Fuji las 4000 image analyzer

Third instar larval brains were manually homogenized in 1.5ml Eppendorf tubes at 65°C using a micropestle in an equal amount of 2X Laemmli buffer and the samples were heated at 95°C for 10 min. The debris was pelleted by centrifugation at 13000×g for 5 min and the protein sample equivalent to 5 heads was resolved on 8% denaturing SDS-PAGE. Proteins were transferred to the Hybond-PVDF-LFP membrane (Amersham, GE Healthcare Life Sciences) and blocked with 5% skimmed milk in 1X Tris-buffered saline, supplemented with 0.1% Tween-20 (TBST) for 1hr at room temperature. The membrane was probed with primary antibodies against Brp Nc82
(1:500) and α-Actin (Cell signaling) in 1X TBST containing 2% BSA at 4°C overnight followed by washing steps in TBST. This was followed by incubation with secondary antibodies conjugated to HRP in 1X TBST, (1:10000) for 1hr followed by washing steps. The membranes were incubated with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and signals were acquired using Fuji LAS-4000 Image Analyzer (Fuji Film, Tokyo, Japan). The images were analyzed using Image Gauge software (Fuji Film).

Proteins were extracted using the Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) per the manufacturer's instructions af-ter epithelial cell differentiation (days 3, 14, and 28). Protein concentration was determined using a BCA protein assay (Thermo Fisher Scientific) per the manufacturer's instructions. For western blot analysis, equal amounts of proteins were separated on 4-15% Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Hercules, CA, USA) and electro-transferred onto polyvinylidene difluoride membranes (Bio-Rad) for 1 h at 50 V. After blocking membranes with 1X Tris-buffered saline (TBS) with 1% casein (Bio-Rad), they were probed for 18 h at 4°C with primary antibodies, including rabbit monoclonal anti-P63 (1:500; Abcam, Cambridge, MA, USA), mouse monoclonal anti-cytokeratin (CK) 14 (1:500; Abcam), mouse monoclonal anti-CK 18 (1:500; Abcam), and mouse monoclonal anti-GAPDH (1:10,000; Thermo Fisher Scientific) antibodies. The membrane was washed with TBS with Tween 20 and then probed for 1 h at 25°C with either preabsorbed goat anti-mouse IgG H&L (HRP; 1:1,000; Abcam) and goat anti-rabbit IgG (H+L; 1:1,000; Thermo Fisher Scientific) used as secondary antibodies. Detection was performed using Clarity Western ECL (Bio-Rad), and a Fuji LAS 4000 image analyzer was used for imaging (Fujifilm, Tokyo, Japan).