Bolt bis tris gel

Frozen Neurospora tissue was ground in liquid nitrogen with a mortar and pestle. Total protein was extracted in buffer (50 mM HEPES (pH 7.4), 137 mM NaCl, 10% glycerol v/v, 0.4% NP-40 v/v, and cOmplete Protease Inhibitor Tablet according to instructions for Roche # 11 836 170 001) and processed as described [130 (link)]. Protein concentrations were determined by Bradford Assay (Bio-Rad # 500–0006). For western blots, equal amounts of total protein (30 μg) were loaded per lane into 4% to 12% Bis-Tris Bolt gels (Invitrogen # NW04125/NW04127) or 8% Bis-Tris Bolt gels (Invitrogen # NW00085/NW00087). Western transfer was performed using the Mini Blot Module (Invitrogen # B1000) and BOLT Transfer Buffer (Invitrogen # BT0006) onto an Immobilon-P PVDF membrane (Millipore # IPVH00010). Primary antibodies used for western blotting were anti-CK1a (1:2,000; rabbit raised) [52 (link)], anti-Tubulin alpha (1:5,000; Sigma # T6199), anti-WC-2 (1:5,000; rabbit raised) [131 (link)], and anti-FRH (1:10,000; rabbit raised) [77 (link)]. The secondary antibodies, goat anti-mouse or goat anti-rabbit HRP, were used at 1:5,000 (Bio-Rad # 170–6516, # 170–6515). SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher # 34578) or Femto Maximum Sensitivity Substrate (Thermo # 34095) was used for detection. Immunoblot quantification and normalization were performed in ImageJ.

For Western blot analysis of sera, 1 μl of serum for each sample was electrophoresed on 12% Bis-Tris Bolt Gels (Thermo Fisher, Italy), transferred to 0.45-μn PVDF membrane, and incubated overnight with the primary antibody rabbit anti-IL-10 (1:500, GeneTex — cod. GTX130513) and the secondary antibody goat anti-rabbit IgG-HRP (1:5000, Thermo Fisher cod. G-21040) 1 h at RT. Immunoreactivity was determined using the enhanced chemiluminescence reaction and captured by iBright CL1500 Imaging System. Densitometric analysis was performed using ImageJ software. Data were expressed as “fold of change” with respect to the nMSPLA group.
For Western blot analysis of PFC, proteins were separated by SDS-PAGE on 10% polyacrylamide gels, transferred electrophoretically to nitrocellulose membranes, and incubated overnight at 4 °C with the anti-GR primary antibody (GR, GeneTex, GTX101120; dilution 1:1000). The detection of hybridization occurs by measuring chemiluminescence to ChemiDoc (Bio-Rad). For this study, we utilized three animals in the nMSPLA group and four animals in the nMSOB, MSOB, and MSPLA groups.

Proteins were resolved by SDS-PAGE on precast gradient gels (4–12% Bis-Tris Bolt gels, Thermo Fisher) and transferred to Hybond-C membrane (Amersham Biosciences) using a Bio-Rad semi-dry transfer apparatus at 25 V for 45 min. The membrane was washed three times with PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄ pH 7.4, 0.1% Tween-20) and blocked with 5% milk-PBST (PBST, 5% skim milk) for 1 hr at RT. The membrane was then incubated overnight at 4°C with 1:2000 M2 Anti-FLAG antibody (Sigma-Aldrich) in 5% milk-PBST. The membrane was washed three times with PBST and then blocked with 5% milk-PBST for 30 min at RT. The membrane was incubated with 1:1000 anti-mouse IgG HRP-linked antibody (Cell Signaling Technology) in 5% milk-PBST for 1 hr at RT. The membrane was washed three times in PBST and then developed using Luminata Forte Western HRP substrate (Millipore).