Figure 4

PRG3 is neuron specific. (A) qRT-PCR analysis revealed a ten times stronger PRG3 mRNA expression in neurons compared to astrocytes and microglia. Data were normalized to GAPDH. (B) WB from total protein lysates of primary brain cells. PRG3 protein was only detected in neurons. The membrane was reprobed with anti-tuj1, anti-GFAP, and anti-Iba1 to verify the quality of the lysates (right). Anti-β-actin was used as loading control. (C) Verification of the PRG3 shRNA and the shRNA resistant PRG3 construct by WB. GFP-mPRG3 was reduced when compared to control or to the PRG3 shRNA-resistant protein levels. Ratios below the WB bands refer to densitometric measurements of the gray intensity of these bands in relation to the control band. Anti-β-actin was used as loading control and for normalization. (D) Quantification of the efficiency of PRG3 shRNA. PRG3 shRNA (n = 28) expression reduced the endogenous PRG3 expression to more than 50% when compared to controls (n = 30). (E) Recordings of membrane voltage in response to a family of 1 second long square current pulses (see inset) from neurons expressing GFP (upper left), GFP-mPRG3 (lower left), control vector (upper right), or PRG3 shRNA (lower right). Irrespective of the PRG3 protein level, voltage responses including repetitive action potentials were roughly similar among all neurons. (F) Population data of intrinsic neuronal characteristics are displayed for the four groups of neurons depicted in D. Individual values for each neuron are shown at the right of each box plot. No differences in the resting membrane potential, input resistance, or capacity were found between GFP (n = 12), GFP-mPRG3 (n = 13), shRNA control (n = 4) or PRG3 shRNA-expressing cells (n = 6). An ANOVA resulted in p values of p = 0.07 (resting membrane potential), p = 0.15 (input resistance), and p = 0.51 (capacity).