4.6. Quantitative dot blot, slot blot, and western blot analysis

The immunoblot analysis was performed as described previously with minor modifications [19]. Briefly, twenty μg of total schwannoma cell homogenate, or the indicated mass, was loaded directly onto a precut 0.2 μm nitrocellulose membrane (Cat. no. 1620168, Bio-Rad Laboratories, Germany) captured within the dot blot or slot blot apparatus (Model no. DHM-96, Scie-Plas, United Kingdom). Standard curves of site-specifically nitrated Hsp90, either Hsp90_NY33, or Hsp90_NY56 (0–200 ng) were included. The negative control consisted of 20 μg total normal Schwann cell homogenate. The nitrocellulose membrane was rinsed three times prior to protein incubation. The membranes were then blocked using TBS Odyssey Blocking Buffer (Cat. no. 927–60001, Li-Cor Biosciences, Lincoln, NE, USA), and incubated overnight at 4 °C with their indicated in-house developed primary antibodies against nitrated Hsp90, or an anti-myc tag antibody [[19], [20], [21]]. IRDdye secondary goat antibodies (Li-Cor Biosciences), anti-mouse (680RD, Cat. no. 925–68070), and anti-rabbit (800CW, Cat. no. 925–32211) were used at a 1:20,000 dilution. All dot blots were visualized and quantified with the Odyssey System (Li-Cor Biosciences). After densitometric analysis of dots, protein content was calculated as percent of total Hsp90 cell content. Western blotting was performed similarly to the dot and slot blots, except 75 μg of total cell homogenate was loaded.