lysates were collected to measure radioactivity. The uptake was expressed as counts in cell lysate. The percent (%) secretion was expressed as [amount released/(amount released + amount in cell lysate)] × 100. Each condition was repeated three times. CHGB-silenced PC12 cells displayed diminished uptake of exogenous catecholamine, as well as reduced release catecholamine when stimulated by nicotine.