NRVMs were cultured for 24 hours prior to treatment with the muscarinic receptor agonist, pilocarpine (10μM), or volume corrected vehicle control, saline, for 48 hours. A and B, western blot analyses of the key proteasome subunits, RPT6 and Beta5. Representative images (A) and the summary of pooled densitometry (B) are shown. β-Tubulin was probed as the loading control. N= 6 per treatment. C, Representative images of 2D western blots for Rpt6 and β5 subunits in cultured NRVMs harvested 15 min after pilocarpine (Pilo, 10μM) or volume corrected saline (Sal) treatment. In gel for the 2nd dimension electrophoresis, the isoelectric focusing gel strip (pH 3-10) was placed in the large central well sandwiched by small wells (denoted by arrowheads) that were loaded with the input of the first dimension.