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. 2015 Oct 20;15(2):493–505. doi: 10.1074/mcp.M115.049288

Fig. 2.

Fig. 2.

Confirmation of differential protein expression by immunoblot analysis. Cell lysates from three independent GCLM(+/+) and GCLM(−/−) astrocyte cultures were analyzed by Western blot to determine the protein expression levels of (A) glutamate-cysteine ligase catalytic subunit (GCLC); (B) catalase (CAT); (C) glucose-6-phosphate dehydrogenase (G6PD); and (D) 6-phosphogluconate dehydrogenase (6PGD). Protein loading was corrected by β-actin levels. The membrane in (A) was stripped and reprobed for G6PD (C) while membrane in (B) was stripped and reprobed for 6PGD (D). Hence, β-actin panels for A and C and B and D, respectively, are the same. Densitometric analysis is expressed as percentage of GCLM(+/+). *Significantly different from GCLM(+/+) (p ≤ .05). (E) Total RNA was extracted from GCLM(+/+) or GCLM(−/−) confluent astrocyte monolayers and GCLC, CAT, G6PD, and PGD mRNA levels were determined by real-time PCR and corrected by actin mRNA levels. mRNA levels are expressed as percentage of GCLM(+/+). *Significantly different from GCLM(+/+) (p ≤ .05). (F) Nuclear fractions were prepared from confluent GCLM(+/+) or GCLM(−/−) astrocyte cultures treated for 48 h with vehicle or glutathione ethyl ester (GSHEE; 5 mm). To determine the level of nuclear Nrf2, samples were analyzed by Western blotting using specific antibodies. As a loading control, membranes were stripped and reprobed for TATA-binding protein (TBP).