** 0.01; error bars = sem. tissues were collected and snap frozen in chilled isopentane for use in later experiments. For immunofluorescence studies of mouse brain, frozen sections were prepared using a cryostat with Optimal Cutting Temperature (OCT) compound embedding (Sakura Finetek, Torrance, CA, USA). After mounting on slides, frozen sections were either fixed and permeabilized with ice-cold acetone or left unfixed and permeabilized with 0.1% Triton X-100 in Tris-buffered saline (TBS), then blocked for 1 to 2 2 h with 10% donkey serum, and 1% bovine serum albumin in Rabbit Polyclonal to Potassium Channel Kv3.2b TBS with 0.025% Triton X-100. Following this, sections were incubated overnight with primary antibodies diluted 1/100 in TBS with 0.025% Triton X-100: rabbit anti-SMIT1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-KCNQ2 (Santa Cruz Biotechnology). After washing 3 times for 5 min, sections were incubated for 1 to 2 2 h in secondary antibodies (1/200 in TBS) raised in donkey (Thermo Fisher Scientific, Waltham, MA, USA) before a final wash, mounting with DAPI-containing antifade Miglustat hydrochloride solution and visualization on an Olympus BX51 microscope with Cell-Sens software (Olympus, Tokyo, Japan). For mouse and rat sciatic Miglustat hydrochloride nerve sections, frozen sections were purchased from Zyagen (San Diego, CA, USA), permeabilized (not fixed) in TBS with 0.1% Triton X-100, blocked for 1 to 2 2 h with 10% donkey serum, and 1% bovine serum albumin in TBS with 0.1% Triton X-100, then incubated overnight with primary antibodies diluted 1/100 in TBS with 0.1% Triton X-100: rabbit anti-SMIT1 (Santa Cruz Biotechnology), rabbit anti-SMIT2 (MBL International, Woburn, MA, Miglustat hydrochloride USA), rabbit antiCankyrin G (Santa Cruz Biotechnology), and goat anti-KCNQ2 or KCNQ3 (Santa Cruz Biotechnology). Other steps were as for the mouse brain sections, as previously described. oocyte preparation and cRNA synthesis was performed using T3, T7, or SP6 mMessage mMachine kits (Ambion, Austin, TX, USA). SMIT1 cDNA was purchased from OriGene Technologies; SMIT2 cDNA was a gift from M. J. Coady and J.-Y. Lapointe [Groupe d’tude des Protines Membranaires (GPROM), Universit de Montral, Montral, QC, Canada]. cRNA quality was verified by spectrophotometry and gel electrophoresis. oocyte microinjection and radiolabeled prepulse voltage and fitted with a single Boltzmann function according to the following equation: where is the normalized tail conductance, 0.05). If multiple comparisons were performed, a Tukeys HSD test was performed after ANOVA. Protein biochemistry CHO cells were transfected using Mirus LT-1 transfection reagent (Mirus Bio LLC, Madison, WI, USA) with a total of 15 g cDNA per 10-cm plate and allowed 36 to 48 h expression at 37C before lysis. Lysis buffer was composed of 1% IGEPAL, 0.1% SDS, 50 mM Tris (pH 8.0), 150 mM NaCl, and a protease inhibitor cocktail tablet (Thermo Fisher Scientific). Total protein was quantified by the bicinchoninic acid method (Thermo Fisher Scientific). Proteins were resolved by SDS-PAGE and transferred onto PVDF membranes for immunoblotting with the following antibodies, as noted: KCNQ2 (Santa Cruz Biotechnology), DDK/FLAG (OriGene Technologies; Sigma-Aldrich), and SMIT1 (Santa Cruz Biotechnology). For secondary detection, horseradish peroxidaseCconjugated antibodies (Bio-Rad, Hercules, CA, USA) were used in conjugation with Luminata Forte horseradish peroxidase substrate (EMD Millipore, Billerica, MA, USA). Imaging was performed by G:Box hardware and software (Syngene, Frederick, MD, USA). For brain tissue biochemistry, tissue was minced and homogenized in a buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% IGEPAL, 1% CHAPS, 1% Triton X-100, 1% SDS, and 1 mini protease inhibitor cocktail tablet per 10 ml (Thermo Fisher Scientific) and mixed at 4C for 2 h. Samples were cleared of insoluble fractions by centrifugation before performing coimmunoprecipitation. For coimmunoprecipitation, all samples were first precleared of nonspecific interaction by incubating the total lysate with protein A/G PlusCcoated agarose beads (Santa Cruz Biotechnology) for 1 h. Beads were then pelleted and discarded. Total protein was quantified by.