Figure 3. Optic nerve crush decreased Kir4.1 protein and mRNA levels. Following retinal injuries with optic nerve crush, retinas were dissected and plasma membrane proteins were isolated. Total RNA was isolated and transcribed into cDNA. Real-time PCR was performed using specific primers (see Methods). mRNA expression of Kir4.1 was adjusted to the mRNA copies of ACTB (reference gene). Thirty microgram protein was loaded into each lane. Immunoreactive bands for Kir4.1 and β-tubulin 2, 7, and 14 days after optic nerve crush showing a significant reduction in Kir4.1 protein levels. Quantitative measurement using western blot showed that elevation of optic nerve crush decreased Kir4.1 protein levels (34±10, 72±6, 22±9, at 2, 7, and 14 day, respectively, A, n=7). Densitometric quantification is shown in B. Data are expressed as a ratio of the control value and each measurement represents mean±SEM *Denote statistical significance of Kir4.1 protein levels in optic nerve crushed retinas versus sham (p<0.005) as determined by one-way ANOVA and Tukey multiple comparison test. Results indicate that mRNA expression level of Kir4.1 was significantly lower in optic nerve crushed retinas compared to sham at 7 and 14 days (55±7%, 41±9%, at 7, and 14 day, respectively, B, n=6). By contrast, optic nerve crush increased Kir4.1 mRNA at 2 days (325±81%, B, n=6). **Denote significant differences compared with sham-retinas at p<0.05. Abbreviations: sham eye (C), and crushed (R).